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WO2018187942A1 - Protéine procaryote destinée au criblage de liant pour le transporteur de glucose (glut) et son procédé de préparation et son utilisation - Google Patents

Protéine procaryote destinée au criblage de liant pour le transporteur de glucose (glut) et son procédé de préparation et son utilisation Download PDF

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WO2018187942A1
WO2018187942A1 PCT/CN2017/080108 CN2017080108W WO2018187942A1 WO 2018187942 A1 WO2018187942 A1 WO 2018187942A1 CN 2017080108 W CN2017080108 W CN 2017080108W WO 2018187942 A1 WO2018187942 A1 WO 2018187942A1
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seq
tryptophan
amino acid
replaced
acid sequence
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颜宁
蒋鑫
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Tsinghua University
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Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor

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  • the present disclosure relates to prokaryotic proteins for screening for binding agents to the glucose transporter GLUT, and methods for their preparation and use. More specifically, the present disclosure relates to a prokaryotic homologous protein of GLUT which can be used in drug development, a method for producing the prokaryotic homologous protein, a method for screening a binding agent against GLUT using the prokaryotic homologous protein, and a prokaryotic core comprising the same A kit of homologous proteins for screening for binding agents to GLUT.
  • GLUT is an important protein that mediates the transport of glucose across the membrane.
  • different types of GLUT can also transport monosaccharides such as galactose, mannose, fructose, and xylose.
  • part of the GLUT can also transport non-saccharide substances such as inositol, uric acid, glucosamine, etc. (Non-Patent Document 1).
  • the GLUT is encoded by the SLC2 gene and consists of approximately 500 amino acids.
  • Fourteen GLUT proteins have been identified in the human genome, all of which are members of the Major facilitator superfamily (MFS).
  • MFS Major facilitator superfamily
  • Members of GLUT have a typical MFS structure, and their primary amino acid sequence is folded to form 12 transmembrane helix (TM), in which the first 6 transmembrane helices form the N-terminal domain, and the last 6 transmembrane helices form C.
  • TM transmembrane helix
  • two transmembrane domains are linked by a soluble domain in the middle of the N/C-terminal transmembrane domain
  • Non-Patent Document 2 As a representative of the members of GLUT, the nucleotide sequence and amino acid sequence of GLUT1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • Non-Patent Document 3 The process of transporting substrates by GLUT can be described by alternate open models (Non-Patent Document 3).
  • the substrate binding site of GLUT is located in the middle of the N/C terminal domain, and some amino acid residues on the N-terminal domain and the C-terminal domain contribute to the binding of the substrate. In the direction perpendicular to the cell membrane, the binding site of the substrate is located approximately in the middle of the cell membrane.
  • the N/C-terminal domain of GLUT opens to the side of the cell membrane, and the hydrophilic channel formed by the substrate enters the substrate binding site located in the center of the cell membrane. .
  • Non-Patent Documents 4 and 5 The N/C-terminal domain then opens the substrate binding site to the other side of the cell membrane by conformational changes.
  • the substrate is released to the other side of the cell membrane by a new hydrophilic channel formed by the other side opening. Thereafter, the N/C terminal domain undergoes a conformational change again, idling back to the initial state without carrying the substrate, thereby completing the transmembrane transport process of the entire substrate.
  • Fig. 1 schematically shows the above process (Non-Patent Document 6).
  • Non-Patent Document 1 Due to the important role of GLUT in the process of cellular glucose transport, inactivating mutations or expression disorders of GLUT are associated with numerous diseases such as GLUT1 deficiency syndrome, Fanconi-Bickel syndrome, type 2 diabetes, and the like (Non-Patent Document 1).
  • overexpression of GLUT has been identified in many cancer cells, such as lymphoma, colorectal cancer, hepatocellular carcinoma, head and neck cancer, stomach cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, pancreatic cancer, sarcoma.
  • Non-Patent Document 7 Non-Patent Document 15. Because cancer cells have altered metabolic pathways, they need to provide energy to cells through glycolysis, an inefficient method of ATP synthesis. This phenomenon is known as the Warburg effect.
