CN101370498A - Targeting GLI proteins in human cancers by small molecules - Google Patents

Abstract

The present invention provides compositions, methods, and kits for diagnosing and treating cancers that express GLI polypeptides, particularly GLI1, GLI2, or GLI3 polypeptides. The present invention also provides small molecule compounds that mimic the transcriptional activation domains of GLI polypeptides. The small molecule inhibitors of the present invention specifically block the activating function of GLI polymorphisms, but not the repressive function of GLI3.

Description

Targeting the GLI protein in human cancer by small molecules

Cross References to Related Applications

This application claims U.S. Provisional Application Serial No. 60/748,968 filed December 9, 2005 and U.S. Provisional Application Serial No. 60/763,829 filed January 30, 2006, U.S. Provisional Application Serial No. 11 filed December 7, 2006 /608, priority of 197, which is incorporated herein by reference in its entirety.

field of invention

The present invention relates generally to compositions and methods for inhibiting tumorigenesis, tumor growth and tumor survival. The composition comprises small molecule compounds that inhibit Hedgehog and GLI signaling pathways.

Background of the invention

Hedgehog (Shh or Hh), WNT, FGF and BMP signaling pathways form a network during embryogenesis, tissue regeneration and carcinogenesis. The pathological consequences of aberrant activation of the Hh signaling pathway are various human tumors, such as gastric and pancreatic cancers. Hedgehog is a secreted glycoprotein that initiates Hh signaling by binding a transmembrane protein complex containing PATCHED1 (ptch1) and SMOOTHENED (smo) and initiating a cascade of cytoplasmic signaling events, including inhibition leading to GLI zinc Refers to protein kinase A transcribed by the transcription factor. Next, the GLI family of zinc finger transcription factors translates extracellular Hh stimulation into a defined transcriptional program in a content-dependent and cell-type-specific manner (Ruiz I Altaba et al., 2002, Nat. Rev. Cancer 2:361-72).

Several proteins, including the GLI protein, are involved in mediating Hh signal transduction (Katoh and Katoh, 2005, Cancer Biol. Ther. 4:1050-4). Vertebrates have at least three different GLI proteins, GLI (also known as GLI1), GLI2 and GLI3. These proteins are members of the GLI family of zinc finger transcription factors, share a highly conserved C ₂ -H ₂ zinc finger domain (containing five zinc finger DNA-binding motifs), and contain Drosophila wing vein interrupt (Cubitus interruptus ) (Ci) and the Caenorhabditis elegans sex-determining gene tra-1 (Hui et al., 1994, Dev. Biol. 162:402-13). In Drosophila, Ci is required to activate hedgehog targets and Ci also acts as a repressor of hedgehog expression.

During vertebrate embryonic development, all genes with partially overlapping expression are transcriptionally activated in response to Hh signaling, mediating most of the effects of activation of this pathway. During vertebrate development, GLI1, GLI2 and GLI3 each have a specific function (Kinzler et al., 1984, Nature 332: 371-374; Ruppert et al., 1988, Mol. Cell. Biol. 8: 3104-3113; Walterhouse et al., 1993, Dev. Dyn. 196:91-102; Hui et al., 1994, Dev. Biol. 162:402-413). For example, Gli2 mutant mice display severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral bodies and intervertebral discs, short limbs and sternum (Mo et al., 1997, Development 124:113-23).

Gli3 is an important control gene in the development and differentiation of several body structures. For example, decreased gene numbers lead to severe disturbances, especially for limb morphogenesis (Vortkamp et al., 1995 DNA Cell Biol. 14:629-34). In addition, mutations in GLI3 have been identified in several human dysmorphic syndromes, such as Gregg's cephalopolydactyly syndrome (GCPS), which affects limb and craniofacial development, and the autosomal dominant form of Pallister-Hall syndrome. (Vortkamp et al., 1991, Nature 352:539-40; Bose et al., 2002, Hum. Mol. Genet. 11:1129-35). In mice, GLI3 mutations have been implicated in the mutant extra digits of mice.

GLI proteins act as transcriptional activators, binding through their zinc finger domains to DNA binding sites within the promoter and/or enhancer sequences of target genes. In recent years, DNA binding sequences to which GLI proteins bind have been identified. For example, the DNA sequence bound by the GLI3 zinc finger consists of 16 nucleotides, which is highly similar to the sequences bound by GLI and tra-1 proteins (Vortkamp et al., 1995, DNA Cell. Biol. 14:629-34). This binding site includes the 9-base pair sequence 5'-GACCACCCA-3' previously identified in the GLI protein (Kinzler and Vogelstein, 1990, Mol. Cell Biol 10:634-42). GLI, GLI2 and GLI3 are believed to bind the same or similar DNA sequences (Yoon et al., 1998, J. Biol. Chem. 273:3496-3501). In recent years, Hallikas et al. verified the 9bp consensus sequence 5'-GACCACCCA-3' as the binding site of GLI1, GLI2 and GLI3 proteins (Hallikas et al., 2006, Cell 124:47-59).

Activation of Hh/GLI target genes by GLI2 and GLI3 proteins may require the transcriptional coactivator Creb Bing protein (CBP), which binds the CBP binding domain (see Figure 1; Dai et al., 1999, J. Biol. Chem. 274:8143- 52; Kasper, Regl, Frischauf and Aberger, 2006, Eur. J. Cancer).

GLI1 expression has been reported to be under the control of the GLI2 and GLI3 proteins (Dai et al., 1999, J. Biol. Chem. 274:8143-52). While the entire GLI1 protein is a transcriptional activator, GLI2 and GLI3 contain both activation and repression domains (see Figure 1). GLI2 and GLI3 can be processed through the action of protein kinase A (PKA) to achieve different functions. Full-length GLI2 acts as a weak transcriptional activator. Truncating the C-terminal activation domain in half produces a protein with repressor activity, whereas removal of the N-terminal repressor domain turns GLI2 into a strong activator. In transgenic mouse embryos, unlike the full-length protein, the N-terminally truncated GLI2 activates, for example, the Sonic hedgehog (Shh) gene HNF3β. This suggests that exposing the activation domain of GLI2 is one of the key mechanisms of the Shh signaling pathway. A similar regulatory mechanism involving the N-terminal region has also been described for GLI3 (but not GLI). (Sasaki et al., 1999, Development 126:3915-24, the entire text of which is incorporated herein by reference).

Comparison of murine and human GLI3 cDNA revealed an overall identity of 85% between the deduced amino acid sequences, with greater conservation (>95%) in certain domains, including zinc fingers (Thien et al., 1996 , Biochim. Biophys. Acta 1307:267-9).

In recent years, a transcriptional activation domain of GLI was identified at the carboxy-terminus comprising amino acid residues 1020-1091. This domain comprises an 18 amino acid α-helix (amino acids 1037-1054) containing 6 aspartic or glutamic acid residues or (Yoon et al., 1998, J. Biol. Chem. 273:3496-3501) . This α-helical region is very similar to the transcriptional activation domain of herpes simplex virus protein 16 (VP16), containing the conserved motif FXXΦΦ (F = phenylalanine; X = any residue; Φ = any hydrophobic residue), called Common recognition element for the acidic activation domain of TAF _II 31. In addition, three conserved amino acid residues (Asp472, Phe479 and Leu483 in VP16 and Asp1040, Phe1048 and Leu1052 in GLI) were found to be in direct contact with TBP-associated factor (TAF) TAF _II 31 (Yoon et al., 1998, J .Biol.Chem.273:3496-3501; Goodrich et al, 1993, Cell 75:519-530; Klemm et al, 1995, Proc.Natl.Acad.Sci.USA 92:5788-5792; Uesugi et al, 1997, Science277: 1310-1313).

Similar domains exist in other GLI family proteins. For example, a putative TAFU31 binding domain has been described comprising NH ₂ -INK D NLRKDL F TVSIKA-COOH (Drosophila Ci, amino acid residues 1044-1060), NH ₂ -DMA D FEFEQM F TDA L GI-COOH (VP16, amino acid residues 469-485), NH ₂ -DSL D LDNTQLDFVAIL DE -COOH (human GLI, amino acid residues 1037-1054), NH ₂ -DSL D LDNTQLD F VAI L DE-COOH (mouse GLI, amino acid residues 1040 -1057), NH ₂ -DSH D LEGVQID F DAI I DD-COOH (human GLI3, amino acid residues 1495-1512) and NH ₂ -DSQLLEPPQID F DAIMDD-COOH (mouse GLI2, amino acid residues 1509-1526). (Yoon et al., 1998, J. Biol Chem. 273:3496-3501). In addition, the putative TAF _II 31 interacting domain _NH2- DSQLLEAPQID F DAIMDD-COOH (amino acid residues 1501-1518) of human GLI2 can be identified, for example, by GenBank accession number AAY87165. Additionally, the putative TAF _II 31 interacting domain NH2 _- ESHD LEGVQIDF DAI I DD-COOH (amino acid residues 1497-1514) of mouse GLI3 can be identified by, for example, GenBank accession number Q61602. Conserved amino acid residues contacting TAF _II 31 are bolded and underlined.

Although much is known about Hh signaling during Drosophila and mouse development, the molecular mechanisms and oncogenic programs that are activated with Hh signaling and GLI activity in human cancers are still very limited. A common feature of Hh-associated cancers is elevated expression of one or more GLI proteins. For example, in recent years, GLI1 was found to be overexpressed in many cancer types, including prostate cancer, pancreatic cancer, and small cell lung cancer (Karhadkar et al., 2004, Nature 431:707-12). Furthermore, Gli was found to be amplified in childhood sarcomas and increased more than 50-fold in malignant gliomas (Roberts et al., 1989, Cancer Res. 49:5407-13; Kinzler et al., 1987, Science 236:70-3). While Hedgehog (Hh) and its receptor Patched (Ptch) have been found to be overexpressed in non-small cell lung cancer (NSCLC), GLI1 expression was not found in this cancer type.

For example, GLI2 overexpression was found in basal cell carcinoma (BCC) lesions compared with normal skin (Ecram et al., 2004, J. Invest Dermatol 122:1503-9; Regl et al., 2002, Oncogene 21:5529-39; Hutchin et al. , 2005, Genes Dev. 19: 214-23). Furthermore, based on the processing of GLI2 and GLI3, GLI2 and/or GLI3 may be more important for the activation of Hh signaling during tumorigenesis in many cancer types, including lung and prostate cancer.

Lung cancer is the leading cause of cancer death in the United States and the world, with more than 170,000 newly diagnosed lung cancer cases in the United States every year, and nearly one million newly diagnosed lung cancer cases worldwide (Minna et al., 2002, Cancer Cell.1(1): 49-52) . Despite some aggressive approaches to the treatment of lung cancer over the past few decades, the 5-year survival rate for lung cancer remains below 15%. There are two types of lung cancer: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC (75-80% of all cancers) consists of three main types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Minna et al., 2002, Cancer Cell. 1(1):49-52). Lung and squamous cell carcinomas account for 60-70% of all lung cancers. Surgery, chemotherapy, and radiation are commonly used in advanced disease, with unsatisfactory results. Improving the efficacy of lung cancer treatment is a major public health goal.

Prostate cancer is probably the most common solid tumor in men (Nelson et al., 2003, N. Engl. J. Med. 349:366-381). For example, 50 percent of men over the age of 50 and essentially all men over the age of 70 have some form of BPH. Prostate cancer is the most frequently diagnosed cancer in the United States, with more than 250,000 new cases diagnosed each year. 40,000 men die from prostate cancer each year.

Therefore, specific targeting of the Hh/GLI-signaling pathway may provide a very effective therapeutic option for the treatment of various lethal tumors involving Hh/GLI signaling (Pasca di Magliano and Hebrok, 2003, Nat. Rev. Cancer 3: 903-911; Sanchez et al., 2005, Cancer Res. 65: 2990-2; Kasper, Regl, Frischauf and Aberger, 2006, Eur. J. Cancer). Treatment of human diseases resulting from activation of ectopic Hh or GLI signaling pathways may require the use of pathway antagonists. To date, ectopic activity has been inhibited by treatment with signaling antagonists that block signaling pathways at various levels. For example, anti-Shh antibodies act extracellularly, while the plant alkaloid, cyclopamine, acts at the Smo level in the cell membrane (see e.g. U.S. Patent Application No. 2005/0130922 A1; Taipale et al., 2000, Nature 406:1005-9 ; Sanchez and Ruiz I Altaba, 2005, Mech. Dev. 122(2): 223-30; Athar et al., 2004, Cancer Res. 64(20): 7545-52). Small molecule modulators of Hedgehog signaling and Smo binding have been described, including the synthetic non-peptidic small molecules Hh-Ag (Frank-Kamenetsky et al., 2002, J. Biol. 1(2):10), HhAntag (Romer et al., 2004, Cancer Cell. 6(3): 229-40) and Cur61414 (Williams et al., 2003, Proc. Natl. Acad. Sci. USA 100(8): 4616-21). In addition, forskolin was used to intracellularly activate protein kinase A (PKA), a cytoplasmic inhibitor of the GLI signaling pathway. However, these methods have disadvantages. For example, administration of therapeutically effective amounts of anti-Shh antibodies is difficult to achieve in patients and may affect otherwise normal pathway-dependent cells. Cyclopamine is very expensive and is only available for the treatment of diseases produced by activation of the Hh signaling pathway at Smo levels or above. In addition, administration of forskolin may have a number of side effects due to the broad spectrum of activities of PKA. In contrast, the use of small molecule compounds that inhibit GLI signaling holds great promise.

The present inventors have addressed a large unmet need in the treatment of cancers overexpressed by GLI proteins, especially GLI3. The present invention provides small molecule compounds, pharmaceutical compositions, kits and methods for detecting and treating many cancers overexpressing GLI3 protein. Such cancers include lung cancer, NSCLC, breast cancer, colon cancer, mesothelioma, melanoma, sarcoma, prostate cancer, ovarian cancer, kidney cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, nasopharyngeal cancer, pancreatic cancer, colloid Tumor etc.

Summary of the invention

In the present invention, the inventors found that the activation domain of GLI3 protein (approximately 70 kDa) is overexpressed in various human cancer cell lines, including lung cancer, NSCLC, breast cancer, colon cancer, mesothelioma, melanoma , sarcoma, prostate cancer, ovarian cancer, renal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, nasopharyngeal cancer, pancreatic cancer, glioma and other cell lines. GLI3 overexpression (>95%) was also found in metastatic colon cancer and sarcoma tissue samples.

The inventors therefore propose that inhibition of the active form of the GLI3 protein induces significant apoptosis in these cancers, but not in normal cells. GLI3 protein signal transduction can be inhibited by different means, including but not limited to (i) directly inhibiting the transcriptional activity of GLI3 protein, (ii) inhibiting the processing of GLI3 precursor protein, (iii) inhibiting the phosphorylation of GLI3 activation domain, or ( iv) Inhibit any cellular protein involved in the production of active GLI3 protein. GLI3 protein signaling can be inhibited in various ways, such as using small molecule compounds, siRNA, peptides or antisense oligonucleotides. The invention provides a small molecule compound for inhibiting GLI protein signal transduction, especially GLI3 protein signal transduction.

In one embodiment, the present invention provides small molecule compounds having the formula:

In the formula, X ¹ and X ² are each independently N or C, in the formula, one of X ¹ and X ² is N, and one of X ¹ and X ² is C, so the ring N forms a double ring with the C of X ¹ or X ² key. In addition, R ¹ , R ² and R ³ are _each independently selected from C ₁ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkane optionally substituted with 0-3 R groups The R ⁶ groups are each independently selected from hydrogen, halogen, C ₁ -C ₆ alkyl, -OR ⁷ , -C(O)R ⁷ , -OC(O)R ⁷ , -N(R ⁷ , R ⁷ ), -NR ⁷ S(O) ₂ R ⁷ , -C(O)N(R ⁷ , R ⁷ ), -N(R ⁷ )C(O)R ⁷ , -N(R ⁷ )C (O)N(R ⁷ , R ⁷ ), -C(NR ⁷ )N(R ⁷ , R ⁷ ), -N(R ⁷ )C(NR ⁷ )N(R ⁷ , R ⁷ ) and S(O ) _m R ⁷ , wherein the subscript m is 0, 1 or 2. Y is a direct bond or C ₁ -C ₄ alkyl, and Z is selected from C ₁ -C ₄ alkyl, aryl and heteroaryl. R ⁴ is hydrogen, halogen, -OR ⁷ , -OC(O)R ⁷ , -C(O)OR ⁷ , -N(R ⁷ , R ⁷ ), -NR ⁷ S(O) ₂ R ⁷ , -C (O)N(R ⁷ , R ⁷ ), -N(R ⁷ )C(O)N(R ⁷ , R ⁷ ), -C(NR ⁷ )N(R ⁷ , R ⁷ ) and S(O) _m R ⁷ , wherein the subscript m is 0, 1 or 2. R ⁵ is hydrogen, C ₁ -C ₆ alkyl, or optionally combined with Y to form a 5-6 membered heterocycle. Each R ⁷ is independently H or C ₁ -C ₆ alkyl.

In addition, salts, hydrates, solvates, isomers and prodrugs of the small molecule compounds of the invention are also contemplated.

In another embodiment, the present invention provides small molecule compounds having the formula, wherein R ¹ , R ² and R ³ are each aryl. In another embodiment, R ¹ , R ² and R ³ are each phenyl.

In another embodiment, the present invention provides small molecule compounds of the formula, wherein R ⁴ is hydroxyl. In another embodiment, Z is C ₁ -C ₄ alkyl. In another embodiment, Z is aryl. In yet another embodiment, Z is phenyl.

In another embodiment, the present invention provides small molecule compounds having the formula, wherein R ¹ , R ² and R ³ are each phenyl, Y is C ₁ -C ₄ alkyl, Z is C ₁ -C ₄ Alkyl or phenyl, ^R4 is hydroxyl.

In another embodiment, the present invention provides small molecule compounds having the formula, wherein X ¹ is C, X ² is N. In yet another embodiment, the small molecule compound of this formula has the following structure:

In yet another embodiment, the small molecule compound of this formula has the following structure:

In other embodiments, the invention provides small molecule compounds having the formula, wherein X ¹ is N, X ² is C. In another embodiment, the small molecule compound of this formula has the following structure:

In yet another embodiment, the small molecule compound of this formula has the following structure:

The invention also provides pharmaceutical composition and medicine. Therefore, in another embodiment, the present invention provides a pharmaceutical composition comprising the small molecule compound of the present invention and a pharmaceutically acceptable carrier.

The invention also provides methods of using the small molecule compounds of the invention. In one aspect of the present invention, a method for inducing apoptosis of tumor cells expressing GLI protein is provided. The method includes the step of contacting tumor cells with a sufficient amount of the small molecule compound of the present invention, wherein said contacting step can induce tumor cell apoptosis. The GLI protein is preferably GLI2 or GLI3.

In one embodiment of the method, the small molecule compound is administered as part of a cancer treatment regimen.

In another aspect of the invention, a method of inhibiting at least one of undesirable growth, excessive proliferation or survival of a cell is provided. The method includes the step of determining whether the cell expresses the GK gene or the GLI protein. The method also includes the step of contacting said cell with an effective amount of a small molecule of the invention, wherein said contacting step results in inhibition of at least one of undesirable growth, hyperproliferation, or survival of said cell.

The cells or tumor cells used in the method of the present invention are selected from colon cancer, melanoma, mesothelioma, lung cancer, renal cell carcinoma, breast cancer, prostate cancer, sarcoma, ovarian cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, nasopharyngeal cancer Carcinoma, pancreatic cancer and glioma cells.

The methods of the invention can be performed in vitro or in vivo. Accordingly, in another aspect of the invention, a method of treating a cancer patient is provided. The method comprises the step of administering to the subject a therapeutically effective amount of the small molecule compound, wherein said cancer is characterized by expression of a GLI polypeptide, preferably a GLI3 polypeptide, said administering step resulting in treatment of the subject.

In another preferred embodiment of the present invention, a method for inhibiting the activity of GLI protein in cells is provided. The method comprises the step of contacting said cell with a small molecule compound of the invention, wherein said contacting step results in inhibition of GLI protein activity in said cell. The inhibited GLI protein activity is preferably a protein interaction between the GLI protein and a cellular or nuclear protein. Preferred nucleoproteins are TAF proteins, TATA box-binding protein-associated factors, preferably TAF _II 31 proteins.

In addition, the present invention provides a method of inhibiting the expression of a gene containing a GLI DNA binding site operably linked to the promoter of the gene. This method comprises the step of contacting a cell expressing a gene having a GLI DNA binding site operably linked to its promoter with a small molecule compound of the invention, wherein said contacting step results in inhibition of expression of the gene. In a preferred embodiment of the invention, said gene is a Wnt2 gene, preferably a human Wnt2 gene.

In addition, the present invention provides the use of the small molecular compound of the present invention in the preparation of a medicament for inhibiting at least one behavior of undesirable growth, excessive proliferation or survival of cells. In another aspect, the use of the small molecular compound in the preparation of a drug for treating cancer is provided. In yet another embodiment, the present invention provides the application of small molecule compounds in the preparation of drugs for inducing apoptosis. In another aspect, the application of the small molecular compound of the present invention in the preparation of a drug for inhibiting GLI protein signal transduction of cells is provided.

In another aspect, the invention provides screening methods. A preferred method of the invention is a method of identifying a candidate compound capable of inhibiting the protein interaction between the GLI protein and the TAF _II 31 protein. In one embodiment, the method comprises the steps of: contacting the candidate compound with a sample comprising the GLI protein and the TAF _II 31 protein; and determining the binding of the GLI protein to the TAF _II 31 protein. A reduced level of binding of the GLI protein to TAF _II 31 in the presence of the candidate compound compared to binding of the GLI protein to TAF _II 31 in the absence of the candidate compound indicates that the candidate compound is capable of inhibiting the interaction of the GLI protein and the TAF _II 31 protein.

Another method provided herein is a method of identifying compounds that modulate the activity of a GLI protein. In one embodiment, the GLI protein activity is a GLI protein-dependent transcriptional activity and the method is used to identify compounds that modulate the GLI protein-dependent transcriptional activity. The method includes the steps of contacting a sample comprising an expression reporter construct having one or more GLI DNA binding sites operably linked to a reporter gene; and, if present, assaying the candidate compound. Effect of candidate compounds on GLI protein-dependent transcriptional activity levels.

Brief description of the drawings

Figure 1 shows the location of the transcriptional activation domain of GLI3. The GLI3 transcriptional activation domain is located near the C-terminus and contains a conserved VP16-like activation region (TAF _II 31-binding domain), which is required for the activation function of GLI3, but not for the repression function of GLI3. This FXXΦΦ motif is thought to be in direct contact with TAF _II 31 . In GLI3, this domain contains the amino acid sequence FDAII.

Figure 2 shows semi-quantitative RT-PCR analysis for detection of Gli3 expression in human cell lines. In this figure, the upper panel is a schematic diagram of GLI3 showing the repressive domain (amino acid residues 1-397), zinc finger domain (amino acid residues 482-637) and CBP-binding domain (amino acid residues 827-1132 ). The approximate positions of PCR primers F2 and R2 are indicated (see Table 1 for details). In this figure, the middle panels (lanes 1-17) show the following cell lines analyzed by RT-PCR: normal liver (lane 1); NSCLC: H1703, H460 and A549 (lanes 2-4); breast cancer: HuL100 , BT474 and MCF-7 (lanes 5-7); mesothelioma: Met5A, H290, REN, H513, H28, 211H and LARK1A (lanes 8-14); colon cancer: SW480 (lanes 15); a pair of primary NSCLC tissue samples (lanes 16 and 17 are normal and cancer tissues, respectively). In this figure, the lower panels (lanes 1-29) show the following cell lines analyzed by RT-PCR: colon cancer: SW480, HCT116, HT29, Lovo and CaCO2 (lanes 1-5); normal colon (lanes 6); Normal kidney cell lines: HRE-152 and HK-2 (lanes 7 and 8); renal cell carcinoma: 786-Om Caki, 769-P, A704, and OMRC3 (lanes 9-13); normal lung (lanes 14); Skin tumors: REN, H290, 211H, H513, H2052, H28 and MS-I (lanes 15-21); NSCLC: A549, H460, H838, H1703, H1229, H1650, H1975 and A427 (lanes 22-29). Most cancer cell lines and primary tissue samples express Gli3. Notably, Gli3 expression was not detected in the colon cancer cell line HT29 (lower panel, lane 2). See embodiment 4 for details.

Figure 3 shows the RMS overlay of the FXXΦΦ motif (amino acid sequence: FDAII) of the small molecule compounds FN1-5 and GLI3 drawn manually. FDAII is shown as an alpha helix. See (eg) Example 2 for details.

Figure 4 shows the preliminary screening of small molecule compounds FN1-1, FN1-2, FN1-3, FN1-5 and FN2-1. This small molecule compound was designed to inhibit GLI3 transcriptional activation (TAF-binding domain). It was observed that the small molecule compound FN1-5 could significantly kill all the detected cancer cells. DMSO was used as a control. See (eg) Example 5 for details.

Fig. 5 is a graph of flow cytometry analysis, which shows that the small molecule compound FN1-5 can induce the apoptosis of the colon cancer cell line SW480. Significant apoptosis (58.6%) was observed at 100 μM FN1-5, which is consistent with the staining results. See (eg) Examples 5 and 6 for details.

Figure 6 shows the screening of small molecule compounds FN1-7, FN1-8 and FN3-5 using a cytotoxicity assay and NSCLC cell lines H1299, A549 and H460, as described herein. Significant cell killing was observed in all cancer cells when treated with the small molecule compound FN1-8. See (eg) Example 5 for details.

Figure 7 shows the results of cytotoxicity assays after treatment of LOX cells (melanoma) with small molecule compounds FN1-5, FN1-8, FN1-7, FN1-9U and FN1-9S. Consistently, significant cell killing was observed upon treatment with FN1-5 and FN1-8. Some killing effects were also observed when incubating LOX cells with the small molecule compounds FN1-7 and FN1-9U. Viable cells were stained with 0.5% crystal violet. DMSO was used as a control. See (eg) Example 10 for details.

Figure 8 shows the effect of different small molecule compounds on primary cultured human mesothelioma cells freshly prepared from tissue samples. Significant cell killing was observed in FN1-5 and FN1-8. At the same dose, the efficacy of the small molecule compound FN1-8 in killing this primary culture was higher than that of FN1-5. DMSO was used as a control. See (eg) Example 11 for details.