  • Non-Patent Literature 8
  • Non-Patent Document 15 8
  • the prokaryotic homologous protein of GLUT is known as follows: GlcPse protein [Staphylococcus epidermidis] (SEQ ID NO: 52), xylose transporter XylE [Escherichia coli] (SEQ ID NO: 4), D-glucose -Proton symporter [Bifidobacterium] Adolescentis) ATCC 15703] (GenBank: BAF40314.1), Bifidobacterium merycicum (GenBank: KFI68887.1), Glucose/mannose: H+ symporter GlcP [Brevibacterium linens] (GenBank: AOP54074.1), glucose/mannose: H+ symporter GlcP [Frigoribacterium sp.
  • prokaryotic proteins are generally easier to express and purify by molecular biological techniques than their eukaryotic homologs, and generally have better stability in solution, consider prokaryotic homologous proteins using GLUT when screening for binding agents to GLUT. To simulate GLUT.
  • the GlcPse protein is derived from Staphylococcus epidermidis, which has a sequence similarity with GLUT of up to 49% to 58%.
  • the resolved three-dimensional structural information indicates that the GlcPse protein has the same MFS structure as GLUT, the amino acid residues of its substrate binding site are highly conserved compared to GLUT, and it also transports the substrate through an alternate open model. Further transport experiments have also shown that GlcPse is capable of specifically transporting glucose, and this transport activity can be inhibited by a partial GLUT inhibitor (Non-Patent Document 10).
  • the xylose transporter XylE is derived from Escherichia coli and has a sequence similarity of 47% to 51% with GLUT.
  • the inventors first analyzed the three-dimensional structure of the XylE protein in the world (Non-Patent Document 9). By analyzing the structural information, XylE has the same MFS structure as GLUT, its substrate binding site amino acid residues are highly conserved with GLUT, and XylE also transports substrates through alternate open models.
  • a major difficulty in the design of drugs for transporters is how to distinguish the different conformations of transporters.
  • different conformations such as the inward opening and the outward opening
  • the spatial position of the amino acid residues associated with substrate binding may change, resulting in different binding properties of the substrate, the drug molecule and the transporter ( Non-patent documents 11 and 12).
  • Non-Patent Document 13 A method of immobilizing LacY to an outward-opening conformation by introducing a mutation of a tryptophan residue into an Escherichia coli-derived lactose transporter LacY is known (Non-Patent Document 13).
  • LacY is neither capable of transporting nor binding to the substrate glucose of GLUT (Non-Patent Document 14), and thus cannot be used as a prokaryotic protein model of GLUT.
  • prokaryotic protein models capable of mimicking the conformational isomer of GLUT, as well as methods for preparing such prokaryotic protein models.
  • Non-Patent Document 1 Mueckler, M. & Thorens, B. The SLC 2 (GLUT) family of membrane transporters. Molecular aspects of medicine 34, 121-138, doi: 10.1016/j.mam. 2012.07.001 (2013).
  • Non-Patent Document 2 Deng, D. & Yan, N. GLUT, SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters. Protein science: a publication of the Protein Society 25, 546-558, doi: 10.1002/pro .2858 (2016).
  • Non-Patent Document 3 Jardeczky, O. Simple allosteric model for membrane pumps. Nature 211, 969-970 (1966).
  • Non-Patent Document 4 Deng, D. et al. Crystal structure of the human glucose transporter GLUT1. Nature 510, 121-125, doi: 10.1038/nature 13306 (2014).
  • Non-Patent Document 5 Deng, D. et al. Molecular basis of of ligand recognition and transport by glucose transporters. Nature 526, 391-396, doi: 10.1038/nature14655 (2015).
  • Non-Patent Document 6 Yan, N. Structural advances for the major facilitator superfamily (MFS) transporters. Trends in biochemical sciences 38, 151-159, doi: 10.1016/j.tibs. 2013.01.003 (2013).
  • MFS major facilitator superfamily
  • Non-Patent Document 7 Szablewski, L. Expression of glucose transporters in cancers Biochimica et biophysica acta 1835, 164-169, doi: 10.1016/j.bbcan. 2012.12.004 (2013).
  • Non-Patent Document 8 Zhao, Y., Butler, E. B. & Tan, M. Targeting cellular metabolism to improve cancer therapeutics. Cell death & disease 4, e532, doi: 10.1038/cddis. 2013. 60 (2013).
  • Non-Patent Document 9 Sun, L. et al. Crystal structure of a journal homologue of glucose transporters GLUT 1-4. Nature 490, 361-366, doi: 10.1038/nature11524 (2012).
  • Non-Patent Document 10 Iancu, CV, Zamoon, J., Woo, SB, Aleshin, A. & Choe, JYCrystal structure of a glucose/H+symporter and its mechanism of action. Proceedings of the National Academy of Sciences of the United States of America 110, 17862-17867, doi: 10.1073/pnas.1311485110 (2013).
  • Non-Patent Document 11 Quistgaard, EM, Low, C., Moberg, P., Tresaugues, L. & Nordlund, P. Structural basis for substrate transport in the GLUT-homology family of monosaccharide transporters. Nature structural & molecular biology 20, 766-768, doi: 10.1038/nsmb.2569 (2013).
  • Non-Patent Document 12 Wisedchaisri, G., Park, MS, Iadanza, MG, Zheng, H. & Gonen, T. Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE. Nature communications 5, 4521, doi: 10.1038 /ncomms5521(2014).
  • Non-Patent Document 13 Irina Smirnova, Vladimir Kasho, Junichi Sugihara, and H. Ronald Kaback. Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation. Proceedings of the National Academy of Sciences of the United States of America 110,8876-8881, doi:10.1073/pnas.1306849110 (2013).
  • Non-Patent Document 14 Hemant Kumar, Vladimir Kasho, Irina Smirnova, Janet S. Finer-Moorea, H. Ronald Kaback, and Robert M. Stroud. Structure of sugar-bound LacY. Proceedings of the National Academy of Sciences of the United States Of America 111,1784-1788, doi:10.1073/pnas.1324141111 (2014).
  • Non-Patent Document 15 Alison N. McCracken, Aimee L. Edinger. Nutrient transporters: the Achilles' heel of anabolism. Trends in Endocrinology & Metabolism. Volume 24, Issue 4, p200-208, April 2013.
  • the technical problem to be solved by the present disclosure is to provide a prokaryotic protein model for determining the binding of GLUT to a candidate molecule.
  • the prokaryotic protein model can mimic a specific conformer of the GLUT.
  • the technical problem to be solved by the present disclosure is to provide a method of preparing and producing a prokaryotic protein model for determining the binding of a GLUT to a candidate molecule.
  • the prokaryotic protein model can mimic a specific conformer of the GLUT.
  • the technical problem to be solved by the present disclosure is to provide a method for determining the binding of GLUT to a candidate molecule by using a prokaryotic homologous protein of GLUT as a model, and using a prokaryotic homologous protein of GLUT as a model to screen and identify against GLUT.
  • the method of binding agent is to provide a method for determining the binding of GLUT to a candidate molecule by using a prokaryotic homologous protein of GLUT as a model, and using a prokaryotic homologous protein of GLUT as a model to screen and identify against GLUT.
  • the technical problem to be solved by the present disclosure is to provide a kit for screening and identifying a binding agent for GLUT, the kit comprising a prokaryotic protein model of GLUT.
  • the technical problem to be solved by the present disclosure is to provide a method of immobilizing the conformation of prokaryotic homologous proteins of GLUT.
  • the prokaryotic protein to which the conformation is immobilized can mimic a specific conformer of the GLUT.
  • a prokaryotic homologous protein of GLUT is used as a prokaryotic protein model of GLUT, and further, by using a large sterically hindered side chain amino acid residue on a prokaryotic homologous protein of GLUT
  • a base-mutated prokaryotic protein that efficiently screens for binding agents (eg, drug molecules) to GLUT can be used as a prokaryotic protein model of the outward-opening GLUT conformer.
  • the inventors have found a method of preparing and producing a prokaryotic protein model of the above GLUT. And a method of immobilizing the conformation of the prokaryotic homologous protein by introducing a large sterically hindered side chain amino acid residue mutation into a prokaryotic homologous protein of GLUT, wherein the prokaryotic homologous protein can be immobilized as an outward open conformational isomer.