Figure 9 shows the regulation of Wnt2 in the NSCLC cell line A549 with small molecule compounds FN1-1, FN1-2, FN1-3, FN1-5, FN1-7, FN1-8, FN2-1, FN2-5 and FN3-5 Promoter activity. Both FN1-5 and FN1-8 could significantly down-regulate the Wnt2 promoter activity. DMSO and "empty vector" are controls. See, eg, Example 27 for details.

Figure 10 shows the modulation of TOP/FOP activity of colon cancer cell line HCT116 by small molecule compounds FN1-5, FN1-7, FN1-8, FN1-9U, FN1-9S, FN2-1 and FN3-5. It was found that FN1-5 and FN1-8 could significantly down-regulate TOP/FOP activity. DMSO was used as a control. See, eg, Example 31 for details.

Figure 11 shows that small molecule compounds FN1-5 and FN1-8 do not regulate the SOCS3 promoter activity of NSCLC A549 cells. Empty vector and DMSO were used as controls. See, eg, Example 32 for details.

Figure 12 shows Western blot analysis of Dvl-3, cytoplasmic β-catenin, survivin and actin (control) in colon cancer cells after treatment with FN1-5 and FN1-8. Small molecule compounds FN1-5 and FN1-8 inhibit canonical Wnt signaling in colon cancer cells. See, eg, Example 33 for details.

Figure 13 shows Western blot analysis of Dvl-3, cytoplasmic β-catenin, survivin and actin (control) in NSCLC cells after treatment with FN1-5 and FN1-8. Small molecule compounds FN1-5 and FN1-8 can inhibit canonical Wnt signaling in NSCLC cells. See, eg, Example 33 for details.

Figure 14 shows the results of time-course experiments, showing that FN1-8 can induce significant apoptosis in Malme-3M cells (A), A375 cells (B) and SK-Mel-5 cells (C), but not in normal human skin. Fibroblast cells (D) apoptosis. See embodiment 10 for details.

Figure 15 shows that small molecule compound FN1-8 can down-regulate GLI1-induced transcription in melanoma cell lines Malme-3M (A), A375 (B), SK-Mel-2 (C) and SK-Mel-5 (D) active. See Example 29 for details.

Figure 16 shows that the small molecule compound FN1-8 can reduce the level of transcriptional activation induced by GLI protein-TAF in the NSCLC cell line A549. See Example 35 for details.

Figure 17 shows the in vivo efficacy of small molecule compounds FN1-8 in the mouse xenograft tumor model NSCLCH460 (arrow) and melanoma LOX (arrow). Tumor growth was significantly inhibited after treatment with FN1-8. See, eg, Examples 36 and 37 for details.

Figure 18 shows the in vivo efficacy study of the small molecule compound FN1-8 using a mouse xenograft tumor model (NSCLC H460). A. Treatment with the small molecule compound FN1-8 significantly inhibited tumor growth. Arrows indicate daily injections of the small molecule compound FN1-8 or DMSO used as a control (twice each for 6 consecutive days). B. Shows tumors excised from mice of each group. DMSO, DMSO treatment; FN1-8, treatment with small molecule compounds FN1-8 described herein. C. Tumor weight was significantly reduced after treatment with FN1-8. Results are mean ± SD (error bars). See, eg, Example 36 for details.

Figure 19 shows the in vivo efficacy study of the small molecule compound FN1-8 using a mouse xenograft tumor model (melanoma LOX). A. Treatment with the small molecule compound FN1-8 significantly inhibited tumor growth. Arrows indicate daily injections of the small molecule compound FN1-8 or DMSO used as a control (twice each for 6 consecutive days). B. Shows tumors excised from mice of each group. DMSO, DMSO treatment; FN1-8, treatment with small molecule compounds FN1-8 described herein. C. Tumor weight was significantly reduced after treatment with FN1-8. Results are mean ± SD (error bars). See, eg, Example 37 for details.

Figure 20 shows the in vivo efficacy study of the small molecule compound FN1-8 using a mouse xenograft tumor model (colon cancer HT29). See Example 35 for details. A. Tumor growth was not affected after treatment with the small molecule compound FN1-8. Arrows indicate daily injections of the small molecule compound FN1-8 or DMSO used as a control (twice each for 6 consecutive days). B. Tumor weight after treatment with FN1-8 was similar to DMSO control group. Results are mean ± SD (error bars). See, eg, Example 38 for details.

Figure 21 shows the pharmacokinetic analysis of small molecule compounds FN1-5 (A) and FN1-8 (B) in mice (intraperitoneal injection). Results are expressed as mean and SD (error bars) of data obtained from three mice. See Example 43 for details.

Figure 22 shows the pharmacokinetic analysis of small molecule compounds FN1-5 (A) and FN1-8 (B) in mice (oral administration). Results are expressed as mean and SD (error bars) of data obtained from three mice. See Example 43 for details.

Figure 23 shows the quantitative results of co-immunoprecipitation analysis, which proves that the small molecule compound FN1-8 can block the protein interaction between GLI protein and TAF _II 31 protein. Binding of the GLI-TAF binding domain peptide (GLI-TAFbd) and TAF _II 31 protein was quantified by measuring the intensity (arbitrary units) of each band in the co-immunoprecipitation experiment. The binding of each GLI-TAFbd peptide after DMSO treatment was set as 100%. FN1-8 interfered with this binding in a dose-dependent manner: after treatment with 20 μM and 50 μM FN1-8, binding of GLI1-TAFbd was 73.6% and 33.5%, GLI2-TAFbd was 73.8% and 60%, GLI3- TAFbd was 71.6% and 28.6%, respectively. See Example 35 for details.

Detailed description of the invention

I. Definition

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled person with common definitions of many of the terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition, 1994); The Cambridge Dictionary of Science and Technology (Cambridge Dictionary of Science and Technology) (Walker, ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), SpringerVerlag (Springer Verlag) (1991); and Hale and Marham, The Harper Collins Dictionary of Biology (1991). The following terms used herein have their respective meanings unless otherwise stated.

As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon group containing the indicated number of carbon atoms (ie, _C1 - _C10 refers to 1-10 carbons), and may include divalent and multivalent groups. Examples of saturated hydrocarbon groups include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, for example n-pentyl, n-hexyl, Analogs and isomers of n-heptyl, n-octyl, etc.

The term "alkenyl" as used herein refers to an unsaturated alkyl group containing one or more double bonds. Examples of alkenyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, and 3-(1,4-pentadienyl) Dienyl) and higher analogs and isomers.

The term "alkynyl" as used herein refers to an unsaturated alkyl group containing one or more triple bonds. Examples of alkynyl groups include ethynyl, 1-propynyl, 1- and 2-butynyl and higher analogs and isomers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are subsequently modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. "Amino acid analogue" means a compound having the same basic chemical structure as a naturally occurring amino acid, e.g., containing an alpha carbon bound to a hydrogen, carboxyl, amino group, and R group, e.g., homoserine, norleucine, methionine sulfoxide , Methylsulfonium methionine. Such analogs may contain modified R groups (eg, norleucine) or modified peptide backbones, but still have the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetic" refers to a compound that differs in chemical structure from the ordinary chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid.

Amino acids may be referred to by either the well-known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter designations.

The term "aryl" as used herein refers to a polyunsaturated aromatic hydrocarbon substituent containing 5-12 ring atoms, which may be a single ring or multiple rings (up to three rings) fused together or linked covalently. Non-limiting examples of aryl include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl and benzyl. Other aryl groups are also contemplated by this invention.

A "biological sample" as used herein is a sample of biological tissue or fluid containing nucleic acids or polypeptides. Such samples are generally from humans, but include tissues isolated from mammals or rodents other than humans, such as mice and rats. Biological samples may also include tissue sections, such as biopsy and autopsy samples, frozen sections for histological purposes, blood, plasma, serum, sputum, feces, tears, mucus, hair, skin, and the like. Biological samples also include explants and primary and/or transformed cell cultures from patient tissue. "Biological sample" also refers to a cell or population of cells or a quantity of tissue or fluid from an animal. A biological sample is often taken from an animal, but the term "biological sample" can also refer to cells or tissues that are analyzed in vivo (ie, without being removed from the animal). A "biological sample" generally contains animal cells, but the term also refers to cell-free biological material that can be used to measure levels of cancer-associated polynucleotides or polypeptides, such as cell-free components of blood, saliva, or urine. Many types of biological samples can be used with the present invention, including but not limited to: tissue biopsy samples, blood samples, oral scrapings, saliva samples, or nipple discharge. As used herein, "tissue biopsy" refers to a quantity of tissue removed from an animal, preferably a human, for diagnostic analysis. In cancer patients, tissue from a tumor may be removed to analyze the cells within the tumor. "Tumor biopsy" may refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, and the like.

"Providing a biological sample" refers to obtaining a biological sample for use in the methods described herein. This is usually done by removing a sample of cells from the patient, but it is also possible to use cells previously isolated (such as from another person, at another time, and/or for another purpose), or to perform the method of the invention in vivo , to complete the process. Archived organizations with treatment or outcome histories are especially useful.

"Cancer cell", "transformed" cell or "transformation" in tissue culture refers to a spontaneous or induced phenotypic change, not necessarily including the uptake of new genetic material. Although transformation can occur through infection and incorporation of new genomic DNA by transforming viruses, or by uptake of exogenous DNA, transformation can also occur spontaneously or through exposure to carcinogens, thereby mutating endogenous genes. Transformation is associated with phenotypic changes such as cell immortalization, abnormal growth control, nonmorphological changes, and/or malignant transformation (see Freshney, Culture of Animal Cell a Manual of Basic Technique (3rd ed., 1994) )).

The term "altered cell growth" refers to any alteration in cell growth and proliferation in vitro or in vivo, such as foci formation, anchorage independence, growth on semi-solid or soft agar, altered contact inhibition and density limitation of growth, lack of growth factors or serum requirements , changes in cell morphology, gain or loss of immortalization, gain or loss of tumor-specific markers, ability to form or inhibit tumors when injected into a suitable animal host, and/or cell immortalization. See, eg, Freshney, Culture of Animal Cell a Manual of Basic Technique (Animal Cell Culture: A Manual of Basic Technique) pp. 231-241 (3rd Ed., 1994).

"Correlating the amount" refers to comparing the amount of a substance, molecule or marker (such as Gli or GLI) measured in one sample with the amount of the same substance, molecule or marker measured in another sample. The amount of the same substance, molecule or marker measured in another sample may be specific for a given cancer.

Also included within the scope of the present invention are synonyms for the term "measured amount", including but not limited to: detecting, measuring, testing or determining the presence, absence, amount or concentration of a molecule such as Gli or GLI.

The term "cycloalkyl" as used herein refers to a saturated cyclic hydrocarbon containing 3-15 carbons and 1-3 fused or covalently linked rings. Cycloalkyl groups used in the present invention include, but are not limited to, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The bicycloalkyl used in the present invention includes but not limited to: [3.3.0] bicyclooctyl, [2.2.2] bicyclooctyl, [4.3.0] bicyclononane, [4.4.0] bicyclodecane ( decalin), spiro[3.4]octyl, spiro[2.5]octyl, etc.

The term "cycloalkenyl" as used herein refers to an unsaturated cyclic hydrocarbon containing 3-15 carbons and 1-3 fused or covalently linked rings. Cycloalkenyl groups used in the present invention include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Bicycloalkenyl groups are also useful in the present invention.

"Determining a functional effect" refers to determining the effect of a compound on increasing or decreasing a parameter directly or indirectly affected by a GLI protein, such as functional, enzymatic, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, such as changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamics (e.g., shape), chromatography, or solubility properties of the protein, detection of induced labeling or Transcription activation of GLI protein; determination of binding activity such as binding activity of TAF protein (such as TAF _II 31), determination of cell proliferation, determination of apoptosis, etc. Assays known to those skilled in the art, such as in vitro assays, such as cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cell proliferation; cell transformation; growth factor or serum dependence; tumor-specific Invasion into matrigel; in vivo tumor growth and metastasis; mRNA and protein expression in metastatic cells and other characteristics of cancer cells to determine the functional effect of compounds (such as small molecule compounds) on cancer. Functional effects can be assessed in many ways known to those skilled in the art, e.g., quantitative or qualitative microscopic determination of changes in morphological characteristics, determination of changes in Gli RNA or protein levels, determination of RNA stability, identification of downstream or reporter gene expression ( CAT, luciferase, β-gal, GFP, etc.), as determined by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible labeling, and ligand binding assays. "Functional effect" includes in vitro, in vivo and ex vivo activity.

The scope of the present invention includes synonyms for the term "determining", including but not limited to: detecting, determining, testing or determining the presence, absence, amount or concentration of a molecule such as a GLI polypeptide, tag, small molecule compound of the present invention. The term designates or quantifies a determination.

In Shh signaling, GLI signaling or Wnt2 signaling, the term "down-regulation" or "inhibition" refers to a known experimental determination of Shh signaling, GLI signaling or Wnt2 signaling, either partially or completely Blocks Shh signaling, GLI signaling, or Wnt2 signaling. Inhibitors are, for example, small molecule compounds of the invention.

"Effective amount", "effective dose", "sufficient amount" or their grammatical equivalents of the small molecule compound of the present invention used for treatment is an amount sufficient to relieve, or alleviate symptoms in some way, or arrest or reverse disease progression. Alleviation of symptoms of a particular disease, such as cancer, by administration of a particular pharmaceutical composition refers to permanent or temporary, persistent or transient relief that may be associated with the administration of the pharmaceutical composition. An "effective amount" can be administered in vivo and in vitro.

An "expression vector" or "expression construct" is a recombinantly or synthetically produced nucleic acid construct containing a series of specialized nucleic acid elements capable of transcribing a specific nucleic acid in a host cell. An expression vector can be part of a plasmid, virus or nucleic acid fragment. Expression vectors generally comprise a nucleic acid to be transcribed operably linked to a promoter.

The term "GLI" refers to the GLI family of proteins. GLI proteins include GLI (also known as GLI1), GLI2, and GLI3. Preferred GLI proteins are GLI1, GLI2 or GLI3.

The term "Gli" refers to the gene encoding the GLI protein. Thus, Gli1, Gli2 and Gli3 are the genes encoding the GLI1, GLI2 and GLI3 proteins, respectively.

The term "GLI protein activity" refers to GLI signal transduction, including, for example, GLI transcriptional activation of downstream genes, binding of GLI proteins to GLI DNA binding sites, and binding of GLI proteins to other proteins such as TAF or co-activators such as CBP (Creb protein binding protein). ) combination.

The term "GLI DNA binding site" refers to a nucleotide sequence to which a GLI protein binds and which activates gene transcription. GLI DNA binding sites see eg Vortkamp et al (1995, DNA Cell. Biol. 14:629-34) Kinder and Vogelstein (1990, Mol. Cell. Biol. 10:634-42) and Yoon et al (998, J Biol Chem 273:3496-3501). For example, the GLI DNA binding site for GLI3 zinc finger binding is found in Vortkamp et al. (1995, DNA Cell. Biol. 14:629-34), which consists of 16 nucleotides and is highly similar to the binding sequence of GLI and tra-1 proteins. This binding site comprises the 9-base pair sequence 5'-GACCACCCCA-3'. It will be appreciated that the GLI3 DNA binding site need not be 100% identical to the GLI DNA binding site identified by Vortkamp et al. (1995, DNA Cell. Biol. 14:629-34). For example, in recent years, Hallikas et al. determined the DNA-binding specificities of GLI1, GLI2, and GLI3, and also found that GLI binds to, for example, 5′- T ACCAACCCA-3′, 5′-G C CCACCCA-3′, 5′- G G CCACCCA-3′ and 5′-GA T CACCCA-3′, where the underlined nucleotides indicate a 9 bp consensus sequence difference (Hallikas et al., 2006, Cell 124(1): 47-59; fully incorporated herein by reference ). Assays for determining the binding of GLI proteins to GLI DNA binding sites are known in the art, including, for example, electrophoretic mobility shift assay (EMSA; Fried and Crothers, 1981, Nucleic Acids Res. 9:6505-6525; Hallikas et al., 2006, Cell 124:47-59), SELEX (Roulet et al., 2002 Nat. Biotechnol. 20:831-835) and chromatographic binding experiments.

"GLI"polypeptides include naturally occurring or recombinant forms. Accordingly, in some embodiments, the GLI polypeptides and GLI subdomain polypeptides described herein may comprise sequences corresponding to human GLI sequences. Accordingly, exemplary GLIs are provided herein, which are known in the art. For example, several vertebrate GLI1, GLI2, and GLI3 proteins were identified, such as human GLI1 (GenBank accession numbers NM_005269, P08151), mouse GLI1 (GenBank accession numbers NM_010296, AB025922, AAC09169, P47806), zebrafish GLI1 (GenBank accession numbers NM_178296), human GLI2 (GenBank accession numbers NM_030381; NM_030380; NM-030379, DQ086814), mouse GLI2 (GenBank accession numbers XM_922107), human GLI3 (GenBank accession numbers NM_000168, AJ250408, M57606, P1007) (chimpanzee GenBank accession numbers NM_001034190, AY665272, Q5IS56), mouse GLI3 (GenBank accession numbers X95255, NM_008130, NP_032156, Q61602), rat GLI3 (GenBank accession numbers XM_225411), zebrafish GLI3 (GenBank accession numbers NM_205728, 2

The GLI protein may be a full-length GLI protein, or may be a partial GLI protein, such as a GLI protein subdomain. For example, a "GLI3" polypeptide refers to polypeptides and polymorphic variants, alleles, mutants of human GLI3: (i) whose amino acid sequence is preferably at least about 100, 150, 200, 250, 300, 500 or more amino acids is greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90% identical to the amino acid sequence of a human GLI3 selected from GenBank accession numbers NM_000168, AJ250408, M57609, P10071 and AAY87165 in a region of Preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher, (ii) comprising the amino acid motif FXXΦΦ (F = phenylalanine; X = any residue; Φ=any hydrophobic residue), preferably the amino acid sequence FDAII, (iii) comprises a transcriptional activation domain, (iv) binds to the GLI DNA binding site and/or (v) binds to TAF.

"Gli nucleic acid" or "gli polynucleotide" refers to a vertebrate gene encoding a GLI, GLI2 or GLI3 protein. "Gli nucleic acid"includes naturally occurring or recombinant forms. Gli polynucleotides or GLI polypeptide coding sequences are generally derived from humans, but may be derived from other mammals, including but not limited to primates other than humans, rodents such as rats, mice or hamsters; cattle, pigs, horses, sheep or other mammals. Gli nucleic acids useful in the practice of the present invention have been cloned and characterized, such as human Gli1 (GenBank accession number NM_005269), mouse Gli1 (GenBank accession number NM_010296, AB025922), zebrafish Gli1 (GenBank accession number NM_178296), human Gli2 (GenBank Accession number NM_030381; NM_030380; NM-030379, DQ086814), mouse GH2 (GenBank accession number XM_922107), human GH3 (GenBank accession number NM_000168, AJ250408, M57609), chimpanzee Gli3 (GenBank accession number NM_0010A3416, mouse) (GenBank accession numbers X95255, NM_008130), rat Gli3 (GenBank accession numbers XM_225411), zebrafish Gli3 (GenBank accession numbers NM_205728, AY377429). The Gli polynucleotide may be a full-length Gli polynucleotide, ie, a polynucleotide encoding a complete GLI protein, or may be a partial Gli polynucleotide encoding a subdomain of a GLI protein.

The terms "GLI pathway", "GLI signal transduction" or "GLI signal transduction pathway" are used interchangeably and refer to the signal transduction pathway initiated by the binding of hedgehog protein to its receptor, leading to the expression and/or activation of GLI protein.

The term "halogen" as used herein refers to elements such as fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "hedgehog" is used interchangeably with the term "Hh", which is a cytokine that binds to the Hh receptor to initiate the Hh signaling pathway, resulting in the expression or activation of the GLI protein. There are three Hh family genes in mammals, they are Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh). Several vertebrate hedgehog proteins are known in the art, eg, human SHH, murine SHH, rat SHH, human IHH, and murine DHH.

The term "heteroaryl" as used herein refers to a polyunsaturated aromatic hydrocarbon substituent containing 5-12 ring atoms, which may be a single ring or multiple rings (up to three rings) fused together or linked covalently, wherein At least one heteroatom such as N, O or S. A heteroaryl can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of heteroaryl include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4 -thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4 -pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5- Quinoxalinyl, 3-quinolinyl and 6-quinolinyl. Other heteroaryl groups used in the present invention include pyridyl N-oxide, tetrazolyl, benzofuryl, benzothienyl, indazolyl, or any of the above groups which are substituted, especially monosubstituted or disubstituted .

The term "heterocycloalkyl" as used herein refers to a saturated cyclic hydrocarbon containing 3-15 ring atoms and 1-3 fused or covalently linked rings with at least one heteroatom such as N, O or S in the ring. In addition, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of heterocycloalkyl groups include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- Morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl and the like.

The term "isomer" as used herein refers to compounds of the invention having asymmetric carbon atoms (optical centers) or double bonds. Included within the scope of the present invention are racemates, diastereomers, geometric isomers and individual isomers.

A "label" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include ³ H, ¹²⁵ I, ³² P, fluorescent dyes, electron-dense reagents, enzymes (such as those commonly used in ELISAs), biotin, digoxigenin or haptens, and proteins or Other entities that are detected by some means, such as the incorporation of a radioactive label into a small molecule compound. The tag can be incorporated anywhere on the small molecule compound.

The term "Gli mRNA level" or "Wnt2 mRNA level" as used in a biological sample refers to the amount of mRNA transcribed from the Gli or Wnt gene, respectively, present in a cell or biological sample. The mRNA usually encodes a functional GLI or WNT protein, but there may be mutations that alter or eliminate the function of the encoded protein. "Gli mRNA levels" or "Wnt2 mRNA levels" need not be quantified, but can be easily detected by human subjective observation (with or without comparison to control sample levels or control sample expected levels).

"GLI polypeptide level" or "Wnt2 polypeptide level" in a biological sample refers to the amount of polypeptide present in a cell or biological sample translated from Gli or Wnt2 mRNA, respectively. A polypeptide may or may not have GLI or WNT2 protein activity. "GLI polypeptide levels" or "WNT2 polypeptides" need not be quantified, but are readily detectable by human subjective observation (with or without comparison to control sample levels or control sample expected levels).

As used herein, a "modulator" of the level or activity of a polypeptide (eg, GLI) includes activators and/or inhibitors of that polypeptide, which term describes a compound that activates or inhibits the expression level of a polypeptide or the activity of a polypeptide. Preferably the polypeptide is GLI1, GLI2 or GLI3. An activator is a compound that, for example, induces or activates expression of a polypeptide of the invention or binds, stimulates, increases, turns on, activates, helps or enhances activation, sensitization or upregulates the activity of a polypeptide of the invention. Activators include naturally occurring and synthetic compounds, small chemical molecules, and the like. Activator assays include, for example, administering a candidate compound to cells expressing a GLI polypeptide and then determining the functional effect. A sample or assay comprising a GLI polypeptide treated with a potential activator is compared to a control sample not treated with the activator to detect the extent of the effect. The relative activity of the control sample (not treated with the candidate compound) was assigned 100%. Polypeptide activation is achieved when the polypeptide activity value relative to the control is 110%, optionally 130%, 150%, optionally 200%, 300%, 400%, 500% or 1000-3000% or higher. Inhibitors are compounds that, for example, inhibit or inactivate expression of a polypeptide of the invention, or bind to, decrease, switch off, inactivate, block or reduce expression, desensitize, or downregulate the activity of a polypeptide of the invention. Inhibitors include nucleic acids that interfere with GLI protein expression, such as siRNA and antisense RNA, as well as naturally occurring and synthetic compounds, small chemical molecules, and the like. Inhibitor assays are described herein. A sample or assay comprising a GLI polypeptide treated with a potential inhibitor is compared to a control sample not treated with the inhibitor to detect the extent of the effect. The relative activity of the control sample (not treated with the candidate compound) was assigned 100%. When the activity value of the polypeptide relative to the control is reduced by 10%, optionally 20%, optionally 30%, optionally 40%, optionally 50%, 60%, 70%, 80% or 90-100%, then the polypeptide is achieved inhibition.

The term "pharmaceutically acceptable" refers to a composition that is physiologically tolerated or generally does not produce allergic or similar adverse reactions when administered to a subject, preferably a human subject. Preferably, the term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopoeia or other generally accepted pharmacopoeia for use in animals, more particularly humans.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, amino acid polymers containing modified residues, and non-naturally occurring amino acid polymers thing.

As used herein, the term "prodrug" refers to a compound that readily undergoes a chemical change under physiological conditions to provide the compound of the present invention. In addition, prodrugs can be converted to compounds of the invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to compounds of the invention when placed in a transdermal patch reservoir containing suitable enzymes or chemical reagents.

A "promoter" is defined as an array of nucleic acid control sequences that mediate the transcription of a nucleic acid. A promoter as used herein includes essential nucleic acid sequences near the site of initiation of transcription, such as the TATA element in a polymerase II type promoter. A promoter also optionally contains distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.

A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence mediates transcription of the nucleic acid corresponding to the second sequence .

The term "recombinant" when used to modify, for example, a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or by altering a native nucleic acid or protein, or that the cell is derived from such modified cells. Thus, for example, a recombinant cell is capable of expressing a gene not found in the native (non-recombinant) form of the cell, or expressing a native gene that is otherwise aberrantly expressed, underexpressed, or not expressed at all. As used herein, the term "recombinant nucleic acid" refers to a nucleic acid not normally found under natural conditions, originally formed in vitro by the manipulation of nucleic acid (eg, using polymerases and endonucleases). In this way, operative linkage of different sequences is achieved. Thus, isolated nucleic acids in linear form, or expression vectors formed in vitro by ligating normally disjoint DNA molecules, are considered recombinant for the purposes of the present invention. It is understood that when a recombinant nucleic acid is produced and introduced into a host cell or organism, it replicates in a non-recombinant form, that is, using the host cell's in vivo cellular machinery, rather than manipulation in vitro; however, once such nucleic acid is produced recombinantly , although subsequently replicated in a non-recombinant form, is still considered recombinant for the purposes of the present invention. Similarly, a "recombinant protein" is a protein produced using recombinant techniques, ie, a protein produced by expression of a recombinant nucleic acid as described above.