  • a method of screening a binding agent for a GLUT comprising the steps of 1) and/or 2) below, and the step comprising 3):
  • the first prokaryotic protein is a prokaryotic-derived homologous protein of GLUT, the first prokaryotic protein capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9 a group consisting of GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the first prokaryotic protein has more than 35% sequence similarity to the GLUT;
  • the second prokaryotic protein is a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in an amino acid sequence of the first prokaryotic protein, which mimics a conformational isomer of GLUT;
  • the large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.
  • step 3 the combination of the candidate molecule and the GLUT is determined as follows: a), b) and/or c):
  • step 1) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule is capable of binding to the GLUT;
  • step 2) if the binding of the candidate molecule to the second prokaryotic protein is detected in step 2), determining that the candidate molecule is capable of binding to the conformational isomer of the GLUT mimicked by the second prokaryotic protein;
  • step 1) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), and the binding of the candidate molecule to the second prokaryotic protein is not detected in step 2), then the candidate molecule is determined to be different from said The conformational isomer of the GLUT of the conformational isomer of the two prokaryotic proteins.
  • the second prokaryotic protein mimics an outward open-ended conformer of the GLUT.
  • step 3 the combination of the candidate molecule and the GLUT is determined as follows: a), b) and/or c):
  • step 1) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule is capable of binding to the GLUT;
  • step 2) if the binding of the candidate molecule to the second prokaryotic protein is detected in step 2), determining that the candidate molecule is capable of binding to the outward open conformational conformation of the GLUT;
  • step 1) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), and the binding of the candidate molecule to the second prokaryotic protein is not detected in step 2), then the candidate molecule can be determined to be inward-open with the GLUT Conformational isomers are combined.
  • the detecting is performed using a micro thermophoresis method and/or an isothermal calorimetric method.
  • kits for screening a binding agent against a GLUT comprising a first prokaryotic protein and a second prokaryotic protein;
  • the first prokaryotic protein is a prokaryotic-derived homologous protein of GLUT, the first prokaryotic protein capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9 a group consisting of GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the first prokaryotic protein has more than 35% sequence similarity to the GLUT;
  • the second prokaryotic protein is a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in an amino acid sequence of the first prokaryotic protein, which mimics a conformational isomer of GLUT;
  • the large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.
  • the second prokaryotic protein mimics an outward open-ended conformer of the GLUT.
  • an isolated prokaryotic protein mimicking a GLUT which is a prokaryotic-derived homologous protein of GLUT, wherein the prokaryotic protein is capable of binding glucose
  • the GLUT is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the prokaryotic protein and the GLUT Has a sequence similarity of more than 35%.
  • an isolated prokaryotic protein which mimics the conformational isomer of GLUT, which is an amino acid sequence of a homologous protein derived from a prokaryote derived from GLUT a protein having a sequence of a large sterically hindered side chain amino acid residue mutated by a prokaryotic-derived homologous protein of the GLUT,
  • the GLUT is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the prokaryotic source of the GLUT a homologous protein having more than 35% sequence similarity to the GLUT, and
  • the large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.
  • the prokaryotic protein mimics an outward open-ended conformer of GLUT.
  • a polynucleotide encoding a prokaryotic protein provided by the third aspect of the present disclosure is provided.
  • an expression vector comprising the above polynucleotide is provided.
  • a transformant obtained by transforming a host with the above expression vector, preferably Escherichia coli.
  • a method for producing a prokaryotic protein simulating a conformational isomer of GLUT or GLUT which is prepared by using the above polynucleotide, expression vector or transformant, is provided.
  • the prokaryotic protein is provided.
  • a method of immobilizing a conformation of a prokaryotic-derived homologous protein of GLUT characterized by a homologue derived from a prokaryote of said GLUT
  • the protein introduces a large sterically hindered side chain amino acid residue mutation to fix the same A conformation of a source protein, wherein the homologous protein is capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUTs a group consisting of subtypes, and the homologous protein has more than 35% sequence similarity to the GLUT, and
  • the large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.
  • the homologous protein is immobilized in an outwardly open conformation.
  • the present disclosure provides prokaryotic protein models of the glucose transporter GLUT, which can serve as a tool protein for efficient screening of binding agents (eg, drug molecules) to GLUTs. Based on these prokaryotic proteins, the present disclosure also provides methods for efficiently screening for binding agents (eg, drug molecules) to GLUTs, as well as kits for screening for binding agents to GLUTs. Compared to previous screening methods, the disclosed method not only provides direct binding information of the protein to the candidate molecule, but also distinguishes the positional information of the binding agent binding to the GLUT. This important information can, for example, enable researchers to further optimize the potential drug molecules that have been initially screened, thereby accelerating drug development efforts.
  • binding agents eg, drug molecules
  • Figure 1 is a schematic representation of a substrate transport model for GLUT proteins. Wherein N represents an N-terminal domain and C represents a C-terminal domain.
  • Figure 2 is a graph showing the alignment of amino acid sequences of XylE with GLUT1, GLUT2, GLUT3 and GLUT4.
  • the site marked by "*" in Figure 2 corresponds to a conserved site in the amino acid sequence of XylE.
  • Figure 3 is a graph showing the results of purification of XylE protein. Wherein a in Fig. 3 is a graph showing the purification results of the XylE protein obtained by size exclusion chromatography, and b in Fig. 3 is a graph showing the results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the purified XylE protein.
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • Figure 4 is a graph showing the results of purification of the XylE-X0 mutant protein.
  • a in FIG. 4 is a map showing the purification result of the XylE-X0 mutant protein by size exclusion chromatography
  • b in FIG. 4 is a SDS-PAGE result diagram of the purified XylE-X0 mutant protein.
  • Figure 5 is a graph showing the atomic-scale resolution structure of the XylE-X0 mutant protein. Mutations in the two large sterically hindered side chain amino acid residues of the XylE-X0 mutant are indicated by G58W and L351W, respectively. TM2 stands for "second transmembrane helix", and other transmembrane helices have the same shorthand way.
  • Figure 6 is a graph showing the structural alignment of the XylE-X0 mutant and the wild-type XylE protein at the substrate binding site.
  • Figure 7 is a graph showing the results of liposome transport experiments of XylE-X0 mutants or wild-type XylE proteins.
  • a in Figure 7 shows the results of a substrate (3H isotope-labeled xylose) transport experiment using liposomes carrying wild-type XylE protein or XylE-X0 mutant, respectively.
  • FIG. 7 is a graph showing the results of SDS-PAGE showing the amount of protein inserted in the liposome, wherein "M” represents a molecular weight marker (Marker), and “control” represents a liposome negative control not loaded with a protein, “WT” is the result of liposome carrying the wild-type XylE protein, and “G58W/L315W” is the result of liposome carrying the XylE-X0 mutant.
  • M represents a molecular weight marker (Marker)
  • control represents a liposome negative control not loaded with a protein
  • WT is the result of liposome carrying the wild-type XylE protein
  • G58W/L315W is the result of liposome carrying the XylE-X0 mutant.
  • Figure 8 is a graph showing the results of a polyethylene glycol labeling experiment.
  • a in Figure 8 is a schematic representation of a polyethylene glycol label (mPEG-Mal-5K) for a Cys-less mutant in wild-type XylE (denoted as "No Cys” a marker pathway for the marker-derived mutant (referred to as "I171C & WT") introduced by single point mutation of Ile171Cys
  • b in Figure 8 schematically represents mPEG-Mal-5K for the "Cys-free” mutant
  • a labeling pathway for a marker mutant (referred to as "I171C & G58W/L315W) in which the Ile171Cys mutation and the Gly58Trp and Leu315Trp mutations were introduced was introduced.
  • c in Fig. 8 is a graph showing the results of Western Blot of each mutant protein after the cross-linking experiment, showing that the ultrasonic destruction is performed without or without In the case of cells, the labeling results of mPEG-Mal-5K for the "no Cys" mutant, the "I171C & WT” mutant, and the “I171C & G58W/L315W” mutant.
  • Figure 9 is a graph showing the binding of wild-type XylE protein ("XylE WT”) or XylE-X0 mutant ("XylE-X0”) to xylose or glucose as determined by isothermal calorimetry. " ⁇ M” in the figure indicates ⁇ mol/L.
  • Figure 10 is a graph showing the binding of wild-type XylE protein ("XylE WT”) or XylE-X0 mutant ("XylE-X0”) to xylose or glucose as determined by microcalorimetry. " ⁇ M” in the figure indicates ⁇ mol/L.