"Chemotherapy resistance" as used herein refers to non-response to chemotherapy treatment, ie failure to kill or inhibit tumor growth by such treatment.

The term "salt" as used herein refers to salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the particular substituents on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (pure ash or a solution of such base in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either pure or in a suitable inert solvent. . Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids including, for example, hydrochlorides, hydrobromides, nitrates, and salts derived from relatively nontoxic organic acids. , carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, hydrogen sulfate, hydroiodide or phosphite, etc., the salts derived from organic acids include, for example, acetic acid Salt, propionate, isobutyrate, maleate, malonate, benzoate, succinate, suberate, fumarate, mandelate, phthalate Salt, benzenesulfonate, p-toluenesulfonate, citrate, tartrate, methanesulfonate, etc. Also included are salts of amino acids such as arginine etc. and organic acids such as glucuronic acid or galacturonic acid etc. (see, e.g., Berge, S.M. et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain particular compounds of the present invention contain both basic and acidic functional groups, so that the compounds can be converted into base or acid addition salts.

The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but the salts are equivalent to the parent form of the compound for the purposes of the present invention.

The term "solvate" as used herein refers to a compound of the invention in complex with a solvent. Solvents that can form solvates with the compounds of the present invention include common organic solvents such as alcohols (methanol, ethanol, etc.), ethers, acetone, ethyl acetate, halogenated solvents (methylene chloride, chloroform, etc.), hexane and pentane. Other solvents include water. When water is used as the complex solvent, the complex is called "hydrate".

The term "subject" or "patient" refers to a mammal, preferably a human, in need of treatment for a condition (eg, cancer), disorder or disease.

The term "TAF" refers to TBP-associated factor. TAF is preferably TAFII, the transcriptionally activating TAF protein of eukaryotic genes involved in mediating RNA polymerase II transcription. TAF protein can interact with other transcriptional activators or repressors (Goodrich and Tjian, Curr Opin Cell Biol 6(3):4O3-9(1994); Albright and Tjian, Gene 242(1-2):1-13 (2000)). TAF is preferably from human, mouse, Drosophila or yeast. The TAF protein that interacts with GLI is preferably TAF _II 31 protein. Klemm et al. cloned the human TF _II D subunit and called it hTAF _II 32 (Klemm et al., 1995, Proc Natl AcadSci USA, 92(13):5788-92). This 32-kD protein was isolated from HeLa cell nuclear extracts and partially sequenced. The identified cDNA has a deduced amino acid sequence of 264 residues and is related to Drosophila TAF _II 40. Klemm et al. have demonstrated that TAF _II 32 can interact with GTF2B and the viral transcriptional transactivator VP16 (Klemm et al. 1995, Proc Natl Acad Sci USA, 92(13):5788-92). The authors demonstrate that recombinantly expressed TAF _II 32 is functional in a partially reconstituted TF _II D complex that mediates activation through the GAL4-VP16 fusion protein. TAF _II 32 and TAF _II 31 are two names for the same protein, now also known as TAF9. cloned TAF9, which they termed TAF _II 31, by Lu et al. TAF9 can encode a protein of 264 amino acids. Immunoprecipitation and binding assays demonstrated that TAF9 interacts with the N-terminal domain of p53 at the same site as that of MDM2, the major negative regulator of p53 activity in cells. (Lu et al., 1995, Proc Natl Acad Sci USA, 92(11):5154-8). Human TAF _II 31 nucleotide and protein sequences can be found, eg, in GenBank Accession Nos. U25112, U21858 and NM_016283.

The term "treating" as used herein includes: (1) preventing a disease (such as cancer), even if a subject predisposed to developing the disease but not yet showing any symptoms of the disease does not develop clinical symptoms of the disease; (2) inhibiting the disease, i.e. Block or reduce the occurrence of the disease or its clinical symptoms; or (3) alleviate the disease, even if the disease or its clinical symptoms subside. Treatment refers to any means by which the symptoms or pathology of a condition, disorder or disease are ameliorated or beneficially altered. Preferably, the subject in need of such treatment is a mammal, more preferably a human.

"Tumor cells" refer to precancerous cells, cancer cells and normal cells in a tumor.

The term "Wnt" refers to a family of mammalian genes that encode proteins related to the Drosophila somite polarity gene wingless. In humans, the Wnt gene family typically encodes a 38-43 kDa cysteine-rich glycoprotein containing a hydrophobic signal sequence and a conserved asparagine-linked oligosaccharide consensus sequence (Shimizu et al., Cell Growth Differ 8 (12): 1349-58 (1997)). The Wnt family includes at least 19 mammalian members. Exemplary Wnt proteins include Wnt1, Wnt2, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt10A, Wnt10B, Wnt11, Wnt12, Wnt13, Wnt14, Wnt15, and Wnt16. A preferred Wnt protein of the invention is Wnt2, preferably human Wnt2 protein.

II. Small Molecule Compounds

A. Selection of GLI3 target sequence

Transcriptional activation domains share little sequence homology and often lack folded structure when not bound to a target protein. GLI, GLI2 and GLI3 described herein are believed to be able to make contact with the TBP-associated factor TAF _II 31 through an alpha-helical region within the transcriptional activation domain (see Figure 3). One aspect of the present invention recognizes that, while the individual GLI, GLI2 and GLI3 transcriptional activation domains are well conserved across many species, they themselves vary widely. For example, the transactivation domains of human GLI1, NH2 _- DSLDLDNTQLDFVAILDE-COOH (human GLI1, amino acid residues 1037-1054) and mouse GLI1, _NH2- DSLDLDNTQLDFVAILDE-COOH (mouse GLI1, amino acid residues 1040-1057) same. Additionally, transactivation of human GLI2, NH ₂ -DSQLLE A PQIDFDAIMDD-COOH (amino acid residues 1501-1518) and mouse GLI2, NH ₂ -DSQLLE P PQIDFDAIMDD-COOH (mouse GLI2, amino acid residues 1509-1526) Domains differ by only one amino acid residue position (italicized and underlined). The transactivation domains of human GLI3, NH ₂ -D SHDLEGVQIDFDAIIDD-COOH (human GLI3, amino acid residues 1495-1512) and mouse GLI3, NH ₂ -E SHDLEGVQIDFDAIIDD-COOH (amino acid residues 1497-1514) also differ only by An amino acid residue position (italics and underline).

However, there are substantial differences between human GLI1, human GLI2, and human GLI3 (asterisks indicate identical amino acid residues between human GLI1, GLI2, and GLI3):

NH ₂ -DSLDLDNTQLDFVAILDE-COOH (human GLI1, amino acid residues 1037-1054),

＊＊＊＊＊＊＊＊＊＊

NH ₂ -DSQLLEAPQIDFDAIMDD-COOH (human GLI2, amino acid residues 1501-1518),

＊＊＊＊＊＊＊＊＊＊

NH ₂ -DSHDLEGVQIDFDAIIDD-COOH (human GLI3, amino acid residues 1495-1512),

The following is a comparison of the TAF _II 31 action domains of human GLI1 and human GLI2 (asterisks represent identical amino acid residues):

NH ₂ -DSLDLDNTQLDFVAILDE-COOH (human GLI1, amino acid residues 1037-1054)

＊＊＊＊＊＊＊＊＊＊

NH ₂ -DSQLLEAPQIDFDAIMDD-COOH (human GLI2, amino acid residues 1501-1518)

The following is a comparison of the TAF _II 31 action domains of human GLI2 and human GLI3 (asterisks represent identical amino acid residues):

NH ₂ -DSQLLEAPQIDFDAIMDD-COOH (human GLI2, amino acid residues 1501-1518)

＊＊＊＊＊＊＊＊＊＊＊＊＊

NH ₂ -DSHDLEGVQIDFDAIIDD-COOH (human GLI3, amino acid residues 1495-1512)

As noted above, specific and redundant functions of GLI2 and GLI3 have been identified during mammalian skeletal development, however, for example, GLI1 but not GLI3 activates the HNF-3β enhancer in tissue culture (Mo et al., 1997, Development 124:113-123; Sasaki et al., 1997, Development 124:1313-1322). Differences in TAF binding or binding affinity may result in different properties of GLI1, GLI2 and GLI3.

Yoon et al. describe that the GLI1 transcriptional activation domain is similar to the VP16d transcriptional activation domain, including an alpha-helical region containing a conserved FXXΦΦ motif. (Yoon et al., 1998, J. Biol. Chem., 273(6):3496-3501). Upon binding to the target protein TAF _II 31, the VP16 activation domain is induced to convert from a random coil to an α-helix, and three residues have been identified as direct contacts with TAF _II 31: D472, F479 and L483 (Uesugi et al., 1997, Science, 277:1310-1313). These three amino acid residues (D1040, F1048 and L1052) were also found in the activation domain of human GLI1 comprising FXXΦΦ(FVAIL).

The FXXΦΦ motif in GLI3 is FDAII. This GLI3 motif is very likely directly involved in contacting TAF, possibly TAF _II 31 .

The FXXΦΦ motif is also found in the activation domain of the tumor suppressor gene p53 and in NF-κB p65. The cell attenuator MDM2 of p53 can distinguish the FXXΦΦ motif of p53 from NF-κB p65 and VP16, and specifically inhibit p53 activity (Uesugi and Verdine, 1999, Proc.Natl.Acad.Sci.USA, 96 (26 ): 14801-14806). Therefore, it is reasonable to assume that the activation domain of polypeptide I containing the FXXΦΦ motif can be distinguished from the activation domain of polypeptide II which also contains the FXXΦΦ motif but has a different amino acid sequence. Therefore, it is also possible to significantly inhibit the activity of polypeptide I (such as GLI3) by designing and using a small molecule compound that mimics the FXXΦΦ motif of polypeptide I, that is, the amino acid sequence of the FXXΦΦ motif of polypeptide I, but does not significantly inhibit the activity of polypeptide II. active.

Therefore, the object of the present invention is to provide small molecule compounds that can significantly inhibit GLI3 signal transduction but not GLI or GLI2 signal transduction. Accordingly, one aspect of the present invention provides small molecule compounds that differentially affect the transcriptional activation potential of GLI1, GLI2, and GLI3.

In one embodiment of the present invention, the small molecule compound inhibits or reduces the binding of GLI3 to TAF, preferably TAF _II 31 .

In another embodiment of the present invention, a small molecule compound that mimics the FXXΦΦ motif of a GLI protein (preferably a human or mouse GLI protein) is provided. In a preferred embodiment, the small molecule compound mimics the FXXΦΦ motif FDAII of GLI3 (see FIG. 3 ).

In other embodiments of the present invention, the small molecule compound mimics the FXXΦΦ motif FDAIM of GLI2. In another embodiment of the present invention, the small molecule compound mimics the FXXΦΦ motif FVAIL of GLI1.

B. Small molecule compounds of the present invention

As mentioned above, one of the objects of the present invention is to provide small molecular compounds that mimic the transcriptional activation domain of GLI polypeptides, preferably GLI3 polypeptides. The prior art describes a number of α-helical mimetics containing triaryl/alkyl heterocycles. However, pyrazoline-based compounds that mimic the α-helical region have not been sufficiently studied. Therefore, one of the objects of the present invention is to provide small molecule compounds based on pyrazoline and mimicking the α-helical region of the GLI transcriptional activation domain, preferably the α-helical region of the GLI3 transcriptional activation domain. Figure 3 shows the RMS overlap of the small molecule compounds of the invention (FN1-5; see below), including the proposed alpha-helical region containing the GLI3 FDAII motif.

Accordingly, compounds of the present invention include small molecule compounds having the formula:

In the formula, X ¹ and X ² are each independently N or C, in the formula, one of X ¹ and X ² is N, and one of X ¹ and X ² is C, so the ring N forms a double ring with the C of X ¹ or X ² key. In addition, R ¹ , R ² and R ³ are _each independently selected from C ₁ -C ⁶ alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkane optionally substituted with 0-3 R groups The R ⁶ groups are each independently selected from hydrogen, halogen, C ₁ -C ₆ alkyl, -OR ⁷ , -C(O)R ⁷ , -OC(O)R ⁷ , -N(R ⁷ , R ⁷ ), -NR ⁷ S(O) ₂ R ⁷ , -C(O)N(R ⁷ , R ⁷ ), -N(R ⁷ )C(O)R ⁷ , -N(R ⁷ )C (O)N(R ⁷ , R ⁷ ), -C(NR ⁷ )N(R ⁷ , R ⁷ ), -N(R ⁷ )C(NR ⁷ )N(R ⁷ , R ⁷ ) and S(O ) _m R ⁷ , wherein the subscript m is 0, 1 or 2. Y is a direct bond or C ₁ -C ₄ alkyl, and Z is selected from C ₁ -C ₄ alkyl, aryl and heteroaryl. R ⁴ is hydrogen, halogen, -OR ⁷ , -OC(O)R ⁷ , -C(O)OR ⁷ , -N(R ⁷ , R ⁷ ), -NR ⁷ S(O) ₂ R ⁷ , -C (O)N(R ⁷ , R ⁷ ), -N(R ⁷ )C(O)N(R ⁷ , R ⁷ ), -C(NR ⁷ )N(R ⁷ , R ⁷ ) and S(O) _m R ⁷ , wherein the subscript m is 0, 1 or 2. R ⁵ is hydrogen, C ₁ -C ₆ alkyl, or optionally combined with Y to form a 5-6 membered heterocycle. Each R ⁷ is independently H or C ₁ -C ₆ alkyl.

In addition, salts, hydrates, solvates, isomers and prodrugs of the small molecule compounds of the invention are also contemplated.

In another embodiment, the present invention provides small molecule compounds having the formula, wherein R ¹ , R ² and R ³ are each aryl. In another embodiment, R ¹ , R ² and R ³ are each phenyl.

In another embodiment, the present invention provides small molecule compounds of the formula, wherein R ⁴ is hydroxyl. In another embodiment, Z is C ₁ -C ₄ alkyl. In yet another embodiment, Z is aryl. In yet another embodiment, Z is phenyl.

In another embodiment, the present invention provides small molecule compounds having the formula, wherein R ¹ , R ² and R ³ are each phenyl, Y is C ₁ -C ₄ alkyl, Z is C ₁ -C ₄ Alkyl or phenyl, ^R4 is hydroxyl.

Therefore, the small molecule compounds of the present invention preferably include but are not limited to:

Other compounds of the present invention include the following small molecule compounds:

Those skilled in the art will understand that other small molecule compounds can also be used in the present invention.

C. Preparation of Small Molecule Compounds

Small molecule compounds of the invention can be prepared by condensation of ethyl cinnamate and benzaldehyde phenylhydrazone followed by saponification of the ethyl ester and reaction with amines to form the desired amide end product (see Example 3). Alternatively, cinnamic acid is reacted with amine to prepare amide, and then condensed with benzaldehyde and phenylhydrazone to prepare the small molecule compound of the present invention. Those skilled in the art recognize that there are other methods of preparing the small molecule compounds of the invention.

In one embodiment of the invention, the small molecule compound of the invention comprises a tag. This is particularly useful for detecting and testing the distribution of small molecule compounds after in vivo administration. For example, ³ H can be used as a label for routine pharmacokinetic/pharmacodynamic studies. Small molecule compounds containing tags can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical methods.

The small molecule compounds of the present invention may also contain unnatural isotopic moieties at one or more atoms constituting the compounds. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ^3H ) or carbon-14 ( ^14C ). The scope of the present invention shall include all isotopic variations (whether radioactive or not) of the compounds of the present invention.

D. Detection of Small Molecule Compounds Using Cellular Assays

Small molecule compounds of the invention can be screened for activity in vitro and in vivo. In in vitro assays, the invention provides cell-based cytotoxicity assays and apoptosis assays, as described herein (see, eg, Example 1). In in vivo assays, the invention provides mouse xenograft tumor assays, as described herein (see eg, Examples 33-35).

III. Pharmaceutical composition

One aspect of the present invention provides a pharmaceutical composition or medicament comprising at least one small molecule compound of the present invention and optionally a pharmaceutically acceptable carrier. A pharmaceutical composition or medicament may be administered to a patient to treat, for example, a condition such as cancer.

A. Formulation and Administration

The small molecule compound of the present invention can be used to prepare a pharmaceutical composition or drug containing an effective amount of the small molecule compound of the present invention, combined or mixed with excipients or carriers suitable for enteral or parenteral administration.

Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein in "Remington's Pharmaceutical Sciences" by E.W. Martin. Small molecule compounds of the invention and their physiologically acceptable salts and solvates may be formulated for administration by any suitable route, including inhalational, topical, nasal, oral, parenteral or rectal. Thus, the drug may be administered by intradermal, subcutaneous, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary, or intratumoral injection with a syringe or other device combination. Transdermal administration is also contemplated, as well as inhalation or aerosol administration. Tablets and capsules can be administered orally, rectally, or vaginally.

In oral administration, the pharmaceutical compositions or medicaments may be administered by conventional means, for example in the form of tablets or capsules prepared with pharmaceutically acceptable excipients. Tablets and gelatin capsules containing the active ingredient, i.e. the small molecule compound of the present invention, together with, for example: (a) diluents or fillers such as lactose, dextrose, sucrose, mannitol, sorbitol, Cellulose (eg ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates and/or dibasic calcium phosphate, calcium sulfate; (b) lubricants such as silicon dioxide, talc, stearic acid , magnesium stearate or calcium stearate, metal stearate, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and/or polyethylene glycol; for tablets also contains (c ) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropylmethylcellulose; if desired, can also be added (d) disintegrating agents such as starch (such as potato starch or sodium starch glycolate), glycolic acid, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents such as sodium lauryl sulfate and/or or (f) adsorbents, coloring agents, flavoring and sweetening agents.

Tablets may be film or enteric coated according to methods known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional methods with pharmaceutically acceptable additives including, for example: suspending agents such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats; emulsifying agents such as lecithin or arabic gums; non-aqueous vehicles, such as almond oil, fat, ethanol, or fractionated vegetable oils; and preservatives, such as methyl or propyl p-hydroxybenzoic acid or sorbic acid. The formulations may also contain buffering, flavouring, coloring and/or sweetening agents as appropriate. Preparations for oral administration may be suitably formulated so as to provide controlled release of the active compound, if desired.

For administration by inhalation, the compound is conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethylene, alkanes, carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered quantity. Capsules and cartridges of gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Small molecule compounds of the invention may be formulated for parenteral injection administration, such as bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The compositions may be sterile and/or contain auxiliary agents, such as preservatives, stabilizers, wetting or emulsifying agents, solubilizers, salts for adjusting osmotic pressure and/or buffers. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile pyrogen-free water, before use. In addition, they may contain other therapeutically valuable substances. The composition is prepared according to conventional mixing, granulating or coating methods, and contains about 0.1-75%, preferably about 1-50%, of the active ingredient.

Formulations suitable for transdermal administration comprise an effective amount of a compound of the invention and a carrier. Preferred carriers include absorbable pharmaceutically acceptable solvents to facilitate passage through the skin of the host. For example, a transdermal device is in the form of a bandage comprising: a liner member, a reservoir containing a compound and optionally a carrier; an optional rate controlling barrier to allow the drug to be released at a controlled and predetermined rate over an extended period of time administering the compound to the skin of the host; and a mechanism for securing the device to the skin. Matrix transdermal formulations may also be used.

Suitable formulations for topical application, eg, to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well known in the art. Such formulations may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

Furthermore, the compounds can be formulated as depot preparations. Such long-acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials or ion exchange resins, eg, as emulsions with acceptable oils, or as sparingly soluble derivatives, eg, as sparingly soluble salts.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispensing device may be accompanied by instructions for administration.

In one embodiment of the invention, a pharmaceutical composition or medicament comprises an effective amount of a compound of the invention as described above and another therapeutic agent, such as a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to: daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, ethrubicin, bleomycin, maphos Amide, Ifosfamide, Cytarabine, Dichloroethylnitrosourea, Busulfan, Mitomycin C, Actinomycin D, Mithramycin, Chlorprednisone, Hydroxy Progesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosourea , nitrogen mustard, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxy Cyclophosphamide peroxide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, paclitaxel, vincristine, vinblastine, etopol glycosides, trimetrexate, teniposide, cisplatin, and diethylstilbestrol (DES). See generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., 1987, pp. 1206-1228, eds. Berkow et al., Rahway, NJ.

When used together with the small molecular compound of the present invention, this type of chemotherapeutic drug can be used alone (such as 5-FU and small molecular compound), continuous administration (such as administration of 5-FU and small molecular compound after a period of time, giving (such as) MTX and small molecule compounds), or in combination with one or more of these chemotherapeutic agents (eg, 5-FU, MTX, and small molecule compounds, or 5-FU, radiotherapy, and small molecule compounds). They can be administered by the same or different routes of administration, or they can be administered together in the same pharmaceutical preparation.

In one aspect of the invention, a therapeutically effective amount of a small molecule compound of the invention is administered in combination with surgery and optionally another chemotherapeutic agent.

B. Therapeutically Effective Amounts and Administration

In one embodiment of the present invention, a therapeutically effective dose of a pharmaceutical composition or medicament is administered to a patient to prevent, treat or manage cancer. A sufficient amount of the pharmaceutical composition or medicament is administered to a patient to produce an effective therapeutic effect in the patient. An effective therapeutic effect is one that at least partially arrests or slows down the symptoms or complications of the disease. The amount capable of accomplishing this is defined as "therapeutically effective dose".

The dose of the active small molecule compound to be administered depends on the species of warm-blooded animal (mammal), body weight, age, individual condition, surface area of the area to be treated and the form of administration. The size of the dose will also depend on the existence, nature and extent of side effects associated with the administration of a particular small molecule compound to a particular subject. A unit dose for oral administration to a mammal of about 50-70 kg may contain about 5-500 mg of active ingredient. Generally, the dosage of the active small molecule compound of the present invention is a dosage sufficient to achieve the desired effect.

The optimal dosage regimen can be calculated from the cumulative measured value of the small molecule compound in the subject. Typically, the dose is 1 nanogram to 1,000 mg/kg body weight, given one or more times daily, weekly, monthly or yearly. One of ordinary skill in the art can easily determine the optimum dosage, method of administration and frequency of administration.

In one embodiment of the present invention, the dosage of the pharmaceutical composition or medicament containing the small molecule compound of the present invention is administered daily in the range of about 1 mg small molecule compound/kg subject body weight (1 mg/kg) to about 1 g/kg. In another embodiment, the daily dosage ranges from about 5 mg/kg to 500 mg/kg. In yet another embodiment, the daily dosage is from about 10 mg/kg to 250 mg/kg. In another embodiment, the daily dosage is about 25 mg/kg-150 mg/kg. The daily dose may be administered once per day, or in sub-doses administered multiple times per day, such as two, three or four times per day.

To achieve the desired therapeutic effect, small molecule compounds must be administered over multiple days in therapeutically effective daily doses. Thus, therapeutically effective administration of small molecule compounds to treat cancer in a subject requires regular (eg, daily) dosing for a period of time ranging from three days to two weeks or more. Generally, the small molecule compound can be administered for at least three consecutive days, often for at least 5 consecutive days, more often for at least 10 consecutive days, and sometimes for 20, 30, 40 or more consecutive days. While continuous daily administration is the preferred route to achieve a therapeutically effective dose, therapeutically beneficial effects can be achieved even if the small molecule compound is not administered daily so long as repeated dosing occurs frequently enough to maintain a therapeutically effective concentration of the small molecule compound in the subject . For example, the small molecule compound may be administered every other day, every third day, or once a week if a higher dosage range is employed and tolerated by the subject. See, eg, Examples 33-35 and Figures 15-17 for dosing regimens.

Optimal dosage, toxicity, and therapeutic efficacy of such small molecule compounds may depend on the relative potency of the individual small molecule compounds, which can be determined by standard pharmaceutical methods used in cell culture or experimental animals, e.g., determining the _LD50 (the amount that is lethal to 50% of the population). dose) and _ED50 (dose effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio _LD50 / _ED50 . Compounds with large therapeutic indices are preferred. While compounds with toxic side effects may be employed, care should be taken in designing delivery systems that target such compounds to diseased tissue sites to minimize damage to normal cells, thereby reducing side effects.

Data obtained, for example, from cell culture assays and animal studies can be used in determining a range of dosage for use in humans. The dosage of such small molecule compounds lies preferably within a range of circulating concentrations that include the _ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any small molecule compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be determined in animal models so that circulating plasma concentrations include the _IC50 (the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Concentrations in plasma can be determined by high performance liquid chromatography (HPLC). Generally, the dose equivalent of a small molecule compound is about 1 ng/kg to 100 mg/kg for a typical subject.

Following successful treatment, the subject may require maintenance therapy to prevent recurrence of the disease being treated, such as cancer.

IV. Identification of Cells Expressing GLI Proteins

A. Detection of GLI protein

In another embodiment, a method of detecting cancer comprises measuring the level of a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, in a biological sample from a subject (eg, a patient). In a preferred embodiment, the level of the activating fragment of the GLI3 protein containing the transcriptional activation domain is determined. Detection of an increased level of a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide or an increased level of an activating fragment of a GLI3 protein (relative to normal levels) indicates that the subject has cancer.

For example, increased expression of GLI1, GLI2, or GLI3 is indicative of cancer, or may be associated with various cancers. Therefore, GLI1, GLI2 or GLI3 polypeptides or Gli1, Gli2 or Gli3 polynucleotides can be used as biomarkers for diagnosing cancer. In a preferred embodiment of the present invention, the content of a GLI protein such as GLI3 in a biological sample is determined. Typically, for example, the level of GLI3 in a biological sample provided by a normal, healthy or non-cancerous subject correlates with the level of GLI3 in a biological sample provided by a cancer patient or a subject suspected of having cancer. The amount of GLI3 detected in a biological sample provided by a cancer patient or a subject suspected of having cancer may be specific for a given cancer.

For example, a GLI polypeptide such as a GLI3 polypeptide can be detected by any particular detection method, including but not limited to: affinity capture, mass spectrometry, traditional immunoassays for GLI3, PAGE, Western blot, or HPLC, as further described herein or by those skilled in the art known.

Examples of detection that can be used for this purpose include optical methods, electrochemical methods (techniques that measure voltage and measure current), atomic force microscopy, and radio frequency methods such as multi-order resonance spectroscopy. Examples of optical methods, in addition to confocal and non-confocal microscopy, are the detection of fluorescence, luminescence, chemiluminescence, light absorption, reflectance, transmittance, and birefringence or refractive index (e.g. surface plasmon resonance, ellipsometry , resonant mirror method, grating coupler waveguide method or interferometry).