  • Figure 11 is a graph showing the inhibitory effects of two GLUT inhibitors on the transport activity of XylE.
  • the transport activity inhibitory effect was determined by a proteoliposome transport assay.
  • Control indicates the result of an empty liposome negative control to which no protein was added
  • WT indicates the result of transport activity of wild-type XylE protein when not added with a GLUT inhibitor (set to 100%)
  • CB cytochalasin B
  • phloretin were added, respectively.
  • Figure 12 is a graph showing the binding of phloretin or CCB to wild-type XylE (“XylE WT”) or XylE-X0 mutant (“XylE-X0”) as determined by microcalorimetry. " ⁇ M” in the figure indicates ⁇ mol/L.
  • Figure 13 is a graph showing the results of purification of GlcPse protein. Wherein a in Fig. 13 is a graph showing the result of purification of the GlcPse protein obtained by size exclusion chromatography, and b in Fig. 13 is a graph showing the results of SDS-PAGE of the purified GlcPse protein.
  • Figure 14 is a graph showing the results of purification of a GlcP-6 mutant protein. Wherein a in Fig. 14 is a graph showing the result of purification of the GlcP-6 mutant obtained by size exclusion chromatography, and b in Fig. 14 is a graph showing the results of SDS-PAGE of the purified GlcP-6 mutant.
  • Figure 15 is a graph showing the binding of wild-type GlcPse protein ("GlcPse”) or GlcP-6 mutant (“GlcP-6”) to glucose as determined by isothermal calorimetry.
  • Figure 16 is a graph showing the binding of wild-type GlcPse protein (“GlcPse”) or GlcP-6 mutant (“GlcP-6”) to glucose as determined by microcalorimetry.
  • 17A-17C are graphs showing the binding of phloretin to mutant proteins of XylE-X1 to XylE-X23 as determined by microcalorimetry.
  • X1 to X23 represents the result of binding of phloretin to a mutant of XylE-X1 to XylE-X23, and the same applies hereinafter.
  • ⁇ M in the figure indicates ⁇ mol/L.
  • Figure 18 is a graph showing the results of screening a small molecule compound test library using wild-type XylE protein ("XylE WT”) and XylE-X0 mutant ("XylE-X0").
  • a in Fig. 18 shows a positive screening result, and Phloridzin and Fasentin which are bound to both the wild type XylE protein and the XylE-X0 mutant are selected.
  • b in Figure 18 shows the negative screening results, and D-ribose does not bind to either the wild-type XylE protein or the XylE-X0 mutant.
  • the binding of each small molecule compound in the library to the XylE or XylE-X0 mutant was determined by microcalorimetry. " ⁇ M" in the figure indicates ⁇ mol/L.
  • Fig. 19 is a graph showing the results of an experiment for inhibiting the transport activity of phlorizin and farestein to XylE.
  • the transport activity inhibitory effect was determined by a proteoliposome transport assay.
  • Control indicates the result of an empty liposome negative control to which no protein was added
  • WT indicates the result of transport activity of XylE when no phlorizin or farestein was added (set to 100%)
  • “root glucoside” and " “Farsentin” indicates the results of the transport activity of XylE when phlorizin and farestein were added, respectively.
  • homologous protein refers to a protein having significant similarity to the GLUT in the amino acid sequence, and the similarity in the amino acid sequence can be used by those skilled in the art, for example. Or an amino acid sequence alignment algorithm or program commonly used in the art such as ClustalW is readily determined.
  • prokaryotic protein model of GLUT prokaryotic homologous protein of GLUT
  • prokaryotic protein model prokaryotic protein model
  • prokaryotic protein prokaryotic homologous protein
  • prokaryotic protein model protein model
  • prokaryotic protein prokaryotic protein
  • prokaryotic homologous protein and the like can be used interchangeably herein, and each represents the present disclosure.
  • Proteins of prokaryote origin that mimic the GLUT or its specific conformer may include wild-type proteins and mutant proteins.
  • wild-type protein as used herein shall be understood in the ordinary manner known to those skilled in the art and may be interpreted as a protein of an organism obtained from nature, i.e., a mutation of an amino acid residue without artificial introduction. Protein.
  • mutant protein refers to a protein in which a mutation in an amino acid residue is introduced into a corresponding wild-type protein. Unless otherwise specified herein, the wild type protein is represented only by the name of the protein. Unless otherwise specified, "wild type XylE protein” herein refers to a protein comprising the amino acid sequence of SEQ ID NO: 4.
  • mutant protein and "a protein mutant” are used interchangeably herein to refer to a protein having a mutation in an amino acid residue on the wild type protein.
  • transmembrane helix denotes a portion of a helical structure spanning a cell membrane in GLUT or its prokaryotic homologous protein.
  • first transmembrane helix from the N-terminus of the prokaryotic homologous protein of GLUT
  • TM1 the first transmembrane helix
  • TM2 the second transmembrane helix from the N-terminus
  • TM2 the rest of the transmembrane helix is the same.
  • GLUT indicates a group selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes. Any one or more of the proteins.
  • large sterically hindered side chain amino acid residue mutation refers to a mutation of an amino acid residue in an amino acid sequence of a protein substituted with an amino acid residue having a large sterically hindered side chain.
  • the large sterically hindered side chain amino acid is selected from the group consisting of tryptophan Trp, tyrosine Tyr, phenylalanine Phe, lysine Lys, arginine Arg, glutamic acid Glu, glutamine Gln, aspartame A group consisting of acid Asp and asparagine Asn.
  • nucleotide refers to ribonucleotides and/or deoxyribonucleotides.
  • glucose refers to D-glucose and xylose refers to D-xylose.
  • the prokaryotic protein model of the GLUT of the present disclosure is not particularly limited as long as it is a prokaryotic homologous protein of GLUT, and is preferably a prokaryotic homologous protein derived from Escherichia coli of GLUT, and more preferably a xylose transporter XylE.
  • the prokaryotic protein model of the GLUT of the present disclosure is preferably a protein that mimics an outward-opening type GLUT conformer, and is preferably a mutant of a prokaryotic homologous protein of GLUT.
  • the mutant of the prokaryotic homologous protein of the present disclosure is not particularly limited as long as it can mimic the conformational isomer of GLUT.
  • the mutant of the prokaryotic homologous protein is capable of mimicking the outward-opening GLUT conformer.
  • the mutant of the prokaryotic homologous protein of the present disclosure is preferably a mutant of a prokaryotic homologous protein derived from Escherichia coli of GLUT, and more preferably a mutant of the xylose transporter XylE.
  • the prokaryotic protein model of the GLUT of the present disclosure preferably has a sequence identity of 15% or more, 16% or more, 17 or more, 18% or more, or 19% or more with the GLUT as a simulation target, and more preferably has 20% or more and 21% or more. More than 22%, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more.
  • the sequence identity can be determined by one skilled in the art by using any algorithm or program known in the art and commonly used to determine the percent identity between two amino acid sequences, for example, can be used Or ClustalW program. When using BLAST or ClustalW, you can use the default parameters of the corresponding program.
  • the prokaryotic protein model of the GLUT of the present disclosure preferably has a sequence similarity of 35% or more with the GLUT as a simulation target, and more preferably has a sequence similarity of 36% or more, 37% or more, 38% or more, or 39% or more, more preferably 40% or more, 41% or more, 42% or more, 43% or more, 44% or more or 45% or more of sequence similarity, more preferably 46% or more, 47% or more, 48% or more, 49% or more, 50% Above, 51% or more, 52% or more, or 53% or more of sequence similarity.
  • sequence similarity can be determined by one skilled in the art by using any algorithm or program for determining the percent similarity between two amino acid sequences known and used in the art, for example, can be used Or ClustalW program. When using BLAST or ClustalW, you can use the default parameters of the corresponding program.
  • the prokaryotic protein model of the GLUT of the present disclosure is capable of binding glucose.
  • the prokaryotic protein model of the GLUT of the present disclosure has a sequence identity of more than 80% with XylE, and further preferably has a sequence identity of 81% or more, 82% or more, 83% or more, 84% or more, or 85% or more.