B. Detection of Gli mRNA

In one embodiment, a method of detecting cancer comprises determining the level of a transcript encoding a GLI polypeptide, preferably a GLI3 polypeptide (see, e.g., Genbank Accession No. NM 007191 and SEQ ID NO: 1 ) in a biological sample of a subject (such as a patient). Detection of an elevated level of Gli mRNA, preferably GLI3 mRNA relative to normal levels indicates that the subject has cancer. In one embodiment, the step of determining the level of GlimRNA comprises an amplification reaction. In another embodiment, the presence of cancer is assessed by measuring the expression level of mRNA encoding a GLI polypeptide in the cells. Gli mRNA levels higher than in corresponding non-cancerous tissue indicate the presence of cancer. Methods for assessing the level of RNA expression of a particular gene are well known to those skilled in the art, including hybridization and amplification assays, and the like.

1. Direct hybridization test

Methods for detecting and/or quantifying the level of Gli gene transcript (mRNA or cDNA prepared therefrom) using nucleic acid hybridization techniques are well known to those skilled in the art. For example, one method of assessing the presence, absence or amount of a Gli polynucleotide, such as a Gli3 polynucleotide, includes Northern blots. Gene expression levels can also be analyzed by techniques known in the art, such as dot blotting, in situ hybridization, RNase protection, detection DNA microchip assays, and the like.

2. Amplification test

In another embodiment, the expression level of the Gli gene, preferably the Gli3 gene, is determined using an amplification assay. In such assays, the Gli nucleic acid sequence is used as a template for an amplification reaction such as polymerase chain reaction or PCR. In quantitative amplification, the amount of amplified product is directly proportional to the amount of template in the original sample. Comparison to an appropriate control measures Gli mRNA levels in the sample. Methods for quantitative amplification are well known to those skilled in the art. Detailed protocols for quantitative PCR are found in (e.g., Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). The known Gli nucleic acid sequence (see, eg, ATCC accession number herein) is sufficient to allow one skilled in the art to select primers to amplify any portion of the gene. See, eg, Example 1 and Figure 2 for semi-quantitative RT-PCR analysis.

In one embodiment, cancer-associated polynucleotides are quantified using a TaqMan-based assay. TaqMan-based assays employ fluorescent oligonucleotide probes containing a 5' fluorescent dye and a 3' quencher. This probe hybridizes to PCR products but cannot extend itself because of the capping agent at its 3' end. The 5' nuclease activity of a polymerase such as AmpliTaq results in cleavage of the TaqMan probe when the PCR product is amplified in subsequent cycles. This cleavage separates the 5' fluorescent dye from the 3' quencher, thereby increasing the level of fluorescence as amplification proceeds (see, for example, literature from Perkin-Elmer at www2.perkin -elmer.com).

Other suitable amplification methods include, but are not limited to: ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989); Landegren et al., Science 241:1077 (1988); and Barringer et al., Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc.Natl.Acad.Sci.USA 86:1173 (1989)), self-sustained sequence replication (Guatelli et al., Proc.Nat.Acad.Sci.USA 87:1874 ( 1990)), dot PCR and linker adapter PCR, etc.

V. method

The small molecule compounds of the invention can be used in a variety of ways. The present invention provides methods for using the small molecule compounds of the present invention to, for example, (i) induce apoptosis of cells expressing GLI polypeptides, (ii) inhibit adverse growth, excessive proliferation or survival of cells expressing GLI polypeptides, (iii) ) treating a condition expressing a GLI polypeptide, such as cancer, (iv) inhibiting the expression of a gene having a GLI DNA binding site and (v) inhibiting the activity of a GLI polypeptide.

Any cell or tumor cell expressing a GLI polypeptide, preferably a GLI3 polypeptide, can be used to practice the methods of the invention. The cell or tumor cell expressing the GLI polypeptide is preferably selected from colon cancer, melanoma, mesothelioma, lung cancer, renal cell carcinoma, breast cancer, prostate cancer, sarcoma, ovarian cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, nasopharyngeal carcinoma , pancreatic cancer and glioma cells.

The methods of the invention can be practiced in vitro or in vivo.

A. Induction of apoptosis with small molecule compounds

Apoptosis plays a central role in the development and homeostasis of multicellular organisms. "Apoptosis" refers to programmed cell death characterized by certain cellular features such as membrane blebbing, chromatin shrinkage and fragmentation, formation of apoptotic bodies, and positive "TUNEL" (terminal deoxynucleotidyl transferase- UTP-mediated nick-end labeling) staining pattern. The next step in the apoptotic process is the degradation of the plasma membrane, allowing the apoptotic cell to leak various dyes (eg, propidium iodide).

Apoptosis may be induced by multiple independent signaling pathways centered on final effector mechanisms by multiple interactions between several death receptors and their ligands (belonging to the tumor necrosis factor (TNF) receptor/ligand superfamily). composed of interactions. The best characterized death receptors are CD95 ("Fas"), TNFRl (p55), death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). The final effector mechanism of apoptosis is the activation of a series of proteases called caspases. Activation of these caspases results in the cleavage of a series of viable cellular proteins and cell death.

The present invention provides methods for inducing apoptosis in cells expressing GLI proteins. In one aspect, the method of inducing apoptosis comprises the step of exposing the cell to a composition or contacting the cell with a composition comprising a small molecule compound of the invention. In a preferred embodiment, the composition comprises small molecule compounds FN1-5. In another preferred embodiment, the composition comprises the small molecule compound FN1-8. Generally, the cells are exposed or contacted with an effective amount of the composition, such contacting being capable of inducing apoptosis. In preferred embodiments, the GLI protein is GLI2 or GLI3.

In another preferred embodiment of the invention, exposing a cell to a composition comprising a small molecule of the invention comprises introducing the composition into the cell.

In another aspect of the present invention, a method for inducing tumor cell apoptosis is provided, which includes the step of administering the small molecular compound of the present invention. In a preferred embodiment, the small molecule compound is FN1-5. In another preferred embodiment, the small molecule compound is FN1-8.

In a preferred embodiment, the cells are exposed or contacted to the composition ex vivo. In another preferred embodiment, the cells are exposed to or contacted with the composition in vivo.

B. Inhibition of Undesired Growth, Proliferation, or Survival of Cells with Small Molecule Compounds

One of the objects of the present invention is to provide novel therapeutic regimens for the treatment of cancer comprising the administration of small molecule compounds of the present invention that prevent signal transduction through the GLI signaling pathway. One aspect of the invention provides a method of inhibiting at least one of undesirable growth, excessive proliferation or survival of a cell. This method includes the step of determining whether the cell expresses a Gli gene or a GLI polypeptide. The method also includes the step of contacting the cell with an effective amount of a small molecule of the invention, wherein said contacting step results in inhibition of at least one of undesirable growth, excessive proliferation or survival of the cell.

Cells or tumor cells whose at least one behavior of undesirable growth, hyperproliferation or survival is inhibited are preferably selected from: colon cancer cells, melanoma cells, mesothelioma cells, lung cancer cells, renal cell carcinoma cells, breast cancer cells, prostate cancer cells cells, sarcoma cells, ovarian cancer cells, esophageal cancer cells, gastric cancer cells, hepatocellular carcinoma cells, nasopharyngeal cancer cells, pancreatic cancer cells, and glioma cells.

In another preferred embodiment of the present invention, a method of inhibiting the proliferation of cells expressing a GLI polypeptide, preferably GLI3, is provided. "Proliferation" refers to cell growth, multiplication or multiplication of cells or pathological cysts. The method includes the step of contacting the cell with an effective amount of the small molecule compound that inhibits cell proliferation. Preferably, the small molecular compound FN1-5 or FN1-8 used alone or in combination can inhibit the proliferation of cells, preferably cancer cells.

In a preferred embodiment of the invention, the method is performed in vitro. As further described herein, the methods of the invention can also be practiced in vivo.

Many tumors metastasize. Thus, in another aspect of the invention, the small molecule compounds of the invention are used to prevent the formation of metastases. The method includes the step of administering a small molecule compound of the invention to tumor cells, such administration preventing metastasis. In a preferred embodiment, the small molecule compound is FN1-5. In another preferred embodiment, the small molecule compound is FN1-8.

In a preferred embodiment of the present invention, the cells expressing the GLI protein and contacting the small molecule compound of the present invention are cancer cells selected from the group consisting of colon cancer, melanoma, mesothelioma, lung cancer, renal cell carcinoma, breast cancer, prostate Carcinoma, sarcoma, ovarian, esophageal, gastric, hepatocellular, nasopharyngeal, pancreatic, and glioma cells. Preferably the cancer cells are lung cancer cells, colon cancer cells or melanoma cells.

C. Treating Cancer with Small Molecule Compounds

The methods of the invention can be performed in vitro and in vivo. Accordingly, in another aspect of the invention, methods of treating a subject suffering from cancer are provided. The method comprises the step of administering to the subject a therapeutically effective amount of the small molecule compound, wherein the cancer is characterized by expression of a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, wherein the administering step results in treatment of the subject.

As shown herein, a majority of cancers express GLI polypeptides, preferably GLI3 polypeptides (Figure 2; data not shown). Thus, the small molecule compounds of the present invention can be used to treat cancer in most subjects. The cancer is preferably selected from colon cancer, melanoma, mesothelioma, lung cancer, renal cell carcinoma, breast cancer, prostate cancer, sarcoma, ovarian cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, nasopharyngeal cancer, pancreatic cancer and glioma .

Therefore, in a preferred embodiment of the invention, a subject suffering from colon cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound.

In another preferred embodiment of the invention, a subject suffering from breast cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound.

In yet another preferred embodiment of the present invention, a subject suffering from nasopharyngeal carcinoma expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound.

In one embodiment of the invention, a subject suffering from lung cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Lung cancers include, but are not limited to: bronchial carcinoma [squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma], pneumocytic carcinoma [bronchiole carcinoma], bronchial adenoma, sarcoma, lymphoma, Chondromatous hamartoma, mesothelioma, SCLC and NSCLC.

In another embodiment of the invention, a subject suffering from a sarcoma expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Sarcomas include, but are not limited to, cancers such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma.

In another embodiment of the invention, a subject suffering from a gastrointestinal cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Gastrointestinal cancers include, but are not limited to: Esophageal cancer [squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma] Gastric cancer [epithelial carcinoma, lymphoma, leiomyosarcoma] Pancreatic cancer [biliary adenocarcinoma, insulinoma, Glucagonoma, gastrinoma, carcinoid tumor, vasodilatory intestinal peptide tumor], small bowel cancer [adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, nerve fibroma, fibroma] and colorectal carcinoma [adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma].

In one embodiment of the invention, a subject suffering from a genitourinary cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Cancers of the genitourinary tract include, but are not limited to: kidney cancer [adenocarcinoma, Wilms tumor (nephroblastoma), lymphoma, leukemia, renal cell carcinoma], bladder cancer, and urethral cancer [squamous cell carcinoma, transitional cell carcinoma , adenocarcinoma], prostate cancer [adenocarcinoma, sarcoma] and testicular cancer [seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor, fibroma, fibroma Tumors, adenoids, lipomas].

In another embodiment of the present invention, a subject suffering from liver cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Liver cancer includes, but is not limited to: hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

In one embodiment of the invention, a subject suffering from a skin cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Skin cancers include, but are not limited to: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles, dysplastic moles, lipomas, hemangiomas, dermatofibromas, keloids, and psoriasis.

In one embodiment of the invention, a subject suffering from a gynecological cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Gynecologic cancers include, but are not limited to: Uterine cancer [endometrial cancer], cervical cancer [epithelial carcinoma of the cervix, preinvasive cervical dysplasia], ovarian cancer [epithelial carcinoma of the ovary (serous cyst adenocarcinoma, mucinous cyst adenocarcinoma, uterine Endometrioid carcinoma, clear cell adenocarcinoma, unclassified epithelial carcinoma], granulosa theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma, and other blasts carcinoma], vulvar carcinoma [squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma], vaginal carcinoma [clear cell epithelial carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma) and fallopian tube carcinoma [ Epithelial Carcinoma].

In yet another embodiment of the invention, a subject suffering from a bone cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Bone cancers include, but are not limited to: osteogenic sarcoma [osteosarcoma], fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing sarcoma, malignant lymphoma [reticulocyte sarcoma], multiple myeloma, malignant giant cell tumor , chordoma, osteochondroma [osteochondral exostosis], benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant cell tumor.

In one embodiment of the invention, a subject suffering from a nervous system cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Nervous system cancers include, but are not limited to: cancers of the skull [osteoma, hemangioma, granuloma, xanthoma, Paget's disease of bone], meningeal cancers [meningioma, meningiosarcoma, gliomatosis] , brain cancer [astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, nerve sheath tumors, retinoblastoma, congenital tumors] and spinal cord cancers [neurofibromas, meningiomas, gliomas, sarcomas].

In one embodiment of the invention, a subject suffering from a hematological cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Cancers of the blood include, but are not limited to: Cancers of the blood [myelogenous leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome], Hodge King's disease and non-Hodgkin's lymphoma (malignant lymphoma).

In one embodiment of the invention, a subject suffering from adrenal cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, is treated with a small molecule compound. Adrenal cancers include, but are not limited to: neuroblastoma.

The present invention provides methods of treating or preventing cancer expressing a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide. In one embodiment of the invention, the method includes the step of administering to the patient a pharmaceutical composition. Such pharmaceutical compositions comprise, for example, small molecule compounds of the invention. In a preferred embodiment, the small molecule compound is FN1-5. In another preferred embodiment, the small molecule compound is FN1-8. The pharmaceutical compositions of the invention are administered alone or in combination with one or more other therapeutic compounds or treatments. Examples of such therapeutic compounds or treatments include, but are not limited to, paclitaxel, cyclophosphamide, tamoxifen, fluorouracil, and doxorubicin. Additionally, other chemotherapeutic agents are as described herein.

The cancer treatment method may optionally include one or more of the following steps: obtaining a biological sample of tissue or fluid from the individual; for example, contacting the biological sample with an antibody to GLI1, GLI2 or GLI3 to screen for expression of a GLI polypeptide, preferably GLI1, GLI2 or a biological sample of Gli3 polypeptide; or screening a biological sample expressing Gli1, Gli2 or Gli3 polynucleotide by detecting Gli1, Gli2 or Gli3 mRNA.

Many cancers are initially treated with the chemotherapeutic agents described herein. However, cancers often develop resistance to these chemotherapeutic drugs, which are then no longer effective. Thus, in one embodiment, the cancer is multidrug resistant or refractory cancer. Therefore, in another aspect of the present invention, the resistance of tumor cells to chemotherapeutic drugs is overcome with the small molecule compounds of the present invention. Such methods comprise the step of administering a small molecule compound of the invention to tumor cells resistant to at least one chemotherapeutic agent, wherein said administration results in subsequent tumor cell death. In a preferred embodiment, the small molecule compound is FN1-5. In another preferred embodiment, the small molecule compound is FN1-8.

D. Inhibition of expression of genes containing GLI PNA binding sites

Because GLI polypeptides, such as GLI1, GLI2 or GLI3 polypeptides, are used as transcriptional activators, the inhibition of GLI transcriptional activity by the methods of the present invention should also inhibit or downregulate downstream genes whose expression is regulated by GLI, preferably GLI1, GLI2 or GLI3 gene expression. GLI generally regulates the expression of downstream genes by binding to its cognate DNA binding site, which is operably linked to the promoter and/or enhancer sequence of the downstream gene. For example, the DNA binding site of GLI3 was described (Vortkamp et al., 1995, DNA Cell. Biol 14:629-34; Hallikas et al., 2006, Cell 124(1):47-59).

In one aspect of the invention, genes downstream of GLI1, GLI2 or GLI3 are identified. According to the present invention, genes downstream of GLI1, GLI2 or GLI3 are identified by DNA sequencing of the promoter and/or enhancer regions of the genes suspected to be regulated by GLI1, GLI2 or GLI3. In cancers expressing or overexpressing GLI1, GLI2 or GLI3, genes downstream of GLI1, GLI2 or GLI3 are expected to be also expressed or overexpressed. GLI downstream genes with GLI DNA binding sites can also be identified by fragmenting the genome and using the resulting fragments as probes in gel shift assays known in the art. Any fragments identified in such assays can be amplified, characterized and/or used as nucleic acid probes for screening and identifying the gene of origin, as is known in the art for screening genomic DNA libraries. (The present invention provides) Another method to identify GLI binding sites in mammalian enhancer elements, thus, GLI target genes are enhancer element locators (EEL; Hallikas et al., 2006, Cell 124:47-59) . For example, Hallikas et al. identified several enhancer elements containing two or more GLI binding sites conserved in mouse-to-human and rat-to-human alignments. These enhancer elements are linked to GLI target genes, such as exocytic complex elements Sec5 (SEC5L1), NULL (NM_018271; NM_144962), early growth response protein 3 (EGR-3) (zinc finger protein pilot) (EGR3), PR -domain zinc finger protein 10 (PRDM10), T-box transcription factor TBX2 (T-box protein 2; TBX2), SOX-13 protein (type 1 diabetes autoantigen ICA12) (island cell antigen 12) (SOX13), patch Patched protein homologue 1 (PTC1) (PTC) (PTCH) and proteins related to DAN and Cerberus (NM_022469). Therefore, the small molecule compounds of the present invention can be used to inhibit the expression of these target genes.

Therefore, in one embodiment of the invention, GLI downstream genes containing a GLI DNA binding site, preferably a GLI1, GLI2 or GLI3 DNA binding site, operably linked to a promoter and/or enhancer sequence are suppressed. Downstream genes can be inhibited using the methods of the present invention that inhibit GLI signal transduction. Specifically, the present invention provides a method for inhibiting the expression of GLI3 downstream genes, the method comprising the step of administering the small molecular compound of the present invention to cells expressing GLI3 downstream genes, wherein the administration leads to inhibition of the expression of GLI3 downstream genes. Optionally, the method includes a step of verifying that GLI3 downstream genes are repressed. This can be achieved using methods known in the art, such as RT-PCR or Western blot analysis.

In addition to the GLI target genes identified by Hallikas et al. (Hallikas et al., 2006, Cell 124:47-59), the present invention also identifies the Wnt2 gene as a GLI target gene, i.e., containing a GLI DNA binding site (see Examples herein). Therefore, in one embodiment of the present invention, the downstream gene is a Wnt gene, preferably a Wnt-2 gene, more preferably a human Wnt2 gene. In this embodiment, inhibiting GLI3 signaling results in inhibition or downregulation of Wnt-2 and Wnt-2 signaling. Inhibition of Wnt-2 signaling can affect, for example, the expression of Dv1 and β-catenin, as described in US Patent Application Nos. 10/678,639 and 11/131,425.

GLI3, located upstream of GLI1 and GLI2, also appears to regulate the transcription of Gli1 and Gli2 genes by binding to GLI3 DNA-binding sites within their promoter/enhancer regions. Thus, the signaling of GLI3, GLI1 and GLI2 is not parallel but more likely within the same signaling pathway. Therefore, in one embodiment of the present invention, the downstream gene is Gli1 gene, preferably human Gli1 gene. In this embodiment, inhibiting GLI3 signaling results in inhibition or downregulation of Gli1 expression and GLI1 signaling.

In another embodiment of the present invention, the downstream gene is Gli2 gene, preferably human Gli2 gene. In this embodiment, inhibiting GLI3 signaling results in inhibition or downregulation of Gli2 expression as well as GLI2 signaling.

E. Inhibition of GLI polypeptide activity

GLI polypeptides possess several of the biological activities described herein. One aspect of the invention provides a method of inhibiting the activity of a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide. The method includes the step of contacting the GLI polypeptide with a small molecule compound of the present invention, thereby inhibiting the activity of the GLI polypeptide. In another embodiment of the present invention, the method comprises the step of contacting the cells expressing the GLI polypeptide with the small molecule compound of the present invention, thereby inhibiting the activity of the GLI polypeptide.

For example, GLI polypeptide is a transcriptional activator protein, and generally exerts its transcriptional activation potential by binding to or acting on TBP-associated factor (TAF). Therefore, in one aspect of the present invention, the method for inhibiting the activity of a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, comprises the step of contacting the GLI polypeptide with a small molecule compound of the present invention, thereby inhibiting the binding of the GLI polypeptide to TAF or the interaction between the GLI polypeptide and TAF . In another embodiment of the present invention, the method comprises the step of contacting the cells expressing the GLI polypeptide with the small molecule compound of the present invention, thereby inhibiting the binding of the GLI polypeptide to TAF or the interaction between the GLI polypeptide and TAF.

GLI polypeptide is a transcriptional activator protein, and generally exerts its transcriptional activation potential by binding to or acting on auxiliary activators such as CBP. Accordingly, in one aspect of the invention, a method of inhibiting the activity of a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, comprises contacting the GLI polypeptide with a small molecule compound of the invention, thereby inhibiting the binding of the GLI polypeptide to a coactivator or the binding of a GLI polypeptide to a coactivator. Interaction steps. In another embodiment of the present invention, the method comprises the step of contacting the cell expressing the GLI polypeptide with the small molecule compound of the present invention, thereby inhibiting the binding of the GLI polypeptide to the coactivator or the interaction of the GLI polypeptide with the coactivator.

The GLI polypeptides described herein are transcriptional activator proteins, and generally exert their transcriptional activation potential by binding to or acting on GLIDNA binding sites. Therefore, in another aspect of the invention, the method of inhibiting the activity of a GLI polypeptide, preferably a GLI1, GLI2 or GLI3 polypeptide, comprises contacting the GLI polypeptide with a small molecule compound of the invention, thereby inhibiting the binding of the GLI polypeptide to the GLI DNA binding site. In another embodiment of the present invention, the method comprises the step of contacting the cell expressing the GLI polypeptide with the small molecule compound of the present invention, thereby inhibiting the binding of the GLI polypeptide to the GLI DNA binding site.

GLI polypeptide activity can be inhibited in vitro or in vivo.

F. Application of Small Molecular Compounds in the Preparation of Pharmaceutical Compositions or Medicines

Another aspect of the present invention provides the use of the small molecular compound of the present invention in the preparation of pharmaceutical compositions or medicines for the treatment and/or preventive treatment of diseases, such as cancers expressing GLI polypeptides, preferably GLI3 polypeptides.

In another embodiment, the present invention provides the application of a small molecular compound in the preparation of a pharmaceutical composition or drug for inducing apoptosis of cancer cells, wherein the cancer cells express GLI polypeptide, preferably GLI3 polypeptide.

In another related aspect, the present invention provides a use of a small molecule compound in the preparation of a pharmaceutical composition or medicament for use in combination with another chemotherapeutic anticancer agent for treating cancer expressing a GLI polypeptide, preferably a GLI3 polypeptide. Pharmaceutical compositions and medicaments provided herein are described herein.

G. Screening for modulators of GLI protein activity

Inhibitors and activators, referred to herein as modulators of GLI protein activity, are identified using methods known in the art and described herein. A number of different screening protocols can be used to identify compounds other than those described herein that are capable of modulating the activity of GLI polypeptides. The term "modulate" includes increasing or decreasing a measured GLI polypeptide activity compared to a suitable control.

These screening methods are applicable to cells, especially mammalian cells, especially human cells, even more preferably human cancer cells. In general, the screening methods include screening various compounds to identify compounds capable of modulating the activity of GLI polypeptides. The method generally comprises the steps of: (a) contacting a candidate compound with a GLI polypeptide, a sample containing the GLI polypeptide, or a mammalian cell expressing the GLI polypeptide; and (b) measuring the activity of the GLI polypeptide in the presence of the candidate compound. The measured increase in activity compared to the activity of the GLI polypeptide in a suitable control (such as the GLI polypeptide in the absence of the candidate compound, a sample containing the GLI polypeptide in the absence of the candidate compound, or a mammalian cell expressing the GLI polypeptide in the absence of the candidate compound) or The decrease indicates that the candidate compound can modulate the activity of the GLI polypeptide. Once a candidate compound or drug candidate is identified using one of the screening methods of the invention, it is generally referred to as a compound or drug rather than a candidate compound or drug candidate.

Compounds that can increase or decrease the activity of the GLI polypeptide to a desired level can be selected for further study, and cell availability, pharmacokinetic analysis, cytotoxicity, biocompatibility, etc. can be evaluated.

In a preferred aspect, the screening method comprises screening candidate compounds to identify compounds that inhibit or reduce the activity of a GLI polypeptide. In another aspect, the screening method comprises screening candidate compounds to identify compounds that increase the activity of a GLI polypeptide.

In one aspect, the invention provides a method of identifying a compound that modulates the activity of a GLI protein. A preferred method of the invention is a method of identifying candidate compounds capable of modulating the protein interaction between the GLI protein and the TAF _II 31 protein. In one embodiment, the method comprises the steps of: contacting a candidate compound with a sample containing GLI protein and TAF _II 31 protein; determining the binding of GLI protein to TAF _II 31 protein. The reduced level of binding of the GLI protein to TAF _II 31 in the presence of the candidate compound compared to the binding of the GLI protein to TAF _II 31 in the absence of the candidate compound indicates that the candidate compound is capable of inhibiting the interaction between the GLI protein and the TAF _II 31 protein . Compared with the binding of GLI protein to TAF _II 31 in the absence of the candidate compound, the increased level of binding of the GLI protein to the TAF _II 31 protein in the presence of the candidate compound indicates that the candidate compound can increase the interaction between the GLI protein and the TAF _II 31 protein. interaction.

In one embodiment, the GLI protein activity is a GLI protein-dependent transcriptional activity and the method is used to identify compounds that modulate the GLI protein-dependent transcriptional activity. The method comprises the steps of: contacting a sample with a candidate compound, wherein the sample comprises an expression reporter construct having one or more GLI DNA binding sites operably linked to a reporter gene; and assaying the candidate compound, if any. Effects of compounds on the level of GLI protein-dependent transcriptional activity.

Many reporter genes known in the art can be used. Suitable reporter genes include, but are not limited to: luciferase, CAT (chloramphenicol-acetyltransferase), GFP (green fluorescent protein), and β-Gal (β-galactosidase).

Modulation of GLI protein binding to TAF _II 31 in the absence or presence of candidate compounds can be assessed using, eg, binding assays as described herein, such as immunoprecipitation assays and transcription assays (see, eg, Example 35). In general, the assays described herein can be used in screening methods by replacing the small molecule compounds analyzed in these assays with candidate compounds to be tested.

As described herein, inhibition of GLI protein activity can lead to apoptosis of cancer cells. Accordingly, in another aspect of the invention, a method of identifying a candidate compound capable of inducing apoptosis is provided. In a preferred embodiment of this method, the method comprises the steps of: (a) contacting the GLI polypeptide with a candidate compound, and (b) determining whether the candidate compound binds to the GLI polypeptide to inhibit the activity of the GLI polypeptide; wherein the binding to the GLI polypeptide Or a candidate compound that inhibits the activity of a GLI polypeptide is a candidate compound that is useful for inducing apoptosis.

Optionally, the method of identifying candidate compounds as described herein includes the step of selecting a compound that binds to a GLI polypeptide or modulates the activity of a GLI polypeptide.

Candidate compounds identified by the methods described herein for inducing apoptosis can be evaluated using the apoptosis assays described herein and assays known in the art.

Using known tests, such as trypan blue dye exclusion test, MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]) test, etc. Evaluate the possible cytotoxic activity of candidate compounds in normal cells (such as non-cancerous cells). Compounds without cytotoxic activity were considered candidate compounds.

a) Screening compounds

In addition to the screening methods described above and the assays described herein, in another embodiment of the screening method of the present invention, a cell-based two-hybrid system ("MATCHMAKER Two-Hybrid system", "Mammalian Matchmaker Two-Hybrid system") is used. Hybridization Assay Kit", "Matchmaker One-Hybrid System" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); see also Dalton and Treisman, 1992, Cell 68: 597-612; Fields and Sternglanz, 1994, Trends Genet 10:286-92).

For example, in a two-hybrid system, a GLI polypeptide is fused to a SRF-binding domain or a GAL4-binding domain and expressed in yeast cells. The TAF _II 31 polypeptide binding GLI polypeptide fused to the VP16 or GAL4 transcriptional activation region was also expressed in yeast cells. Alternatively, a TAF _II 31 polypeptide that binds a GLI polypeptide can be fused to a SRF binding region or a GAL4 binding region, and a GLI polypeptide can be fused to a VP16 or GAL4 transcriptional activation region. When the test compound cannot inhibit the combination of GLI polypeptide and TAF _II 31 polypeptide, the combination of the two can activate the reporter gene, so positive clones can be detected. In addition to the HIS3 gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene, etc. can be used as a reporter gene. Alternatively, when the test compound inhibits the binding of the GLI polypeptide to the TAF _II 31 polypeptide, non-binding of the two polypeptides can also be detected (eg, no expression of the lazZ gene produces white yeast colonies, whereas expression of the lacZ gene produces blue colonies) .

Any test compound can be used in the screening method of the present invention, such as cell extracts, cell culture supernatants, microbial fermentation products, marine organism extracts, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic Small molecule compounds and natural compounds. Many of the combinatorial library methods known in the art can also be used to obtain test compounds of the invention, including (1) biological libraries, (2) space-addressable parallel solid-phase or solution-phase libraries, (3) requiring Deconvoluted synthetic library approaches, (4) "one-bead-one-compound" library approaches and (5) synthetic library approaches using affinity chromatography selection. The biological library method using affinity chromatography selection is limited to peptide libraries, while four other methods can be used for peptide, non-peptide oligomer or small molecule compound libraries (Lam, 1997, Anticancer Drug Des. 12:145). Examples of methods for synthesizing molecular libraries can be seen in the art (DeWitt et al., 1993, Proc Natl Acad Sci USA 90:6909; Erb et al., 1994, Pro. Natl Acad Sci USA 91:11422; Zuckermann et al., 1994, J Med Chem 37 : 2678; Cho et al., 1993, Science 261: 1303; Carell et al., 1994, Angew Chem Int.Ed Engl.33: 2059; Carell et al., 1994, Angew Chem Irtt Ed.Engl.33: 2061; Med Chem 37:1233). Compound libraries can exist in solution (see Houghten, 1992, Bio/Techniques 13:412) or on beads (Lam, 1991, Nature 354:82), on chips (Fodor, 1993, Nature 364:555), on bacteria ( U.S. Patent No. 5,223,409), on spores (U.S. Patent Nos. 5,571,698; 5,403,484 and 5,223,409), on plasmids (Cull et al., 1992, Proc Natl Acad Sci USA 89:1865) or on phages (Scott and Smith, 1990, Science 249:386 ; Devlin, 1990, Science 249:404; Cwirla et al., 1990, Proc Natl Acad Sci USA 87:6378; Felici, 1991, J Mol Biol 222 301; US Patent Application 20020103360). The test compound contacted with the cell or protein according to the screening method of the present invention may be a single compound or a group of compounds. When a panel of compounds is used in the screening methods of the invention, the compounds may be contacted sequentially or simultaneously.

Compounds isolated using the screening methods of the invention are those that modulate the activity of GLI polypeptides to induce apoptosis and treat or prevent disorders such as cancer (as described herein). The compounds obtained by the screening method of the present invention also include compounds in which part of the structure of the compounds obtained by the screening method of the present invention is transformed by addition, deletion and/or substitution.

Both naturally occurring GLI polypeptides and recombinant GLI polypeptides can be used to practice the methods of the invention.

Provided herein are methods of detecting and assaying compounds, drugs, or antagonists identified using the methods described herein, including determining whether a given compound, drug, or small molecule compound can be used in the practice of the methods of the invention in various accepted test. The methods of the invention may optionally include detection of nucleic acids such as mRNA (e.g. by nucleic acid hybridization or PCR), polypeptides (e.g. by Western blot, immunoassay or mass spectrometry), enzymatic activity (e.g. of a reporter gene product such as luciferase), or induced or the step of inhibiting apoptosis. These detection methods are well known in the art.

VI. Kit

The present invention also provides kits for the above-mentioned diagnostic, research and therapeutic applications. In diagnostic and research applications, such kits may include any or all of the following: assay reagents, buffers, small molecule compounds of the invention, GLI polypeptides, Gli nucleic acids, anti-GLI antibodies, hybridization probes and/or primers, Gli expression constructs and the like. Therapeutic products may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.

In addition, the kit can include instructional material containing directions for practicing the methods of the invention (ie, protocols). The instructions may be present in a subject kit in a variety of forms, one or more of which may be present in the kit.

While instructions generally comprise handwritten or printed material, they are not limited to such. The present invention contemplates any medium capable of storing instructions and delivering them to end users. Such media include, but are not limited to: electronic storage media (such as magnetic disks, tapes, tapes, chips), optical media (such as CD ROM), etc. Such media may include Internet addresses where such instructional materials are provided.

Various kits and components can be prepared according to the invention, depending on the intended user of the kit and the specific needs of the user.

In a preferred embodiment of the present invention, the kit is a kit, comprising a pharmaceutical composition comprising the following components: (i) a small molecule compound, preferably FN1-5 or FN1-8 and (ii) a pharmaceutically acceptable carrier. The kit optionally contains instructions that the pharmaceutical composition can or should be used to treat a cancer expressing a GLI polypeptide or Gli nucleic acid, preferably a GLI3 polypeptide or Gli3 nucleic acid.

Kits of the invention may also comprise reagents for performing mass spectrometry. Such reagents are well known to those skilled in the art and include, for example, probes or chips.

Other kit embodiments of the invention include optional functional components that allow one of ordinary skill in the art to perform any of the method variations described herein.

Although the above invention has been described in detail by way of illustration and example for the purpose of illustration and understanding, those of ordinary skill in the art can understand through the content of the present invention that some changes and changes can be made without departing from the concept and scope of the present invention. , modification and equivalent substitution. As a result, various modifications, changes, etc., of the embodiments described herein may be made within the scope of the invention to be determined solely by the appended claims. Those skilled in the art will readily recognize various noncritical parameters that can be varied, altered, or modified to produce substantially similar results.

Although various embodiments of the invention are described herein as containing various embodiments, it is to be understood that, unless otherwise indicated, each embodiment of a given component of the invention can be used with various embodiments of other components of the invention for various applications. shall constitute distinct embodiments of the invention.

All publications, patents and patent applications cited in this specification are herein incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

As can be understood from the above disclosure, the present invention has various applications. The invention is also illustrated by the following examples, which are illustrative only and should not limit the definition and scope of the invention in any way.

VII. Embodiment

Example 1: General method

A. Cell lines

Most human cell lines are obtained from the American Type Culture Collection (A.T.C.C.; Manassas, Virginia). These cell lines include: non-small cell lung cancer (NSCLC) cells A549, H1703, H460, H358, H322, H838, H1299, H1650, H1975, and A427; mesothelioma cells 21IH, H513, H2052, H28, and Met5A; colon cancer cells SW480, HCT116, HT29, Lovo, and CaCO2; breast cancer cells MCF7, HuL100, SKBR-3, and BT474; sarcoma MG-63, MNNG, A-204, and SJSA-I; melanoma cells LOX, FEM, FEMX, A375, G361 , SK-Mel-2, SK-Mel-5, SK-Mel-28 and Malme-3M; human normal skin fibroblasts, Malme-3; renal cell carcinoma cells 786-O, Caki, 769-P, A704 and OMRC3; prostate cancer cell line LnCAP; glioma cell line U87; hepatocellular carcinoma cells HepG2 and SK-Hep-1; normal muscle cell line; normal kidney cell line HRE152 and HK-2. Other human mesothelioma cancer cell lines H290 and MS-I were obtained from the National Institute of Health (NIH, Frederick, Maryland), and LRK1A and REN were obtained from Penn, Philadelphia, PA. Courtesy of Dr. Steven Albelda's laboratory, University of Pennsylvania, Philadelphia, Pennsylvania. The normal mesothelial cell line LP-9 was obtained from the Cell Culture Core Facility, Harvard University, Boston, Massachusetts. Human pancreatic cancer cell lines BxPC3, Panc4.21, CFPAC-1 and L3.6sl were kindly provided by the laboratory of Dr. Matthias Hebrok, University of California, San Francisco (San Francisco, CA). Human nasopharyngeal carcinoma cell lines HNE-I and HONE-I were kindly provided by Dr. Ronald Glaser, Ohio State University, Columbus, Ohio. Human gastric cancer cell lines NUGC3, MNK28, and AGS were kindly provided by Dr. Xin Chen, University of California, San Francisco (San Francisco, CA); esophageal cancer cell lines SEG-I, TE-7, BIC-I, and OE21 Kindly provided by Dr. Michael Korn, University of California, San Francisco (San Francisco, CA); Ovarian carcinoma cell line AZ23247 was provided by Karen Smith, University of California, San Francisco (San Francisco, CA) - Courtesy of Dr. McCune.

Most cell lines were routinely cultured with RPMI1640 supplemented with 10% fetal bovine serum, penicillin (100IU/ml) and streptomycin (100μg/ml), but LP-9 was cultured with 15% CS plus 10ng/ml EGF plus 0.4μg The M199 culture of /ml HC; The ovarian cancer cell line AZ23247 is cultivated with the M15 medium supplemented with 10% fetal calf serum (UCSF Cell Culture Facility (UCSF Cell Culture Facility)); The normal kidney cell line HRE152 is supplemented with 10% fetal calf serum αMEM culture of serum, penicillin (100 IU/ml) and streptomycin (100 μg/ml); normal human small airway epithelial cells obtained from Clonetics, Walkersville, Maryland ( SAEC) and bronchial epithelial cells (NHBE) (primary culture) were cultured with the Clontech SAGM ^™ Bullet kit. All cell lines were cultured in a humidified incubator at 37 °C with 5% _CO2 .

B. Tissue samples

Fresh cancer tissues and adjacent normal tissues (IRB approval number H8714-15319-040) from patients undergoing curative tumor resection for the first time were obtained at the time of surgery, and immediately snap-frozen with liquid nitrogen. These tissue samples were stored at -170°C in liquid nitrogen until use. Primary tissue cultures were prepared as follows: Fresh cancer tissue was obtained from the patient undergoing resection with consent, cut into small pieces (1-2 mm in diameter), and treated with collagenase A (Indianapolis, Indiana) according to the manufacturer's protocol. Digestion was performed at room temperature for 2 hours at Roche Applied Science, Indianapolis, Indiana. The obtained single cells were digested by centrifugation, and the cell pellet was washed twice with RPMI1640 supplemented with 10% fetal bovine serum, penicillin (100 IU/ml) and streptomycin (100 μg/ml). Then, cells were resuspended in the same medium and cultured in a 6-well plate in a 37 °C humid incubator with 5% _CO2 until they were ready for further treatment.

C. Cell Viability Assay (Cell Based Cytotoxicity Assay)

The small molecule compound of the present invention is generally dissolved in DMSO at a concentration of 30 mM. Different concentrations of small molecule compounds ranging from 0, 10, 30, 50 to 100 [mu]M were then assayed under cell culture conditions. To determine cell viability after treatment, cells were incubated with the small molecule compound in 6-well plates for approximately 3 days. After removing the cell culture medium, add 1 ml of 0.5% crystal violet solution (prepared with 20% ethanol and 20% methanol) and stain the cells for 5 minutes. Then, rinse off the crystal violet solution with tap water. Cell viability was estimated from the density of crystal violet-stained plates.

产物，该产物在组织培养基中可溶。可通过代谢活性细胞中的脱氢酶产生的完成这种转化。经490nm吸光度测定的甲

产量与培养基中的活细胞数成正比。因为MTS甲

产物在组织培养基中可溶，所以此试验需要的步骤比使用四唑鎓化合物如MTT要少。MTT还原产生的甲

产物是结晶沉淀，需要额外步骤来溶解这些晶体，然后记录570nm的吸光度读数。Another assay for determining the number of viable cells in a proliferation or cytotoxicity assay is the colorimetric MTS assay. MTS assay reagents are commercially available (Promega Corp., Madison, WI). This reagent contains the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- Tetrazolium, inner salt; MTS] and electron coupling reagent (phenazine ethyl sulfate; PES). The increased chemical stability of PES allows it to be mixed with MTS to form a stable solution. MTS tetrazolium compound (Owen's reagent (Owen's reagent)) is bioreduced by cells to colored formazan

product, which is soluble in tissue culture medium. This conversion can be accomplished by the production of dehydrogenases in metabolically active cells. Formazan measured by absorbance at 490nm

Yield is directly proportional to the number of viable cells in the medium. Because MTS A

The product is soluble in tissue culture medium, so this assay requires fewer steps than using tetrazolium compounds such as MTT. Formazan produced by MTT reduction

The product was a crystalline precipitate and additional steps were required to dissolve these crystals and then record absorbance readings at 570 nm.

B. Apoptosis Analysis

Apoptosis was analyzed using the Annexin V FITC Apoptosis Detection Kit (Biosource, Camarillo, CA) according to the manufacturer's protocol. After approximately 3 days of treatment with various concentrations of different small molecule compounds, cells were harvested by trypsinization, stained, and immediately analyzed by flow cytometry (FACScan; Decton Dickinson, Franklin Lake, NJ). New Jersey)). Early apoptotic cells with exposed phosphatidylserine but with intact membranes will bind annexin V-FITC but not propidium iodide (PI). Cells in the necrotic or late apoptotic stages are labeled with both Annexin V-FITC and PI. "% apoptotic cells" or "% apoptosis" refers to the sum of early and late apoptotic cells.

E. Semi-quantitative RT-PCR

Total RNA from cell lines or tissue samples was isolated using the Qiagen RNeasy Mini Kit (Valencia, California). RT-PCR was performed on a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, California) with a one-step RT-PCR kit from Invitrogen Biotechnology (Carlsbad, California). PCR. RT-PCR primers were obtained from Operon Technologies Inc., Alameda, California. The primer sequences for amplifying different genes in the Hedgehog and Wnt signaling pathways are shown in the table below (Table 1; F, forward primer; R, reverse primer). The expected size of the PCR product produced by each primer pair is indicated. GAPDH was used as internal standard.

Table 1. Primer sequences in semi-quantitative RT-PCR

Gene Primer sequence size (bp) Gli1 F: 5'-TACTCACGCCTCGAAAACCT-3'R: 5'-GTCTGCTTTCCTCCCTGATG-3' 340 Gli2 F: 5'-CACCAACCAGAACAAGCAGA-3'R: 5'-ACCTCAGCCTCCTGCTTACA-3' 246 (α and γ) 195 (β and δ) Gli3 F: 5'-GGCCATCCACATGGAATATC-3'R: 5'-TGAAGAGCTGCTACGGGAAT-3' 196 Sh F: 5'-CAGTGGCCAGGAGTGAAACT-3'R: 5'-CCAGGAAAGTGAGGAAGTCG-3' 380 Ptch1 F: 5'-CGCCTATGCCTGTCTAACCATGC-3'R: 5'-TAAATCCATGCTGAGAATTGCA-3' 448 Wnt-2 F: 5'-GGGAATCTGCCTTTGTTTATGCCA-3'R: 5'-GAACCGCTTTACAGCCTTCCTGCC-3' 289 Dvl-3 F: 5'-GCTAAATGGAACTGCGAAGG-3'R: 5'-CCGCTTGTGTCTTCTCATCA-3 187 GAPDH F: 5′-ATGGGGAAGGTGAAGGTCGG-3′F: 5′-GACGGTGCCATGGAATTTGC-3′ 180

F. Transient transfection of cDNA constructs

In transient transfection experiments, cells (2 x ¹⁰⁵ ) were seeded in six-well plates 24 hours before transfection in growth medium without antibiotics. Transfections were mediated by Lipofectamine ^™ 2000 (Invitrogen; Carlsbad, CA) according to the manufacturer's protocol, using 2.0 μg of cDNA in pCDNA3 vector or 2.0 μg of pCDNA3 empty vector as controls. Transfected cells were then cultured in a 37 °C humidified _incubator with 5% CO until they were ready for analysis. Gli2 cDNA was kindly provided by Dr. Fritz Aberger, University of Salzburg, Salzburg, Austria. Gli1 and Gli3 cDNA were kindly provided by Dr. Bert Vogelstein and Dr. Kenneth W. Kinzler, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland. TAF _II 31 cDNA was kindly provided by Dr. Gregory L. Verdine, Harvard University, Cambridge, MA.

G. Promoter Activity Analysis

One day before transfection, cells (1 x ¹⁰⁵ ) were seeded in 12-well plates containing growth medium without antibiotics. When the cells reached 80-90% confluency, each promoter construct in the pGL3Basic vector and 0.025 μg of the pRL-TK vector (Promega; Madison, Wisconsin) containing the flower insect luciferase were added. ) co-transfected them, and the pRL-TK vector containing flower insect luciferase was used as an internal standard for transformation efficiency. Transfection was mediated by Lipofectamine 2000 ^™ (Invitrogen Biotechnology) for about 6 hours. The original medium was then replaced with fresh medium containing various small molecule compounds at a concentration of 30 μM, and the resulting cells were cultured for another 18-24 hours, and luciferase assays were performed. Briefly, cells were lysed with lysis buffer, and firefly and flower bug luciferase activities of cells in each well were measured using a dual luciferase assay system (Promega) and a luminometer. The assayed activity of luciferase was normalized to the pRL-TK vector activity and its activity was given relative to the basal activity of the pGL3-Basic empty vector (set as unity). Data shown represent mean values (±SD). All assays were performed in triplicate, repeated in at least three independent experiments. The laboratory of Dr. Jablons at the University of California, San Francisco (San Francisco, CA) cloned and characterized the human Wnt-2 promoter-luciferase construct, the human SOCS3 promoter-luciferase construct and Human Gremlin promoter-luciferase construct (He et al., Biochem Biophy Res Comm, 2003; and unpublished data). The 6GLI-TKO-luciferase construct (containing 6 repeats of the consensus GLI DNA binding sequence) was kindly provided by the laboratory of Dr. Matthias Hebrok at the University of California, San Francisco (San Francisco, CA).

H.TOPFLASH test

Cells were seeded in 12-well plates. TOPFLASH or FOPFLASH reporter plasmids (Upstate; Charlottesville, Virginia) were transiently transfected into cells as described above. Tcf-mediated gene transcription was determined by the pTOPFLASH:pFOPFLASH luciferase activity ratio, with each activity value normalized to the luciferase activity of the pRL-TK reporter (co-transfected internal standard). All assays were performed in triplicate with at least three repetitions.

I. Western blot

Total proteins were obtained from cell cultures treated with or without small molecule compounds (30 μM for 2-3 days) using standard protocols (Pierce Biotechnology, Rockford, Illinois). -Rad) protein assay reagent measures protein concentration. Whole cell lysate albumen (5-20 μ g) is boiled 5 minutes, separates with 10-20% SDS-PAGE. Use semi-dry transfer cell (Bio-Rad company (Bio-Rad) of Benicia, California -Rad; Benicia, California)) to transfer proteins to Immobilon-P membranes (Millipore Corp., Bedford, Massachusetts). Tris-buffered saline Block the membrane with 5% nonfat dry milk and 0.1% Tween 20 overnight at 4°C, then incubate with primary antibody for 1 hour at room temperature. Wash the membrane three times with 5% nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline , 10 minutes each. Horseradish peroxidase-coupled goat anti-rabbit or donkey anti-mouse antibody was used as a secondary antibody. Chemiluminescent luminol reagent (Santa Cruz Bio-technology )) to observe the protein. Anti-Dvl-3 antibody and anti-survivin antibody were purchased from Santa Cruz Biotechnology, Santa Cruz, California. Anti-caspase 3 antibody was from Massachusetts Oncogene, Cambridge, Massachusetts. Anti-β-actin antibodies were purchased from Sigma-Aldrich Corp. (St.Louis, Missouri). -β-catenin antibody was purchased from Transduction Laboratories, Lexington, Kentucky. Anti-cytochrome c antibody was from BD Biosciences (San Diego, CA). To detect β -Catenin alterations, cytosolic extracts were prepared and assayed as described in previous publications from our laboratory.

J. In vivo anti-tumor development study

Small molecule compounds FN1-8 were tested in vivo in a mouse xenograft tumor model bearing human cancer cells. Briefly, female athymic nude mouse line NCRNU-M (5-10 weeks old, 20-25 g body weight; Taconic, Germantown, NY) was bred under pyrogen-free conditions. Three human cancer cell lines were used: NSCLC H460, melanoma LOX (all showed Gli3 activation) and colon cancer HT29 (no Gli3 activation, used as negative control). Using 10 mice in each group, 3 x ¹⁰ cancer cells were injected subcutaneously in a volume of 100 μl in the dorsal region. After inoculation, human cancer cells were allowed to grow in mice for 7 days to become visible tumor masses. Then the animals were injected with the small molecule compound FN1-8 at a dose of 50 mg/kg body weight (1 mg/mouse/day). DMSO alone was used as a control. Compound and control volumes were adjusted to 40 μl for intraperitoneal injections in the abdomen of mice. One mouse was sacrificed within 1 hour after compound injection (6 injections) and blood was collected. Compound levels in the blood samples were then analyzed using mass spectrometry to verify which compounds were being absorbed into the bloodstream of the mice. Injections were given at approximately the same time each day for 14 days. Tumors were allowed to grow an additional 1-2 weeks after completion of compound treatment. Tumor size was measured every three days, and tumor volume (x ² y/2, where x<y) was calculated using width (x) and length (y). Data are presented as mean (±SD).

K. TUNEL staining analysis of apoptosis on tumor xenografts

At the end of the in vivo experiments, tumors were excised from mice of the different treatment groups after sacrifice. The excised tumors were cryopreserved immediately, and then the tumor mass of each sample was measured. Tumor samples were stained for TUNEL using the ApopTag Peroxidase In Situ Oligonucleotide Ligated Apoptosis Detection Kit (Chemicon International, Temecula, California) according to the manufacturer's protocol.

L. Histological Examination for Assessing Toxicity

After the in vivo studies were completed, the different organs of the mice were excised. These organs include the liver, lungs, heart, kidneys, intestines, ovaries, brain, spleen, skin and muscles. Samples were fixed with 4% buffered formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histological analysis. HE-stained slides of all organs were examined by a mouse pathologist for evidence of toxicity compared to DMSO controls.

M. Statistical Analysis

Data shown represent mean values (±S.D.). The different treatments and cell lines were compared using Excel's unpaired t-test.

Example 2: Peptide FDAII that mimics the transcriptional activation domain of Gli3

To mimic the FDAII motif of the transcriptional activation domain of GLI3 with small molecules, a 3D structural model of FDAII was constructed. The FVAIL motif of the GLI1 polypeptide generates an α-helical structure as described by Yoon et al. (1998, J Biol Chem 273:3496-3501). The alpha-helical structure of the FDAII motif of the GLI3 polypeptide should also be modeled. Therefore, the α-helical structure of the FDAII motif of the GLI3 polypeptide was generated, and small molecular compounds that mimic the motif structure were designed through rational chemical design. Small molecular compounds containing pyrazoline structure were designed. Figure 3 shows the RMS overlap between the small molecule compounds FN1-5 (described herein) and the FDAII motif of the GLI3 polypeptide (modeled as an α-helix).

Example 3: Preparation of Small Molecular Compounds

a) Preparation of ethyl 1,3,5-triphenyl-4,5-dihydropyrazole-4-carboxylate (FN1-1)

A mixture of ethyl cinnamate (7, 2.55 g), benzaldehyde phenylhydrazone (2, 4.21 g), chloramine-T trihydrate (6.45 g) and methanol (45 mL) was heated at reflux for 22 hours. The reaction mixture was diluted with 50% ethyl acetate in n-hexane (400 mL), filtered and evaporated. The residue was purified by column chromatography (silica gel 200 g, eluent: gradient of 0-10% ethyl acetate in hexane) and evaporated to give FN1-1 (8, 1.25 g) as colorless crystals. ¹ H NMR (CDCl ₃ , 400 MHz) δ 7.66-7.64(m, 1H), 7.46-7.22(m, 13H), 6.88(t, J=7.2Hz, 1H), 4.81(d, J=4.0 Hz, 1H), 4.70 (d, J=4.0 Hz, 1H), 4.07 (quartet, J=7.2 Hz, 2H), 0.97 (t, J=7.2 Hz, 3H). MS: 371.9 (M+H), 325.5, 297.5.

b) Preparation of 1,3,5-triphenyl-4,5-dihydropyrazole-4-carboxylic acid (FN1-2)

A mixture of FN1-1 (53 mg), methanol (0.5 mL), 1,4-dioxane (0.5 mL) and 10% (w/v) aqueous sodium hydroxide solution (0.1 mL) was incubated at room temperature for 1 hour. The reaction mixture was diluted with water (30 mL), washed with 30% ethyl acetate in n-hexane. The aqueous phase was acidified with 2N hydrochloric acid and extracted with ethyl acetate. The extract was washed twice with water (5 mL each), then brine (5 mL), dried ( _{Na2SO4 )} and evaporated to give FN1-2 (38 mg). MS: 297.9 (M- _CO2H ).

c) Preparation of n-butyl-1,3,5-triphenyl-4,5-dihydro-pyrazole-4-carboxamide (FN1-3)

FN1-3 was prepared in a similar manner to FN1-5, substituting n-butylamine for 4-(hydroxyphenyl)ethylamine. MS: 420.9 (M+Na), 398.9 (M+H), 325.5, 297.5.

d) Preparation of N-(5-hydroxypentyl)-1,3,5-triphenyl-4,5-dihydro-pyrazole-4-carboxamide (FN1-5)

A mixture of FN1-1 (22 mg), 5-aminopentanol (100 µl) and NN-dimethylformamide (200 µl) was heated at 95°C for 20 hours. The reaction mixture was diluted with ethyl acetate (10 mL), washed twice with water (5 mL each), then brine (5 mL), _dried ( _Na2SO4 ) and evaporated. The residue was purified by column chromatography (silica gel 10 mL, eluent: gradient 0-70% ethyl acetate in hexanes) and evaporated to give pale yellow amorphous FN1-5 (12 mg). ¹ H NMR (CDCl ₃ , 400MHz) δ7.69-7.67 (m, 1H), 7.34-7.14 (m, 13H), 6.94 (t, J=7.6Hz, 1H), 6.37 (broad t, 1H), 4.82 (d, J=4.8Hz, 1H), 4.48(d, J=4.8Hz, 1H), 3.53(t, J=6.4Hz, 2H), 3.40-3.33(m, 1H), 3.22-3.14(m, 1H), 1.56-1.41 (m, 6H). MS: 450.6 (M+Na), 428.6 (M+H), 325.5, 297.5. UV absorption peak at 348nm (0.05% TFA-MeOH-water 8:2).

e) Preparation of N-(3-hydroxypropyl)-1,3,5-triphenyl-4,5-dihydro-pyrazole-4-carboxamide (FN1-7)

FN1-7 was prepared in a similar manner to FN1-5, substituting 3-aminopropanol for 4-(hydroxyphenyl)ethanamine. MS: 422.9 (M+Na), 400.9 (M+H), 325.5, 297.5.

f) Preparation of N-(2-(4-hydroxyphenyl)ethyl)-1,3,5-triphenyl-4,5-dihydro-pyrazole-4-carboxamide (FN1-8)

A mixture of FN1-1 (11 mg), 4-(hydroxyphenyl)ethylamine (132 mg) and NN-dimethylformamide (100 µl) was heated at 95°C for 20 hours. The reaction mixture was diluted with ethyl acetate (10 mL), washed twice with water (5 mL each), then brine (5 mL), _dried ( _Na2SO4 ) and evaporated. The residue was purified by column chromatography (silica gel 10 mL, eluent: gradient 0-50% ethyl acetate in hexanes) and evaporated to give pale yellow amorphous FN1-8 (9.5 mg). ¹ H NMR (CDCl ₃ , 400 MHz) δ 7.66-7.63(m, 1H), 7.34-7.16(m, 14H), 7.07(d, J=8.4Hz, 1H), 6.94(t, J=6.8Hz, 1H), 6.84(d, J=8.4Hz, 1H), 6.52(d, J=8.4Hz, 1H), 6.35(wide t, J=5.6Hz, 1H), 4.66(s, 1H), 4.65(d , J=4.8Hz, 1H), 4.41(d, J=4.8Hz, 1H), 3.56-3.42(m, 2H), 3.40-3.33(m, 1H), 2.76-2.57(m, 2H). MS: 484.5 (M+Na), 462.5 (M+H), 325.5, 297.5. UV absorption peak at 351nm (0.05% TFA-MeOH-water 8:2).

The preparation of small molecule compound FN1-8 was analyzed by liquid chromatography-mass spectrometry and found to be 90-95% pure (data not shown).

g) 5-(1,3,5-triphenyl-4,5-dihydro-pyrazole-4-carbonyl)aminovaleric acid (FN1-9U) and 5-(1,3,4-triphenyl - Preparation of 4,5-dihydro-pyrazole-5-carbonyl)aminovaleric acid (FN1-9S)

Acetyl chloride (5 mL) was added to methanol (150 mL) to prepare a methanolic hydrogen chloride solution, and then 5-aminovaleric acid (1 g) was added. The mixture was incubated overnight at room temperature and evaporated to isolate methyl 5-aminovaleric acid hydrochloride. Methyl 5-aminovalerate hydrochloride (251 mg), cinnamic acid (74 mg), HBTU (168 mg), diisopropylethylamine (0.87 mL) and dimethylformamide (2 mL) were incubated at room temperature for 1 hour . The reaction mixture was diluted with ethyl acetate (20 mL), washed twice with water (10 mL each), then brine ( ₁₀ mL), dried ( _Na2SO4 ) and evaporated to isolate (4-methoxycarbonyl)butylcinnamon Amide (1, 100 mg). A mixture of (4-methoxycarbonyl)butylcinnamic amide (1, 100 mg), benzaldehyde phenylhydrazone (2, 113 mg), chloramine-T trihydrate (160 mg) and methanol (2 mL) was heated at reflux for 22 hours. The reaction mixture was diluted with 50% ethyl acetate in n-hexane (40 mL), filtered and evaporated. The residue was purified by column chromatography (silica gel 20 g, eluent: gradient solution of 0-10% ethyl acetate in hexane) to isolate 5-(1,3,5-triphenyl-4,5-di Hydrogen-pyrazole-4-carbonyl)aminovaleric acid methyl ester (3, 13 mg) and 5-(1,3,4-triphenyl-4,5-dihydro-pyrazole-5-carbonyl)aminovaleric acid Methyl ester (4, 103mg). The orientational chemistry of each isomer was identified by 1H- and 13-C NMR chemical shifts of the CH adjacent to the amide carbonyl. 5-(1,3,5-Triphenyl-4,5-dihydro-pyrazole-4-carbonyl)aminovaleric acid (5,FN1 -9U). MS: 464.9 (M+Na), 442.9 (M+H), 325.8, 297.9. 5-(1,3,4-Triphenyl-4,5-dihydro-pyrazole-5-carbonyl)aminovaleric acid (6, FN1-9S) was obtained from 4 in a manner analogous to the preparation of FN1-2. MS: 464.9 (M+Na), 442.9 (M+H), 325.8, 297.9.

The chemical assignments of the 5 and 6 positions were fairly consistent with their ESI-MS fragments: ie, 5 gave a larger decarbonylation peak (m/z = 297) than 6, since its β-ketoimino structure favors decarbonylation.

ESI-MS of FN1-9U (upper right panel) and FN1-9S (upper left panel). FN1-9U has a larger peak at m/z=297 than FN1-9S due to the formation of decarbonylated fragments (lower panel).

h) Preparation of 1,3-diphenyl-5-isobutyl-4,5-dihydro-pyrazole-4-carboxylic acid ethyl ester (FN2-1)

A 60% dispersion of sodium hydride in mineral oil (0.24 g) was briefly washed with n-hexane to remove the oil, and the dispersion was suspended in anhydrous dimethylformamide (5 mL). The suspension was cooled in an ice-water bath, and triethyl phosphoacetate (1.0 mL) was added. After stirring the mixture for 0.5 hr, 3-methyl-n-butyraldehyde (0.65 mL) was added at ice-bath temperature. The mixture was stirred at room temperature overnight, treated with water (10 mL), extracted with 50% ethyl acetate in n-hexane (30 mL), washed twice with water (20 mL each), then brine (20 mL), _dried ( _Na2SO4 ) and evaporate. The residue was purified by column chromatography (silica gel 40 mL, eluent: gradient 0-5% ethyl acetate in hexanes) and evaporated to give ethyl 5-methyl-hex-2-enoate as a colorless oil. FN2-1 was prepared by substituting ethyl 5-methyl-hex-2-enoate for ethyl cinnamate in Example 2 in a manner similar to FN1-1. ¹ HNMR (CDCl ₃ , 400MHz) δ 7.35-7.29(m, 3H), 7.25(dt, J=2.0Hz, 6.8Hz, 2H), 7.13(dd, J=1.6Hz,

6.4Hz, 2H), 7.06(d, J=8.0Hz, 2H), 6.84(t, J=7.6Hz, 1H), 4.52(d, J=5.6Hz, 1H), 4.26(d, J=5.6Hz , 1H), 4.21 (quartet, J=7.2Hz, 2H), 2.14 (dd, J=9.2Hz, 14.8Hz, 1H), 2.05 (dd, J=5.6Hz, 15.2Hz, 1H), 1.95 ( Double septet, J = 2.8 Hz, 6.0 Hz, 1H), 1.21 (t, J = 7.2 Hz, 3H), 0.94 (d, J = 6.4 Hz, 6H). MS: 351.9 (M+H), 277.9, 194.7.

i) Preparation of N-(5-hydroxypentyl)-1,3-diphenyl-5-isobutyl-4,5-dihydro-pyrazole-4-carboxamide (FN2-5)

FN2-5 was prepared by substituting FN2-1 for FN1-1 in a similar manner to FN1-5. ¹ H NMR (CDCl ₃ , 400MHz) δ 7.31-7.25 (m, 5H), 7.09-7.03 (m, 4H), 6.92 (t, J=7.2Hz, 1H), 6.63 (broad t, J=5.6Hz, 1H), 4.30(d, J=6.0Hz, 1H), 4.20(d, J=6.0Hz, 1H), 3.59(t, J=6.0Hz, 2H), 3.31(dd, J=4.4Hz, 6.8Hz , 1H), 3.28(dd, J=4.4Hz, 6.8Hz, 1H), 2.12(dd, J=8.8Hz, 14.8Hz, 1H), 2.05(dd, J=6.0Hz, 15.6Hz, 1H), 1.93 (double septet, J=2.4Hz, 6.8Hz, 1H), 1.53 (quintet, J=6.4Hz, 2H), 1.50 (quintet, J=6.4Hz, 2H), 1.32 (quintet, J=6.4Hz, 2H), 0.92(d, J=6.8Hz, 3H), 0.90(d, J=6.8Hz, 3H). MS: 431.0 (M+Na), 409.0 (M+H), 305.9, 277.9.

j) Preparation of N-(5-hydroxypentyl)-1-phenyl-3,5-bis(isobutyl)-4,5-dihydro-pyrazole-4-carboxamide (FN3-5)

In a manner similar to Example 2, replace ethyl cinnamate in Example 2 with ethyl 5-methyl-2-hexanoate and replace benzaldehyde phenylhydrazone in Example 2 with 3-methyl-n-butyraldehydephenylhydrazone Preparation of ethyl 1-phenyl-3,5-bis(isobutyl)-4,5-dihydropyrazole-4-carboxylate. FN1-1 in Example 4 was replaced by ethyl 1-phenyl-3,5-bis(isobutyl)-4,5-dihydropyrazole-4-carboxylate in a manner similar to Example 4, Preparation of FN3-5. MS: 411.0 (M+Na), 389.1 (M+H), 285.9, 257.9.

Example 4: Most cancer cell lines express Gli3

Several cancer cell lines and primary tissue samples were analyzed by semi-quantitative RT-PCR to study the expression of Gli3. Figure 2 shows an exemplary analysis of RT-PCR to detect Gli3 expression. Cell lines analyzed to express detectable levels of Gli3 mRNA included: normal liver (Figure 2, middle panel, lane 1); NSCLC: H1703, H460, and A549 (Figure 2, middle panel, lanes 2, 3, 4); mammary gland Carcinoma: HuL100, BT474 and MCF-7 (Figure 2, middle panel, lanes 5, 6, 7); Mesothelioma: Met5A, H290, REN, H513, H28 and 211H (Figure 2, middle panel, lanes 8-13 ); colon cancer: SW480 (Figure 2, middle panel, lane 15); a pair of primary NSCLC tissue samples (Figure 2, middle panel, lanes 16 and 17 are normal tissue and cancer tissue, respectively). LARK1A did not show detectable Gli3 mRNA expression levels (Figure 2, middle panel, lane 14).

In another set of similar RT-PCR experiments, the expression of Gli3 was analyzed and expressed in colon cancer cells SW480, Lovo and CaCO2 (Figure 2, lower panel, lanes 1, 4, 5); normal colon (Figure 2, lower panel, lane 6); normal kidney cell lines: HRE-152 and HK-2 (Figure 2, lower panel, lanes 7 and 8); renal cell carcinoma: 786-O, Caki, 769-P, A704 and OMRC3 (Figure 2, Lower panel, lanes 9-13); normal lung (Figure 2, lower panel, lane 14); mesothelioma: REN, H290, 211H, H513, H2052, H28 and MS-1 (Figure 2, lower panel, lane 15 -21); NSCLC: A549, H460, H838, H1703, H1299, H1650 and H1975 (Figure 2, lower panel, lanes 22-28). Thus, most cancer cell lines express Gli3. No or low expression of Gli3 was found in the cell lines HCT116 (Figure 2, lower panel, lane 2), HT29 (Figure 2, lower panel, lane 3) and A427 (Figure 2, lower panel, lane 29).

Example 5: Screening for Small Molecule Inhibitors of GLI3 Signaling Transduction

Small molecule compounds of the invention were initially screened using the cellular assays described herein. Figure 4 shows the results of this analysis. Two NSCLC cell lines H1299 and A549 and two colon cancer cell lines SW480 and HCT116 were incubated with small molecule compounds FN1-1, FN1-2, FN1-3, FN1-5 and FN2-1 at a concentration of 100 μM. DMSO was used as a control. Significant cell killing by FN1-5 was observed in all cancer cell lines tested (live cells stained with 0.5% crystal violet). This assay identified the small molecule compounds FN1-5, which inhibit the GLI transcriptional activation domain (TAF-binding domain, TAFbd), as active hits.

Figure 6 shows the results of cytotoxicity assays of small molecule compounds FN1-7, FN1-8 and FN3-5 tested with three NSCLC cell lines (H1299, A549 and H460) and one colon cancer cell line (SW480). Cancer cell lines were seeded in 6-well plates as described above and incubated with 50 and 100 [mu]M of each compound (stock solution in DMSO, 30 mM) for 3 days. Significant cell-killing effects of FN1-8, a modified form of the small molecule compound FN1-5, were observed in all cancer cell lines tested. Apoptosis analysis was also performed by flow cytometry with consistent results (data not shown). Thus, using the rational design-screening approach described herein, another small molecule compound, FN1-8, was identified that inhibits the transcriptional activation domain (TAF-binding domain) of GLI3.

Example 6: Small molecules FN1-5 and FN1-8 can induce apoptosis of colon cancer cell lines CaCO2 and SW480

After the colon cancer cells were treated with the small molecular compound of the present invention, cell apoptosis was detected by flow cytometry. In the representative example shown in Figure 5, significant apoptosis (58.6%) in the colon cancer cell line SW480 was observed after treatment with 100 μM FN1-5 for 3 days (Table 2). This result is consistent with the staining results.

When the small molecule compounds FN1-5 and FN1-8 of the present invention were used for the cytotoxicity test, significant cell killing effects on human colon cancer cell lines LoVo and CaCO2 were observed (data not shown). FN1-8 appears to be more effective at killing cells than FN1-5. Both the small molecule compounds FN1-5 and FN1-8 could induce CaCO2 apoptosis (Table 2 and 3).

Two colon cancer cell lines SW480 and HCT116 were also incubated with 10, 30, 50 and 100 [mu]M compounds FN1-9U and FN1-9S. No significant cell killing or apoptosis was observed (data not shown).

Example 7: Small molecule FN1-5 but not FN1-8 can induce cell killing and apoptosis in colon cancer cell line HT29

When the small molecule compound FN1-5 was used in the cytotoxicity assay, a significant cell killing effect was observed in the human colon cancer cell line HT29 (data not shown). However, no cell killing effect was observed with the small molecule compound FN1-8 even at a higher dose (50 [mu]M; data not shown).

After the cells were treated with small molecule compounds FN1-5 and FN1-8, the colon cancer cell line HT29 apoptosis was detected by flow cytometry. While FN1-5 was able to induce apoptosis in HT29 cells (35% apoptotic cells observed at 50 μM; Table 2), no apoptosis was observed after treatment with FN1-8 (Table 3). These results are consistent with the above staining results. Thus, the results that the small molecule compound FN1-8 neither affects survival nor induces apoptosis in HT29 cells demonstrates that the FN1-8-mediated effects detected in other cell lines are not due to the general toxicity of FN1-8.

Notable in this regard is that while HT29 cells express Gli1 and Gli2 (data not shown; Zhu et al., 2004, Cancer Lett. 207 (20:205-214), Gli3 expression was not observed in HT29 cells (Fig. 2 , bottom panel, lane 3).

Example 8: Small molecule compounds FN1-5 and FN1-8 induce apoptosis in NSCLC cells

After treating several NSCLC cell lines with the small molecular compound of the present invention, apoptosis was detected by flow cytometry. For example, after 3 days of treatment with 100 μM FN1-5, significant apoptosis was observed in NSCLC cell lines H1299 (55.9% apoptosis) and A549 (30.0% apoptosis) (see Table 2 for other data). These results are consistent with the staining results.

After 3 days of treatment with 30 μM and 50 μM FN1-8, significant apoptosis was also observed in the NSCLC cell lines H460 (75.2% apoptosis at 50 μM; Table 3) and H838 (82.3% apoptosis at 50 μM; Table 3). These results are consistent with the staining results. FN1-5 also induced apoptosis in H460 and H838 cells (Table 2). Consistently, it was observed that FN1-8 was more potent in inducing apoptosis than FN1-5 at the same dose (Tables 2 and 3).

When the small molecule compounds FN1-5 and FN1-8 of the present invention were used for cytotoxicity test, significant cell killing effects on NSCLC cell lines H460, H838, H1975, H1650 and H1703 were observed. FN1-8 appeared to be more potent than FN1-5 against these cell lines when comparing the same concentrations (data not shown).

NSCLC cell lines A549, H358 and H322 were used for dose titration experiments of small molecule compounds FN1-8. A549, H358 and H322 cells were seeded in 6-well plates and incubated with DMSO (control) or 7.5 μM, 10 μM, 15 μM, 20 μM and 30 μM FN1-8 for 3 days, respectively. Significant and dose-dependent cell killing against these cell lines was observed when FN1-8 was added at concentrations of 15 μM or higher. FN1-8 induced cell killing at slightly lower concentrations in A549 and H368 cells than in H322 cells (data not shown). After 3 days of incubation with 20 μM FN1-8, about 28% of A549 cells and 58% of H358 cells underwent apoptosis (Table 3).

The time course of cell survival in NSCLC cells A549, H358 and H322 was analyzed after administration of the small molecule compound FN1-8. A549 cells were incubated with 10 μM and 20 μM FN1-8, and H358 and H322 cells were incubated with 15 μM and 30 μM FN1-8, respectively. The percentages of live and dead cells at different time points were then detected by flow cytometry. After 3 days, FN1-8 showed a dose-dependent cell killing effect on A549 cells. The cell killing effect of FN1-8 was already evident after 1 day in H358 cells and after 2 days in H322 cells (data not shown).

The small molecule compound FN1-8 can also significantly inhibit the cell proliferation of NSCLC cells, including A549, H358 and H322 cells. This was demonstrated by incubating cells with 7.5 μM or 15 μM FN1-8, followed by MTS assays at various time points after compound addition according to the manufacturer's protocol. It was found that after 1 day, FN1-8 was able to inhibit cell proliferation of these cells in a dose-dependent manner (data not shown).

Based on the screening results obtained with small molecule compounds FN1-5 and FN1-8, the effects of other small molecule compounds on H1299 and A549 cells were tested using the cytotoxicity and apoptosis assays described herein. No significant cell killing or apoptosis was observed with 10, 30, 50 and 100 [mu]M compounds FN1-9U and FN1-9S (data not shown).

Example 9: Small molecule compounds FN1-5 and FN1-8 induce cell killing and apoptosis in primary cultured NSCLC

Apoptosis induced by small molecule compounds of the present invention in primary cultured NSCLC cells (freshly prepared from tissue samples) was also detected by flow cytometry. For example, after 3 days of treatment with 10, 30, and 50 μM FN1-8 or 30 and 50 μM FN1-5, significant apoptosis was observed in primary cultured NSCLC cells (data not shown). For example, approximately 60% apoptotic cells were observed at 50 μM FN1-8. These results are consistent with those obtained in cytotoxicity assays with primary cultured NSCLC cells and compounds FN1-5 and FN1-8 (data not shown). Again FN1-8 was found to be more potent than FN1-5 at the same concentration.

Example 10: Small molecule compounds FN1-5 and FN1-8 can induce apoptosis of melanoma cells

The effects of small molecule compounds FN1-5, FN1-8, FN1-7, FN1-9U and FN1-9S were detected by cytotoxicity assay and melanoma cell line LOX. LOX cells were seeded in 6-well plates and incubated with 100 [mu]M of each compound (stock solution in DMSO, 30 mM) for 3 days, as described herein. Consistently, significant cell killing was observed for FN1-5 and FN1-8 (Figure 7). Some killing effects were also observed when LOX cells were incubated with small molecule compounds FN1-7 and FN1-9U. Viable cells were stained with 0.5% crystal violet.

The cell killing effect was also tested with human melanoma cell line LOX and different doses of small molecule compounds FN1-5 and FN1-8. LOX cells were seeded in 6-well plates and incubated with 10, 30 and 50 [mu]M of each compound for 3 days as described herein. Significant cell killing was observed for both compounds at 50 μM. It appeared that FN1-8 was more potent in killing LOX cells than FN1-5, as cell killing was higher with 30 μM FN1-5 than 30 μM FN1-8 (data not shown).

The cell killing effect of LOX cells treated with small molecule compounds FN1-5 and FN1-8 was analyzed in more detail with different concentrations of these two compounds and by flow cytometry. After 3 days of treatment, both compounds at all tested concentrations could induce significant apoptosis in LOX cell lines: 10 μM small molecule compound FN1-5 induced 14.0% apoptosis, 30 μM induced 16.6% apoptosis, 50 μM induced 58.1% apoptosis; control DMSO induced 10.9% (Table 2); (b) 10 μM small molecule compound FN1-8 induced 16.7% apoptosis, 30 μM induced 19.0% apoptosis, 50 μM induced 78.9% apoptosis (control DMSO induced 10.9% apoptosis) (Table 3 ). Again, the small molecule compound FN1-8 appeared to have slightly higher apoptosis-inducing ability than FN1-5 at equivalent doses (Tables 2 and 3).

The dose titration experiment of small molecule compound FN1-8 was carried out with melanoma cell lines A375, G361, SK-Mel-2, SK-Mel-5 and SK-Mel-28. A375 cells were seeded in 6-well plates and incubated with DMSO (control) or 2 μM, 5 μM, 10 μM, 20 μM and 40 μM FN1-8 for 3 days, respectively. Significant and dose-dependent cell killing was observed for this cell line when FN1-8 was added at concentrations of 10 [mu]M or higher (data not shown). Similar cell killing was observed in the melanoma cell lines G361, SK-Mel-2, SK-Mel-5 and SK-Mel-28 (data not shown). After 4 days of incubation with 20 μM FN1-8, about 20% of Malme-3M cells, 28% of SK-Mel-2 cells, 36% of SK-Mel-5 cells, 41% of A375 cells and 17% of G361 cells underwent apoptosis, while in Less than 3% of normal skin fibroblasts underwent apoptosis under the same conditions (Table 3).

The small molecule compound FN1-8 also significantly inhibited the proliferation of melanoma cells, including Malme-3M, A375, G361, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cell lines. This was demonstrated by incubating cells with 5 μM or 10 μM FN1-8 and performing MTS assays at various time points following the addition of the compound according to the manufacturer's protocol. In Malme-3M, G361, SK-Mel-2 and SK-Mewl-28, 10μM FN1-8 can inhibit the proliferation of these cells after 1-2 days, in A375 and SK-Mel-5 cells, after 1 day This effect has already occurred (data not shown).

The time course of cell survival of melanoma cell lines (Malme-3M, A375, G361, SK-Mel-2, SK-Mel-5 and SK-Mel-28) was analyzed after administration of the small molecule compound FN1-8. These melanoma cells were incubated with 10 μM or 20 μM FN1-8. The percentages of viable and dead cells at different time points were then detected by flow cytometry. In all melanoma cells tested, 20 μM FN1-8 had a significant cell killing effect after 2 days (Fig. 18A, B and C, data not shown).

In order to investigate whether the small molecule compounds of the present invention such as FN1-8 can specifically kill cancer cells, after administration of 10 μM and 20 μM FN1-8, a time-course test of cell survival was also carried out with human normal skin fibroblast cells (Malme-3). When these cells were incubated with 10 μM and 20 μM FN1-8, no cell killing was observed after 6 days ( FIG. 18D ). Therefore, administration of FN1-8 to normal skin fibroblasts could not affect the viability of these normal skin cells. This finding suggests that the cell-killing effects of FN1-8 are specific to cancer cells.

Example 11: Small molecules FN1-5 and FN1-8 can induce apoptosis of mesothelioma cell lines 211H and H290 and primary cultured mesothelioma cells

Cell killing was also tested with the mesothelioma cell lines H290, 211H, MS1 and REN and different doses of the small molecule compounds FN1-5 and FN1-8. Cells were seeded in 6-well plates and incubated with 1, 10 and 30 [mu]M of each compound for 3 days as described herein. Significant cytocidal effects of these two compounds on H290 and 211H cells were observed at 30 [mu]M (data not shown). However, these two compounds had no significant effect on MS-1 and REN cells at the concentrations tested (data not shown).

The apoptosis of mesothelioma cell line 211H treated with small molecule compounds FN1-5 and FN1-8 was detected by flow cytometry. After 3 days of treatment with 30 μM FN1-5 (37.5%; Table 2) or 30 μM FN1-8 (30.7%; Table 3), significant apoptosis in the mesothelioma cell line 211H was observed.

In the next step, the effects of different small molecule compounds were tested on primary cultured human mesothelioma cells freshly prepared from tissue samples. Primary cultures were seeded in 6-well plates as described herein and incubated with 50 and 100 μM of each compound (FN1-5, FN1-7, FN1-8 and FN3-5; stock solution in DMSO, 30 mM) for 3 days. Significant cell killing effects of FN1-5 and FN1-8 were observed. Again, at the same dose, the small molecule compound FN1-8 was more potent than FN1-5 in killing this primary culture. No significant cell killing effect was found for compounds FN1-7 and FN3-5 (Figure 8).

In the next step, primary cultured human mesothelioma cells freshly prepared from tissue samples were treated with different doses (10 μM, 30 μM and 50 μM) of the small molecule compound FN1-5 for 3 days, and apoptosis was detected by flow cytometry. At moderate doses (30 and 50 [mu]M) of FN1-5 and FN1-8 (see Tables 2 and 3, respectively), increased apoptosis was observed. Consistent with the staining results shown in Figure 8, the apoptosis-inducing potency of FN1-8 was higher than that of FN1-5 at the same dose (Tables 2 and 3).

The mesothelioma cell line MS-1 was also incubated with 10, 30, 50 and 100 [mu]M compounds FN1-9U and FN1-9S. While some cell killing was observed with 100 μM FN1-9U, no cell killing or apoptosis was observed with FN1-9S (data not shown).

Example 12: Effects of Small Molecular Compounds FN1-5 and FN1-8 on Human Prostate Cancer Cell Line LnCAF

The cytotoxicity assay described herein was used to determine the effect of the small molecule compounds FN1-5 and FN1-8 on the human prostate cancer cell line LnCAP. LnCAP cell viability was analyzed after 30 and 50 μM FN1-5 treatment and after 10, 30 and 50 μM FN1-8 treatment. Significant cell killing was observed at 50 μM FN1-8 (data not shown). Much less cell killing was observed for FN1-5 (data not shown).

The apoptosis induced by small molecule compounds FN1-5 and FN1-8 in LnCAP cells was also detected by flow cytometry. After 3 days of treatment with 10, 30 and 50 μM FN1-8 or 30 and 50 μM FN1-5, significant apoptosis of LnCAP cells was observed (Tables 2 and 3). For example, approximately 42% apoptotic cells were observed at 50 μM FN1-8. These results are consistent with the results of the cytotoxicity assay described above. Again, FN1-8 was found to be more potent than FN1-5 at the same concentration.

Example 13: Effects of small molecule compounds FN1-5 and FN1-8 on human pancreatic cancer cell lines

The effects of small molecule compounds FN1-5 and FN1-8 on human pancreatic cancer cell lines L3.6sl, Panc4.21, BxPC3 and CFPAC-1 were determined using the cytotoxicity assay described herein. The viability of these cells after treatment with 10, 30 and 50 μM FN1-5 or FN1-8 was analyzed. Significant cell-killing effects of 50 μM FN1-5 or FN1-8 were observed on all human pancreatic cancer cell lines analyzed (data not shown). However, FN1-5 mediated cell killing was lower in Panc4.21 compared with the other three pancreatic cancer cell lines.

The apoptosis induced by small molecule compounds FN1-5 and FN1-8 in human pancreatic cancer cell lines L3.6sl, Panc4.21, BxPC3 and CFPAC-1 cells was also detected by flow cytometry. After 3 days of treatment with 10, 30 and 50 μM FN1-8 or 30 and 50 μM FN1-5, significant apoptosis was observed in all human pancreatic cancer cell lines analyzed except Panc4.21, where FN1-5 Apoptosis does not appear to be induced (Tables 2 and 3). For example, approximately 45% apoptotic cells were observed at 50 μM FN1-5 and 50 μM FN1-8. These results are consistent with the results of the cytotoxicity assay described above. However, the degree of apoptosis induced by FN1-5 and FN1-8 in L3.6sl was similar. At the same concentration, the efficacy of FN1-5 in inducing apoptosis in CFPAC-1 cells was slightly higher, and that of FN1-8 in Panc4.21 and BxPC3 cells The potency of inducing an apoptotic response was higher than that of FN1-5 (Tables 2 and 3).

Example 14: Effects of Small Molecular Compounds FN1-5 and FN1-8 on Human Breast Cancer Cell Line MCF-7

The effects of the small molecule compounds FN1-5 and FN1-8 on the human breast cancer cell line MCF-7 were determined using the cytotoxicity assay described herein. Viability of MCF-7 cells was analyzed after treatment with 30 and 50 μM FN1-5, and after treatment with 10, 30 and 50 μM FN1-8. Significant cell killing was observed at 50 μM FN1-8 and 50 μM FN1-5 (data not shown). In this test, FN1-8 was more potent than FN1-5.

Example 15: Effects of small molecule compounds FN1-5 and FN1-8 on human ovarian cancer cell line AZ23247

The effects of the small molecule compounds FN1-5 and FN1-8 on the human ovarian cancer cell line AZ23247 were determined using the cytotoxicity assay described herein. AZ23247 cell viability was analyzed after treatment with 10, 30 and 50 μM FN1-5 or FN1-8. Significant cell killing was observed with both compounds at 30 μM and 50 μM (data not shown).

Example 16: Effects of Small Molecule Compounds FN1-5 and FN1-8 on Human Renal Cell Carcinoma Cell Lines

The effects of the small molecule compounds FN1-5 and FN1-8 on the human renal cell carcinoma cell lines 786-O, Caki and OMRC3 were determined using the cytotoxicity assay described herein. The viability of these cells after treatment with 10, 30 and 50 [mu]M FN1-5 or FN1-8 was analyzed. Significant cell killing effects of 50 μM FN1-8 and 50 μM FN1-5 were observed in all human renal cell carcinoma cell lines analyzed (data not shown). FN1-8 was more potent than FN1-5 in this assay.

Example 17: Effects of Small Molecular Compounds FN1-5 and FN1-8 on Human Esophageal Cancer Cell Lines

The effects of the small molecule compounds FN1-5 and FN1-8 on the human esophageal cancer cell lines SEG-1, TE-7, BIC-1 and OE21 were determined using the cytotoxicity assay described herein. The viability of these cells after treatment with 10, 30 and 50 μM FN1-5 or FN1-8 was analyzed. Significant cell killing effects of 50 μM FN1-8 and 50 μM FN1-5 were observed in all analyzed human esophageal cells (data not shown). FN1-8 was more potent than FN1-5 in this assay.

Activation of Hh signaling in human esophageal cancer cell lines SEG-I, TE-7, BIC-I and OE21 detected by semi-quantitative RT-PCR. The expression of the Hh pathway, including Ptch1, Gli1, Gli2, and Gli3, was found to be activated in these cell lines (data not shown).

Example 18: Effects of small molecule compounds FN1-5 and FN1-8 on human gastric cancer cell lines

The effects of the small molecule compounds FN1-5 and FN1-8 on the human gastric cancer cell lines NUGC3, MNK28 and AGS were determined using the cytotoxicity assay described herein. The viability of these cells after treatment with 10, 30 and 50 μM FN1-5 or FN1-8 was analyzed. Significant cell killing effects of 50 μM FN1-8 and 50 μM FN1-5 were observed in all human gastric cells analyzed (data not shown). FN1-8 was more potent than FN1-5 in this assay.

Example 19: Effects of Small Molecule Compounds FN1-5 and FN1-8 on Human Sarcoma Cell Lines

The effects of the small molecule compounds FN1-5 and FN1-8 on the human sarcoma cell lines MG63 and MNNG were determined using the cytotoxicity assay described herein. The viability of these cells after treatment with 10, 30 and 50 μM FN1-5 or FN1-8 was analyzed. Significant cell killing by 50 μM FN1-8 was observed in all sarcoma cells analyzed (data not shown). No significant cell killing effect of FN1-5 was observed in these sarcoma cell lines (data not shown).

Example 20: Effects of small molecule compounds FN1-5 and FN1-8 on human hepatocellular carcinoma cell lines

The effects of the small molecule compounds FN1-5 and FN1-8 on the human hepatocellular carcinoma cell lines HEPG2 and SK-Hep-1 were determined using the cytotoxicity assay described herein. The viability of these cells after treatment with 10, 30 and 50 μM FN1-5 or FN1-8 was analyzed. Significant cell killing effects of 50 μM FN1-8 and 50 μM FN1-5 were observed in all human gastric cells analyzed (data not shown). FN1-8 was more potent than FN1-5 in this assay.

Example 21: Effects of small molecule compounds FN1-5 and FN1-8 on human nasopharyngeal carcinoma cell lines

The effects of the small molecule compounds FN1-5 and FN1-8 on the human nasopharyngeal carcinoma cell lines HNE1 and HONE1 were determined using the cytotoxicity assay described herein. The viability of these cells after treatment with 10, 30 and 50 μM FN1-5 or FN1-8 was analyzed. Significant cell killing effects of 50 μM FN1-8 and 50 μM FN1-5 were observed in all human gastric cells analyzed (data not shown). FN1-8 was more potent than FN1-5 in this assay.

Example 22: Effects of small molecule compounds FN1-5 and FN1-8 on human glioma cell line U87

The effects of the small molecule compounds FN1-5 and FN1-8 on the human glioma cell line U87 were determined using the cytotoxicity assay described herein. The viability of U87 cells treated with 30 and 50 μM FN1-5 or 10, 30 and 50 μM FN1-8 was analyzed. Significant cell killing was observed with 50 μM FN1-8, to a lesser extent obtained with 50 μM FN1-5 (data not shown).

Example 23: Effects of Small Molecular Compounds on Normal Human Muscle Cells

The cell killing effects of different doses of small molecular compounds FN1-8, FN1-7 and FN1-5 were detected in normal human muscle cells. FN1-7, which is structurally similar to the other two compounds but was not highly cytotoxic in the cancer cell lines tested, was used as a negative control. Normal muscle cells were seeded in 6-well plates and incubated with 1, 10, 30 and 50 [mu]M of each compound for 3 days (as in other similar experiments described herein). At all doses of all compounds, no significant cell killing was observed, suggesting that FN1-5 and FN1-8 are not toxic to normal muscle cells. FN1-5 and FN1-8 appear to specifically kill cancer cells.

In addition, apoptosis induction was detected by flow cytometry after normal human muscle cells were treated with different doses of FN1-5 or FN1-8. Normal muscle cells were seeded in 6-well plates and incubated with 1, 10, 30, and 50 [mu]M of each compound for 3 days, as described above. There was no significant difference in the number of apoptotic cells after treatment with these two compounds at all doses compared to DMSO control, further demonstrating that neither FN1-5 nor FN1-8 was toxic to normal muscle cells (Tables 2 and 3). This result also indicated that FN1-5 and FN1-8 could specifically induce cancer cell apoptosis.

Example 24: Effects of Small Molecular Compounds FN1-5 and FN1-8 on Normal Human Kidney Cells

The cytotoxic effects of different doses of small molecular compounds FN1-5 and FN1-8 on normal human kidney cells were tested. Normal kidney cells were seeded in 6-well plates and incubated for 3 days with 30 and 50 μM compound FN1-5 and 10, 30 and 50 μM FN1-8 (in the same manner as other similar experiments described herein). At all doses of all compounds, no significant cell killing was observed, suggesting that FN1-5 and FN1-8 are not toxic to normal kidney cells (data not shown).

In addition, apoptosis induction was detected by flow cytometry after normal human kidney cells were treated with different doses of FN1-5 (30 and 50 μM) or FN1-8 (10, 30 and 50 μM). There was no significant difference in the number of apoptotic cells after treatment with these two compounds at all doses compared to DMSO control, further demonstrating that neither FN1-5 nor FN1-8 was toxic to normal kidney cells (Tables 2 and 3).

Example 25: Summary of the apoptosis-inducing effect of small molecule FN1-5

The table below summarizes the percentage of apoptotic cells observed after treatment with the indicated concentrations of the small molecule compound FN1-5. Experiments were performed as described herein. N.d., not performed.

Table 2: Induction of apoptosis with small molecule compounds FN1-5

cell line DMSO (control) 10μM 30μM 50μM 100μM CaCO2 (colon cancer) 7.2 8.2 19.8 30.3 nd HT29 (colon cancer) 5.2 5.8 15.5 32.9 nd SW480 (Colon cancer) 6.2 10.8 nd nd 58.6 A549 (NSCLC) 10.2 9.4 nd nd 30.0 H460 (NSCLC) 7.3 nd 11.9 15.7 nd H838 (NSCLC) 6.7 nd 14.2 20.0 nd H1299 (NSCLC) 5.9 6.7 nd nd 55.9 Primary cultured NSCLC cells 10.0 nd 11.2 19.8 nd LOX (melanoma) 10.9 14.0 16.6 58.1 nd 211H (Mesothelioma) nd 7.7 37.5 nd nd primary cultured mesothelioma nd 12.3 15.8 18.3 nd BxPC3 (pancreatic cancer) 10.1 nd 11.2 30.0 nd CFPAC-1 (pancreatic cancer) 12.8 nd 17.4 30.6 nd L3.6sl (pancreatic cancer) 11.8 nd 27.5 44.8 nd Panc4.21 (pancreatic cancer) 9.6 nd 9.3 10.3 nd LnCAP (prostate cancer) 8.2 nd 7.7 16.3 nd normal muscle cells 7.2 7.5 7.6 8.8 nd normal kidney cells 4.2 nd 5.6 6.6 nd

Example 26: Summary of the apoptosis-inducing effect of small molecule FN1-8

The table below summarizes the percentage of apoptotic cells observed after treatment with the indicated concentrations of the small molecule compound FN1-8. Experiments were performed as described herein. N.d., not performed.

Table 3: Induction of apoptosis with small molecule compounds FN1-8

cell line DMSO (control) 10μM 20μM 30μM 50μM 100μM CaCO2 (colon cancer) 7.2 nd nd 19.9 39.4 nd HT29 (colon cancer) 5.2 nd nd 3.1 5.3 nd H460 (NSCLC) 7.3 nd nd 12.1 75.2 nd H838 (NSCLC) 6.7 10.6 nd 18.3 82.3 nd A549 (NSCLC) 12.2 12.5 27.4 nd nd nd H358 (NSCLC) 8.9 15.2 58.1 nd nd nd Primary cultured NSCLC cells 10.0 9.8 nd 13.1 58.0 nd LOX (melanoma) 10.9 16.7 nd 19.0 78.9 nd Malme-3M (melanoma) 7.7 10.5 19.4 nd nd nd SK-Mel-2 (melanoma) 7.2 9 25.3 nd nd nd SK-Mel-5 (melanoma) 6.9 7.9 34.9 nd nd nd SK-Mel-28 (melanoma) 7.3 7.7 11.8 nd nd nd A375 (melanoma) 3.0 35.8 39.7 nd nd nd G361 (Melanoma) 7.1 7.5 15.5 nd nd nd Malme-3 (normal skin cells) 1.2 2.1 3.2 nd nd nd 211H (Mesothelioma) nd 5.6 nd 30.7 nd nd primary cultured mesothelioma nd 13.2 nd 14.7 33.6 nd BxPC3 (pancreatic cancer) 10.1 11.4 nd 20.4 77.4 nd CFPAC-1 (pancreatic cancer) 12.8 15.4 nd 15.0 19.8 nd L3.6sl (pancreatic cancer) 11.8 12.8 nd 25.1 45.7 nd Panc4.21 (pancreatic cancer) 9.6 11.2 nd 10.6 23.4 nd LnCAP (prostate cancer) 8.2 6.9 nd 11.0 41.5 nd normal muscle cells 8.0 7.8 nd 9.3 9.7 nd normal kidney cells 4.3 3.1 nd 4.1 6.2 nd

Example 27: Small molecules regulate Wnt2 promoter activity in NSCLC cell line A549

In order to test the specificity of the small molecule compound of the present invention, the ability of the compound to regulate the expression of target genes downstream of Gli is tested. Gli activation can activate downstream Wnt signal transduction, and Hh/Gli signal transduction can regulate Wnt gene expression. Cloning of the human Wnt2 promoter was found to contain a putative Gli-DNA binding site in its promoter region (data not shown). Therefore, the Wnt2 promoter-luciferase reporter activity was determined following treatment of the NSCLC cell line A549 (expressing Wnt2) with several small molecule compounds of the present invention. After treatment with 30 μM for 18-24 hours, both FN1-5 and FN1-8 could significantly down-regulate the Wnt2 promoter activity ( FIG. 9 ). Small molecule compounds without potent cell killing effects similar to FN1-5 and FN1-8 had little or no effect on the downregulation of Wnt2 promoter activity ( FIG. 9 ). This result is consistent with cytotoxicity and apoptosis assays, strongly suggesting that both FN1-5 and FN1-8 can induce apoptosis by repressing Wnt2-expressed Gli transcription.

Example 28: Small molecule compounds FN1-5 and FN1-8 can regulate the Hedgehog pathway of cancer cells

In order to further detect the mechanism of action of the small molecule compounds of the present invention, the effects of compounds FN1-5 and FN1-8 on the Hedgehog (Hh) signaling pathway were detected in NSCLC cell lines H460 and A549. H460 and A549 cells were incubated with 30 [mu]M of each compound (FN1-5 or FN1-8) for 2 days before total RNA was isolated for semi-quantitative RT-PCR analysis. Key components and direct target genes of the Hh pathway, such as Ptch1 and GH1, were found to be downregulated (data not shown). GAPDH was used as a control.

Similar to the above, Wnt2 expression was also down-regulated after FN1-8 treatment using Wnt2 promoter-luciferase reporter activity assay (data not shown). These results suggest that small molecule compounds FN1-5 and FN1-8 may be dual inhibitors of Hh and Wnt pathways. Furthermore, it appeared that FN1-8 inhibited both pathways more than FN1-5, which is consistent with the analysis of apoptosis in these cell lines (data not shown).

Small molecule compounds FN1-5 and FN1-8 also modulated the hedgehog pathway in melanoma cell line LOX, colon cancer cell line SW480, and primary cultured human mesothelioma cells freshly prepared from tissue, as shown by semiquantitative RT-PCR (data not shown). Interestingly, no significant changes were observed in HT29 cells (data not shown).

Example 29: Small molecule compound FN1-8 can down-regulate GLI1-induced transcription in melanoma cells

In order to further test the specificity of the small molecule compound FN1-8, specifically, GLI-dependent transcription was detected in the absence and presence of the small molecule compound FN1-8 to determine the effect of FN1-8 on regulating the Hh/Gli signaling pathway. effect. Specifically, the 6 GLI-binding site repeat unit (6xGLI BS; Sasaki et al., Development 124(7):1313-22 (1997)) linked to a luciferase reporter gene was used as a plasmid expression for GLI-dependent transcriptional surrogate assays. constructs to detect 6xGLI BS-luciferase activity after administration of FN1-8 to several melanoma cell lines including Malme-3M (Fig. 15A), A375 (Fig. 15B), SK-Mel- 2 (FIG. 15C) and SK-Mel-5 (FIG. 15D). Co-expression of GLI1 (transfection of the Gli1 expression plasmid described herein into melanoma cells) significantly increased 6xGLI BS-luciferase activity in all four melanoma cell lines tested (Fig. 15A-D, "Gli +DMSO" column). This verifies that overexpressed (ie exogenously added) GLI1 can specifically bind the GLI-binding site and functionally induce transcription of the luciferase reporter gene. A control vector not expressing GLI1 was also transfected into these melanoma cells, but did not produce high levels of luciferase activity (Figure 15A-D, "Control Vector + DMSO" columns). After 16-20 hours of 20 μM FN1-8 treatment, a significant reduction in luciferase activity levels was observed (Fig. 15A-D, "Gli+20 μM FN1-8" column). Therefore, the small molecule compound FN1-8 can significantly reduce GLI1-induced transcription in these melanoma cells. This result indicates that FN1-8 is a repressor of GLI-dependent transcription. This finding also provides a means to inhibit GLI-dependent transcription in cells.

Example 30: Small molecule compounds FN1-5 and FN1-8 can down-regulate GLI1- and GLI2-induced transcription in pancreatic cancer cells

To further test the specificity of the small molecule compounds FN1-5 and FN1-8 in inhibiting the Hh/Gli signaling pathway, GLI-dependent transcription in pancreatic cancer cells in the absence and presence of these compounds was also assayed. Specifically, GLI1 alone (transfected into Panc4.21 cells with the Gli1 expression plasmid described herein) or GLI2 alone (transfected with the Gli1 expression plasmid described herein) were assayed using the (6xGli BS) luciferase reporter gene construct described in Example 29. Gli2 expression plasmid transfected into Panc4.21 cells) overexpression of 6xGLIBS-luciferase can significantly increase 6xGLIBS-luciferase activity (data not shown). This verifies that overexpressed (ie exogenously added) GLI1 or GLI2 polypeptides can specifically bind to the GLI-binding site and functionally induce transcription of the luciferase reporter gene. Transfection of control vectors not expressing Gli1 or Gli2 or Gli3 expression vectors into these pancreatic cancer cells did not produce high levels of luciferase activity (data not shown). The fact that Gli3 expression did not result in increased luciferase activity may be due to the presence of both activator and repressor domains in GLI3.

Significant reduction in luciferase activity levels was observed after 20 μM FN1-5 or FN1-8 treatment for 16-20 hours (data not shown). Thus, small molecule compounds FN1-5 and FN1-8 could significantly reduce GLI1- and GLI2-induced transcription in pancreatic cancer cells. These results suggest that FN1-5 and FN1-8 may act as repressors of GLI1- and GLI2-dependent transcription. This finding also provides a means to inhibit GLI1- and GLI2-dependent transcription in cells.

In a separate experiment, it was found that co-transfection of TAF _II 31 expression plasmids with GLI1 or GlLI2 expression plasmids also significantly increased 6xGLI BS-luciferase activity (data not shown). 6xGLI BS-luciferase activity was significantly reduced after addition of FN1-5 or FN1-8 (20 [mu]M, 16-20 hours) (data not shown). The TAF _II 31 expression plasmid contains full-length TAF _II generated by PCR cloning into the EcoRI (5′) and BamHI (3′) sites of a c-Myc-tagged mammalian expression vector (pCDNA3.1(-)B) 31 cDNA. The sequence of TAF _II 31 corresponds to GenBank accession number U25112.

Example 31: Modulation of TOP/FOP activity in colon cancer cell lines HCT116 and SW480 by small molecule compounds

As another specific detection of the small molecule compounds of the present invention, the small molecule compounds FN1-5, FN1-7, FN1-8, FN1-9U, FN1-9S, FN2-1 and FN3-5 were tested using the TOP/FOP assay Can regulate transcriptional activity of canonical Wnt signaling. In these experiments, colon cancer cell lines with high TCF transcriptional activity as determined by the TOP/FOP assay were used. Figure 10 shows the results for HCT116 cells. After treatment with 30 μM for 18-24 hours, it was found that FN1-5 and FN1-8 could significantly down-regulate TOP/FOP activity. No significant downregulation was observed in this assay with compounds that did not induce significant cell killing. This result is also consistent with the analysis of cytotoxicity, apoptosis and Wnt2 promoter activity, indicating that FN1-5 and FN1-8 can induce apoptosis by inhibiting the Gli transcription of Wnt2 expression, thereby inhibiting Wnt signal transduction-dependent transcription.

The profile of the same compounds above in modulating TOP/FOP activity in the colon cancer cell line SW480 was also analyzed. Although compounds FN1-5 had less effect on this cell line, small molecule compounds FN1-8 reduced TOP/FOP activity by about 32% in SW480 cells compared to about 36% in HCT116 cells (data not shown ) (see Figure 10).

Example 32: Regulation of SOCS-3 and Gremlin promoter activity by small molecule compounds

As a third specificity test for small molecule compounds of the invention, the ability of these compounds to modulate gene expression of non-Gli-activated downstream targets was analyzed. An inhibitor of the JAK/STAT pathway and a downstream target gene SOCS3 were selected for this experiment. Expression of SOCS3 is not controlled by Gli and/or Wnt activation. Furthermore, no GLI and TCF binding sites were found in the human SOCS3 promoter cloned in Dr. Jablons' laboratory (University of California, San Francisco; data not shown). Therefore, SOCS3 promoter-luciferase reporter activity was measured after treatment of NSCLC cell line A549 with small molecule compounds FN1-5, FN1-7, FN1-8 and FN1-9U. After 30 μM treatment for 18-24 hours, it was found that neither FN1-5 nor FN1-8 could modulate SOCS3 promoter activity, similar to other compounds that had no cell killing effect (such as FN1-7 and FN1-9U; FIG. 11 ). This result indicates that FN1-5 and FN1-8 are specific small molecule inhibitors of GLI transcriptional activity.

As the fourth specificity test of the small molecule compounds of the present invention, the ability of the compounds FN1-5 and FN1-8 to regulate the expression of another gene that is not a Gli-activated downstream target was tested. Gremlin, an inhibitor of BMP and TGF-β signal transduction, was chosen for this experiment. Gremlin expression is not controlled by Gli and/or Wnt activation. Therefore, human Gremlin promoter-luciferase reporter activity was detected after HEK293T cells were treated with compounds FN1-5, FN1-8 or DMSO (control) or empty vector (control). After 30 [mu]M treatment for 18-24 hours, neither FN1-5 nor FN1-8 were found to regulate Gremlin promoter activity (data not shown). This result also indicated that FN1-5 and FN1-8 are specific small molecule inhibitors of GLI transcriptional activity. Human Gremlin was cloned in the laboratory of Dr. Jablons (University of California, San Francisco; data not shown).

Example 33: Small molecule compound inhibitors of GLI signal transduction can inhibit canonical Wnt signal transduction in colon cancer cells, NSCLC cells and melanoma cells

In order to detect whether the small molecule compound of the present invention can be used as a small molecule inhibitor of the GLI3 activation domain and inhibit the canonical Wnt signal transduction, after 2 days of treatment with 30 μM small molecule compounds FN-15 and FN1-8 and DMSO (control), Key components of canonical Wnt signaling (such as Dvl-3 and cytoplasmic β-catenin) in colon cancer cells SW480, HT29, and CaCO2 were analyzed by Western blotting, and β-actin was used as a loading control. Consistent with the apoptosis induction results, a significant downregulation of Dvl-3 and cytoplasmic β-catenin in these cells was observed following compound treatment (Figure 12). Survivin, inhibitor of apoptosis protein and Wnt downstream target genes (Kim et al., 2003) were also downregulated (Figure 12). It is interesting to note that in HT29 cells, no downregulation of Dvl-3 and cytoplasmic β-catenin was observed after FN1-8 treatment, which is in agreement with the results that HT29 cell viability was not affected by FN1-8 compounds unanimous.

In order to detect whether the small molecule compound of the present invention can be used as a small molecule inhibitor of the GLI3 activation domain and inhibit canonical Wnt signal transduction, after 2 days of treatment with 30 μM small molecule compounds FN-15 and FN1-8 and DMSO (control), with The key components of canonical Wnt signal transduction (such as Dvl-3 and cytoplasmic β-catenin) in NSCLC cells H460, A549 and H838 were analyzed by Western blotting, and β-actin was used as loading control. Consistent with the apoptosis induction results, a significant downregulation of Dvl-3 and cytoplasmic β-catenin in these cells was observed following compound treatment (Figure 13). Survivin, an inhibitor of apoptosis protein and Wnt downstream target genes (Kim et al., 2003) was also downregulated (Figure 13).

In another experiment, we used Western blot analysis to examine whether the small molecule compound FN1-8 could inhibit the growth of melanoma cells by inhibiting the Wnt canonical pathway (see above). Cytoplasmic proteins were isolated from two melanoma cell lines A375 and SK-Mel-5 and normal human skin fibroblasts (Malme-3) after treatment with DMSO (control), 10 μM or 20 μM FN1-8. β-actin was used as a control. These experiments demonstrated that cytoplasmic β-catenin levels, one of the key indicators of the canonical Wnt activation pathway, were downregulated in melanoma cells after treatment with FN1-8 (data not shown). However, no significant changes in cytoplasmic β-catenin levels in human normal skin fibroblasts were observed (data not shown). These results indicate that the small molecule compounds of the present invention such as FN1-8 can specifically inhibit the growth of melanoma cells and induce apoptosis of these cancer cells by inhibiting the canonical Wnt pathway. As described herein, these results further suggest that FN1-8 is a dual inhibitor of Hh and Wnt signaling pathways.

Example 34: Overexpression of GLI2 can rescue colon cancer cell line SW480 treated with small molecule compound inhibitors FN1-5 and FN1-8

As described herein, GLI3 appears to activate transcription of the Gli1 and Gli2 genes. Therefore, the inhibition of GLI3 activity by the small molecular compound of the present invention should also inhibit the signal transduction of GLI1 and/or GLI2. If so, transfection of GLI1 and/or GLI2 expression constructs into cells treated with a small molecule compound of the invention should at least partially rescue the small molecule compound-mediated apoptosis. Therefore, in order to further test the specificity of the small molecule compound FN1-5 and its inhibitory mechanism, it was tested whether the overexpression of GLI2 protein could alleviate the cell killing effect of FN1-5. In this experiment, the colon cancer cell line SW480 was transfected with either the empty pCDNA3 vector or the Gli2 cDNA expression construct. After selection, the same number of transfected cells were seeded again in a 6-well plate, and then FN1-5 was added to the culture medium at a final concentration of 50 μM. After 5 days, FN1-5 was found to kill fewer Gli2-transfected cells than empty vector-transfected cells (data not shown). This result indicated that FN1-5 could kill cancer cells by inhibiting GLI3 transcriptional activation.

In order to further study the specificity of the small molecule compound FN1-8 and its inhibitory mechanism, we detected whether overexpression of GLI2 protein could alleviate the cell killing effect of FN1-8. Transfection experiments were performed as described above. After 5 days, apoptosis assays were performed by flow cytometry to quantify the assay. Significantly more apoptotic cells were found in SW480 cells transfected with empty vector (48.1%) compared to SW480 cells transfected with Gli2 expression construct (27.7%). This result also indicated that FN1-8 could induce cancer cell apoptosis by inhibiting GLI3 transcriptional activation.

In order to verify the result that GLI2 overexpression can reduce the apoptosis-inducing ability of FN1-5 and FN1-8 in colon cancer cell SW480, Western blot analysis was performed to detect the key components of canonical Wnt signaling (cytoplasmic β-catenin), Expression of its downstream target (survivin) and some key factors in the apoptotic pathway (cytochrome c and active PARP (cleaved form) as positive regulators of apoptosis; survivin as inhibitor of apoptosis). β-actin was used as a loading control. The results obtained were consistent with the flow cytometry analysis of apoptosis described above (data not shown). These data further support other data that both FN1-5 and FN1-8 can induce apoptosis in some cancer cells by inhibiting the transcriptional activation function of GLI3.

In order to verify that overexpression of Gli2 can reduce the apoptosis-inducing ability of FN1-5 and FN1-8 in colon cancer cells by reactivating the Hh signaling pathway, semi-quantitative RT-PCR was used to analyze the key components of Hh signal transduction and its downstream Targets (Shh, Ptch1, Gli1, Gli3). GAPDH was used as a control. This result was found to be consistent with the flow cytometry analysis of apoptosis described above (data not shown).

Example 35: Small molecule compounds inhibit protein interaction between GLI polypeptide and TAF protein resulting in inhibition of GLI-TAF-induced transcriptional activity

In addition, as described above, the specificity of the compounds of the invention to inhibit the activity of GLI was tested, and it was determined whether these compounds could block the interaction between GLI and TAF _II 31 . Specifically, in this assay, an expression construct that links six GLI-binding site repeats (6xGLI BS) to a luciferase reporter gene as a GLI-dependent transcriptional surrogate assay was utilized following treatment of NSCLC A549 cells with compounds FN1-8. to detect 6xGLI BS-luciferase activity. The results of this experiment showed that the overexpression of GLI1 or GLI2 alone could significantly increase the activity of 6xGLI BS-luciferase ( FIG. 16 ). Co-expression of TAF _II 31 with GLI1 or GLI2 further enhanced 6xGLI BS-luciferase activity to a significantly higher level than that of GLI1 or GLI2 alone, while overexpression of TAF _II 31 alone had no effect ( FIG. 16 ). This finding reconfirms that both GLI1 and GLI2 can specifically bind to the GLI-binding site and are functionally active in transcription. This result also indicated that TAF _II 31 may interact with GLI protein as a co-activator. 6xGLI BS-luciferase transcription induced by GLI alone and GLI/TAF _II 31 was reduced in A549 cells treated with 20 μM FN1-8 for 16-20 hours. Interestingly, while the transcriptional activity of GLI alone was reduced by about 30%, the transcriptional activity of A549 cells was reduced by about 50% when co-transfected with TAF _II 31 and GLI2, and the transcriptional activity was reduced by about 50% when co-transfected with TAF _II 31 and GLI1. 70%. This result indicated that FN1-8 may be a specific inhibitor of the interaction between GLI protein and TAF _II 31 .

In co-immunoprecipitation experiments, it has been demonstrated that the small molecule compound FN1-8 can block the protein interaction between the TAF binding domain of the GLI protein and the TAF _II 31 protein ( FIG. 23 ). Specifically for this experiment, the biotinylated TAF binding domains of the GLI proteins GLI1, GLI2 and GLI3 were custom synthesized by Genemed Synthesis, Inc (see below). The GLI-TAF binding domain (GLI-TAFbd) peptide sequence is as follows:

GLI1-TAFbd: (NH ₂ )-LDSLDLDNTQLDFVAILDEPQG-(COOH);

GLI2-TAFbd: (NH ₂ )-VDSQLLEAPQIDFDAIMDDGDH-(COOH); and

G1LI-TAFbd: (NH ₂ )-LDSHDLEGVQIDFDAIIDDGDH-(COOH)

TAF _II 31 protein was prepared using the TNT Rapid Coupled Transcription/Translation System (Promega) according to the manufacturer's protocol. The full-length TAF _II 31 cDNA insert cloned into the (c-myc-tag) pCDNA3.1 expression vector was used as a template for this in vitro translation system. An empty (c-myc tagged) pCDNA3.1 expression vector was used as a control. Biotinylated GLI peptides (ie TAF binding domains of three different GLI proteins) were incubated with TAF _II 31 protein in the presence or absence of small molecule compounds FN1-8 (20 μM and 50 μM). Then, the biotinylated GLI-peptide-TAF _II 31 complex was precipitated with streptavidin agarose, followed by anti-c-myc antibody (A-14; Santa Cruz Biotech, Inc. Cao et al., 2001, Science 293: 115-120) for immunoblotting. In this experiment it was found that the addition of 50 [mu]M FN1-8 reduced the formation of the GLI-peptide-TAF _II 31 complex (Figure 23). In particular, it appeared that FN1-8 blocked the protein interaction of TAF _II 31 with the TAF binding domains of GLI3 and GLI1 more efficiently than TAF _II 31 with the TAF binding domain of GLI2 ( FIG. 23 ). As a control, all three GLI peptides did not pull-down TAF _II 31 when the (c-myc-tag) pCDNA3.1 empty expression vector was used in an in vitro translation system ( FIG. 23 ).

Taken together, these data demonstrate that the small molecule compounds FN1-8 can block the protein interaction between the TAF-binding domains of GLI proteins (more preferably GLI1 and GLI3) and the TAF _II 31 protein, consistent with the GLI/TAF _II 31 -dependent transcription reported above consistent with the experiment. Substances that inhibit the protein interaction between GLI proteins (or the aforementioned GLI peptides) can also be screened using this GLI-peptide/TAF _II 31 protein-protein interaction assay. In this method, substances to be validated or screened are incubated with GLI protein (or GLI peptide) and TAF _II 31 protein, as described above.

Example 36: Small molecule compound FN1-8 inhibits tumor growth in vivo (mouse xenograft tumor model: NSCLC H460)

The in vivo efficacy study of the small molecule compound FN1-8 was carried out using a mouse xenograft tumor model (NSCLC H460). Inject 3 x ¹⁰⁶ cells in a volume of 100 µl subcutaneously in the dorsal region of the mouse. (Fig. 17, arrow). Seven days after inoculation, mice began to receive FN1-8 at a daily dose of 50 mg/kg body weight for 2 x 6 days (n=6). DMSO alone was used as a control (n=7). FN1-8 and DMSO were adjusted to a volume of 40 μl and injected intraperitoneally into the peritoneal cavity of mice. Arrows in Figure 18A indicate the time points of each injection. Tumor size (length and width based on its shape) was measured every three days with vernier calipers. Tumor volumes were calculated using the equation x ² y (where x<y). As shown in Figures 17 and 18, tumor growth was significantly inhibited after treatment with the small molecule compound FN1-8.

Three weeks after the initial tumor inoculation and the above treatment, the tumors were excised from the mice in each group and weighed with a balance. Tumor weight was significantly reduced after treatment with the FN1-8 compound (Figures 18B and 18C).

Example 37: Small molecule compound FN1-8 inhibits tumor growth in vivo (mouse xenograft tumor model; melanoma LOX)

The in vivo efficacy of the small molecule compound FN1-8 was studied using a mouse xenograft tumor model (melanoma Lox). Inject 3 x ¹⁰⁶ cells in a volume of 100 µl subcutaneously in the dorsal region of the mouse. Figure 17, arrows). Seven days after inoculation, mice began to receive FN1-8 at a daily dose of 50 mg/kg body weight for 2 x 6 days (n=9). DMSO alone was used as a control (n=10). FN1-8 and DMSO were adjusted to a volume of 40 μl and injected intraperitoneally into the peritoneal cavity of mice. Arrows in Figure 19A indicate the time points of each injection. Tumor size and tumor volume were calculated as described above. As shown in Figures 17 and 19, tumor growth was significantly inhibited after treatment with the small molecule compound FN1-8.

Three weeks after the initial tumor inoculation and the above treatment, the tumors were excised from the mice in each group and weighed with a balance. Tumor weight was significantly reduced after treatment with the FN1-8 compound (Figures 19B and 19C).

In vivo efficacy studies of the small molecule compounds FN1-5 and FN1-8 were repeated using lower doses of the compounds in a mouse xenograft tumor model (NSCLC H460). Mice were injected as described above. Nine days after inoculation, mice began to receive compound treatment at a daily dose of 15 mg/kg body weight for 2 x 6 days (n=7). DMSO alone was used as a control (n=7). FN1-8 and DMSO were adjusted to a volume of 50 μl and injected intraperitoneally into the peritoneal cavity of mice. Twenty-four days after the initial tumor inoculation, tumors were excised from mice in each group and weighed with a balance. Tumor weight decreased after treatment with the small molecule compound FN1-8 (data not shown). However, mice treated with low doses of compound FN1-5 had tumor weights similar to those of the DMSO control group. This low dose in vivo result is consistent with the in vitro data presented herein.

Example 38: Small molecule compound FN1-8 does not affect tumor growth in vivo (mice xenograft tumor model: colon cancer HT29)

In vivo efficacy studies of the small molecule compound FN1-8 were also performed using a negative mouse xenograft tumor model (colon carcinoma HT29). Inject 3 x ¹⁰⁶ cells in a volume of 100 µl subcutaneously in the dorsal region of the mouse. Seven days after inoculation, mice began to receive FN1-8 at a daily dose of 50 mg/kg body weight for 2 x 6 days (n=2). DMSO alone was used as a control (n=4). FN1-8 and DMSO were adjusted to a volume of 40 μl and injected intraperitoneally into the peritoneal cavity of mice. Arrows in Figure 20A indicate the time points of each injection. Tumor size was measured every three days with vernier calipers. Tumor volumes were calculated using the equation x ² y (where x<y). As shown in Figure 16A, small molecule compound FN1-8 did not affect tumor growth after treatment. This result is consistent with in vitro data showing that FN1-8 has no effect on GLI3 activation in HT29 cells and no Gli3 RT-PCR product (see Figure 12, data not shown). Tumor weight after FN1-8 treatment was similar to that of the DMSO control group (Fig. 20B). Results are expressed as mean ± SD (error bars).

In a separate experiment, 7 days after inoculation, mice began to receive daily doses of 15 mg/kg bw of FN1-8 or FN1-5 for 2 x 6 days (n=7), followed by another round of daily doses 30 mg/kg body weight treatment for 1 x 6 days. DMSO alone was used as a control (n=7). Compounds and DMSO were adjusted to a volume of 50 μl and injected intraperitoneally into the abdominal cavity of mice. 43 days after the initial tumor inoculation, tumors were excised from mice in each group and weighed with a balance. The tumor weight of HT29 after FN1-8 treatment was found to be similar to that of the DMSO control group (data not shown). FN1-5 treatment was also found to slightly reduce tumor weight (data not shown). These results are consistent with in vitro data in HT29 cells that FN1-5, but not FN1-8, can induce cell killing and apoptosis in the colon cancer cell line HT29 (see Example 7).

Example 39: Small molecule compound FN1-8 can inhibit tumor growth in vivo (mouse xenograft tumor model: NSCLC A549

The in vivo efficacy of the small molecule compound FN1-8 was studied using a mouse xenograft tumor model (NSCLC A549). Inject 3 x ¹⁰⁶ cells in a volume of 100 µl subcutaneously in the dorsal region of the mouse. Ten days after inoculation, mice began to receive the small molecule compound FN1-8 at a daily dose of 15 mg/kg body weight for 2 x 6 days (n=5), followed by another round of daily doses of 30 mg/kg body weight. Treatment, 1x6 days in total. DMSO alone was used as a control (n=5). FN1-8 and DMSO were adjusted to a volume of 50 μl and injected intraperitoneally into the peritoneal cavity of mice. 43 days after the initial tumor inoculation, tumors were excised from mice in each group and weighed with a balance. Tumor weight was reduced after FN1-8 compound treatment compared to the DMSO control group (data not shown). This low dose in vivo result is consistent with the in vitro data presented herein.

Example 40: Effects of small molecule compound FN1-8 on tumor Hedgehog pathway in vivo

To test whether the lead compound FN1-8 inhibits tumor growth in vivo by inhibiting the Hedgehog signaling pathway, total RNA isolated from xenograft tumors was used to analyze key components of the Hh pathway and direct target genes (such as Shh, Ptch1) by semi-quantitative RT-PCR. , Gli1, Gli2, Gli3, Dvl-3) expression. GAPDH was used as a control. This RT-PCR analysis was performed on tumor "pools" from two randomly selected mice with tumors of appropriate size in each group. Hh signaling was found to be inhibited by FN1-8 in NSCLC tumors (H460) (data not shown). However, it was observed that Hh signaling was not significantly altered in negative control HT29 tumors following FN1-8 in vivo treatment. These results are consistent with the in vitro results (see eg Tables 2 and 3, induction of apoptosis in H460 and HT29 cells), suggesting that the small molecule compounds FN1-8 can specifically inhibit tumor growth in vivo by inhibiting the Hh pathway.

Example 41: The effect of the small molecule compound FN1-8 on the canonical Wnt pathway of tumors in vivo

To test whether the lead compound FN1-8 can inhibit tumor growth in vivo by inhibiting Wnt signaling pathway, cytoplasmic proteins isolated from H460 and HT29 xenografts were analyzed by Western blot. GAPDH was used as a control. The Western blot analysis was performed on tumor "pools" from two randomly selected mice with tumors of appropriate size in each group. Expression of key markers of canonical Wnt activation, Dvl-3, and cytoplasmic β-catenin was found to be downregulated in FN1-8-treated H460 tumors compared with the DMSO-treated group (data not shown). However, no significant changes in Dvl-3 and cytoplasmic β-catenin levels were observed in negative control HT29 tumors following treatment with FN1-8 in vivo (data not shown).

Example 42: Effect of small molecule compound FN1-8 on body weight of mice after in vivo treatment

As a preliminary in vivo toxicity study of FN1-8, it was examined whether the body weight of mice decreased after FN1-8 treatment. After initial tumor inoculation and treatment for 3 weeks as described in previous examples, the body weights of the DMSO control group (n=11) and the FN1-8 treatment group (n=8) were weighed with a balance. No significant body weight loss was observed following FN1-8 treatment (data not shown).

Example 43: Pharmacokinetic analysis of small molecule compounds FN1-5 and FN1-8 in mice

Three mice were intraperitoneally injected with small molecule compound FN1-5 at 30 mg/kg body weight. Plasma was collected from the tail vein of each mouse at 10 minutes, 2 hours, 6 hours and 24 hours after the injection of FN1-5. The concentration of FN1-5 in each plasma sample was determined by mass spectrometry, see Figure 21A. This analysis revealed rapid uptake of FN1-5 into plasma within 10 min following compound injection. The concentration of FN1-5 in serum disappeared within 24 hours, which indicated that FN1-5 could be absorbed in vivo.

Similarly, the pharmacokinetic properties of the small molecule compound FN1-8 were analyzed ( FIG. 21B ). Comparing the uptake of FN1-8 to FN1-5, the uptake of FN1-5 after intraperitoneal injection appears to be better, about 2-fold higher.

In another pharmacokinetic study, three mice were orally administered small molecule compound FN1-5 at a dose of 30 mg/kg body weight. Then, as described above, plasma was collected from the tail vein of each mouse, and the concentration of FN1-5 in each plasma sample was measured by mass spectrometry. The results are shown in Figure 22A. The results of this assay demonstrate that FN1-5 is rapidly uptaken into plasma (within 10 minutes after compound injection) and disappears within 24 hours. This result again demonstrates that FN1-5 can be formulated for oral administration to treat cancer.

Similarly, the pharmacokinetic properties of orally administered small molecule compound FN1-8 were analyzed ( FIG. 22B ). Comparing the uptake of FN1-8 to FN1-5, the uptake of FN1-5 appears to be better after oral administration, increasing about 7-fold.

Claims (35)

1. micromolecular compound with following formula, and salt, hydrate, solvate, isomer and prodrug:

X in the formula ¹And X ²Be N or C independently of one another, X in the formula ¹And X ²One of be N, X ¹And X ²One of be C, therefore encircle N and X ¹And X ²In be that of C forms two keys;

R ¹, R ²And R ³Be selected from independently of one another with 0-3 R ⁶The optional C that replaces of group ₁-C ₆Alkyl, aryl, heteroaryl, cycloalkyl and Heterocyclylalkyl, described R ⁶Group is selected from hydrogen, halogen, C independently of one another ₁-C ₆Alkyl ,-OR ⁷,-C (O) R ⁷,-OC (O) R ⁷,-N (R ⁷, R ⁷) ,-NR ⁷S (O) ₂R ⁷,-C (O) N (R ⁷, R ⁷) ,-N (R ⁷) C (O) R ⁷,-N (R ⁷) C (O) N (R ⁷, R ⁷) ,-C (NR ⁷) N (R ⁷, R ⁷) ,-N (R ⁷) C (NR ⁷) N (R ⁷, R ⁷) and S (O) _mR ⁷, wherein subscript m is 0,1 or 2;

Y is direct key or C ₁-C ₄Alkyl;

Z is selected from C ₁-C ₄Alkyl, aryl and heteroaryl;

R ⁴Be hydrogen, halogen ,-OR ⁷,-OC (O) R ⁷,-C (O) OR ⁷,-N (R ⁷, R ⁷) ,-NR ⁷S (O) ₂R ⁷,-C (O) N (R ⁷, R ⁷) ,-N (R ⁷) C (O) N (R ⁷, R ⁷) ,-C (NR ⁷) N (R ⁷, R ⁷), NR ⁷C (O) R ⁷And S (O) _mR ⁷, subscript m is 0,1 or 2 in the formula;

R ⁵Be H, C ₁-C ₆Alkyl, or optional be combined to form the first heterocycle of 5-6 with Y;

R ⁷Be H or C independently of one another ₁-C ₆Alkyl.

2. micromolecular compound as claimed in claim 1 is characterized in that R ¹, R ²And R ³Each is aryl naturally.

3. micromolecular compound as claimed in claim 2 is characterized in that R ¹, R ²And R ³Each is phenyl naturally.

4. micromolecular compound as claimed in claim 2 is characterized in that R ⁴It is hydroxyl.

5. micromolecular compound as claimed in claim 4 is characterized in that Z is C ₁-C ₄Alkyl.

6. micromolecular compound as claimed in claim 4 is characterized in that Z is an aryl.

7. micromolecular compound as claimed in claim 6 is characterized in that Z is a phenyl.

8. micromolecular compound as claimed in claim 1 is characterized in that,

R ¹, R ²And R ³Each is phenyl naturally;

Y is C ₁-C ₄Alkyl;

Z is C ₁-C ₄Alkyl or phenyl; With

R ⁴It is hydroxyl.

9. micromolecular compound as claimed in claim 8 is characterized in that X ¹Be C, X ²Be N.

10. micromolecular compound as claimed in claim 9 has following structure:

11. micromolecular compound as claimed in claim 9 has following structure:

12. micromolecular compound as claimed in claim 8 is characterized in that, X ¹Be N, X ²Be C.

13. micromolecular compound as claimed in claim 12 has following structure:

14. micromolecular compound as claimed in claim 12 has following structure:

15. a pharmaceutical composition, it comprises: (i) the described micromolecular compound of claim 1; (ii) pharmaceutically acceptable carrier.

16. the method for the proteic tumor cell generation of an abduction delivering GLI apoptosis said method comprising the steps of:

(a) described tumor cell is contacted with the described micromolecular compound of the claim 1 of capacity;

Wherein said contact can be induced described apoptosis of tumor cells.

17. method as claimed in claim 16, it is characterized in that described tumor cell is selected from colon cancer, melanoma, mesothelioma, pulmonary carcinoma, renal cell carcinoma, breast carcinoma, carcinoma of prostate, sarcoma, ovarian cancer, esophageal carcinoma, gastric cancer, hepatocarcinoma, nasopharyngeal carcinoma, cancer of pancreas and glioma cell.

18. method as claimed in claim 16 is characterized in that, described GLI albumen is GLI1, GLI2 or GLI3.

19. method as claimed in claim 16, described method is in external enforcement.

20. method as claimed in claim 16, described method is implemented in vivo.

21. method as claimed in claim 16 is characterized in that, gives the part of described micromolecular compound as modality of cancer treatment.

22. the method for at least a behavior in a bad growth that suppresses cell, hyper-proliferative or the survival said method comprising the steps of:

(a) determine whether cell expresses the Gli gene; With

(b) described cell is contacted with the described micromolecular compound of the claim 1 of effective dose;

Wherein step (b) causes the bad growth, hyper-proliferative of cell or at least a behavior in the survival to be suppressed.

23. there is the method for the object of carcinous disease in a treatment, said method comprising the steps of:

(a) give the described micromolecular compound of claim 1 that described object is treated effective dose;

The feature of wherein said carcinous disease is to express GLI albumen; Wherein step (a) causes described object to obtain medical treatment.

24. a method that suppresses GLI protein active in the cell said method comprising the steps of:

(a) the described micromolecular compound of described cell and claim 1 is contacted;

Wherein said contact causes that the proteic activity of GLI is suppressed in the described cell.

25. method as claimed in claim 24 is characterized in that, described GLI activity is the protein-protein interaction between GLI albumen and the TAF albumen.

26. one kind is suppressed the method that GLI protein D NA binding site operability is connected in the gene expression of this gene promoter, said method comprising the steps of:

(a) cell of expressing said gene is contacted with the described micromolecular compound of claim 1;

Wherein said contact causes described expression of gene to be suppressed.

27. method as claimed in claim 26 is characterized in that, described gene is the Wnt2 gene.

28. method as claimed in claim 27 is characterized in that, described Wnt2 gene is a people's gene.

29. the application in the medicine of micromolecular compound at least a behavior in preparation suppresses bad growth, the hyper-proliferative of cell or survives according to claim 1.

30. the application of the described micromolecular compound of claim 1 in the medicine of preparation treatment cancer.

31. the application of the described micromolecular compound of claim 1 in the medicine of preparation cell death inducing.

32. the application of the described micromolecular compound of claim 1 in the medicine of the GLI protein signal transduction of preparation inhibition cell.

33. an evaluation can suppress GLI albumen and TAF _IIThe method of the candidate compound of protein-protein interaction between 31 albumen said method comprising the steps of:

(a) make candidate compound and contain GLI albumen and TAF _II31 proteic samples contact; With

(b) determine GLI albumen and TAF _II31 proteic combinations,

Wherein, GLI albumen and TAF when not having candidate compound _II31 combination is compared, GLI albumen and TAF in the presence of candidate compound _II31 bound water pancake is low to show that this candidate compound can suppress GLI albumen and TAF _IIInteraction between 31 albumen.

34. an evaluation can be regulated the method for the chemical compound of GLI-protein dependent transcriptional activity, said method comprising the steps of:

(a) sample is contacted with candidate compound, wherein said sample comprises and has the expression report construction that operability is connected in one or more GLI DNA binding sites of reporter gene; With

(b), then measure of the influence of described candidate compound to GLI protein dependent transcriptional activity level if having.

35. method as claimed in claim 34 is characterized in that, described sample comprises cell.

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