  • it has a sequence identity of 86% or more, 87% or more, 88% or more, 89% or more, or 90% or more, and more preferably has a sequence of 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • sequence identity of 96% or more, 97% or more, 98% or more, or 99% or more, and particularly preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, and 99.6. Sequence identity above %, above 99.7%, above 99.8%, above 99.9%.
  • the prokaryotic protein model of the GLUT of the present disclosure is capable of transporting xylose.
  • the prokaryotic protein model of the present disclosure is preferably a prokaryotic protein that mimics the conformational isomer of GLUT.
  • the prokaryotic protein of the conformer of the mimetic GLUT comprises two or more, preferably two, three, four, five, six, seven amino acid sequences of XylE represented by SEQ ID NO: a protein of 8, 9, 10, or more amino acid sequences having a large sterically hindered side chain amino acid residue mutation.
  • At least one mutation is located in the extracellular region of TM2, and at least one other mutation is located in the extracellular region selected from the extracellular region of TM1, TM5 The region of the group consisting of a portion and an extracellular region of TM8.
  • the extracellular region of TM2 corresponds to the amino acid residues 52 to 68 of the amino acid sequence shown in SEQ ID NO: 4
  • the extracellular region of TM1 corresponds to the sequence shown by SEQ ID NO:
  • the amino acid residues at positions 25 to 40 of the amino acid sequence, the extracellular region of TM5 corresponds to the amino acid residues from positions 172 to 190 of the amino acid sequence shown in SEQ ID NO: 4, and the extracellular region of TM8 Partially corresponds to amino acid residues from positions 311 to 326 of the amino acid sequence shown by SEQ ID NO: 4.
  • the mutation of the large sterically hindered side chain amino acid residue may be, for example, a mutation produced by Gly58, Ala62, and Leu65 located in the extracellular region of TM2; Ala29 and Ser32 located in the extracellular region of TM1.
  • the large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, aspartic acid
  • the group of the amide composition is more preferably selected from the group consisting of tryptophan, tyrosine and phenylalanine, and more preferably tryptophan.
  • At least one of the mutations is located at a site selected from the group consisting of the 58th, 62nd, and 65th positions, at least one other mutation Located at a site selected from the group consisting of the 29th, 32nd, 36th, 176th, 315th, 318th, 319th, and 322th positions.
  • At least one of the mutations is selected from the group consisting of Gly58Trp (the label indicates that the glycine at position 58 is replaced by tryptophan, the same applies hereinafter), Ala62Trp and Leu65Trp, and at least one other mutation is selected from the group consisting of Ala29Trp, Ser32Trp, Glu36Trp, Leu176Trp, A group consisting of Leu315Trp, Thr318Trp, Ile319Trp, Gly322Trp.
  • the amino acid number therein corresponds to the number of the amino acid in the amino acid sequence of XylE shown in SEQ ID NO: 4.
  • the prokaryotic protein model of the GLUT of the present disclosure may further comprise a mutation that adds, deletes and/or replaces one or more amino acids at other sites, as long as the Mutation does not affect the nature of the prokaryotic protein mimicking GLUT, for example, without affecting the binding of the prokaryotic protein to glucose, is included within the scope of the present disclosure.
  • prokaryotic proteins that mimic the conformational isomer of GLUT as long as the above-described mutations at other sites do not affect the conformation of the mimetic outward-opening GLUT protein conformer possessed by the prokaryotic protein, it is included in the present disclosure. Within the scope.
  • the prokaryotic protein model of the GLUT of the present disclosure is, for example, the following proteins (a) to (x):
  • (n) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan and the isoleucine at position 319 is replaced.
  • (u) includes the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan and the threonine at position 318 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO:46.
  • the prokaryotic protein model of the GLUT of the present disclosure may also be affixed to a label useful for isolation, purification or identification, detection at its N-terminal side and/or C-terminal side, such as an oligopeptide represented by polyhistidine. .
  • a label useful for isolation, purification or identification, detection at its N-terminal side and/or C-terminal side such as an oligopeptide represented by polyhistidine.
  • the length and structure of the label is no particular limitation on the length and structure of the label as long as it does not impair the properties of the prokaryotic protein as a model of the GLUT, such as substrate binding and conformation.
  • a polynucleotide encoding the aforementioned tag can be prepared and genetically added to the end of the polynucleotide encoding the prokaryotic protein of the present disclosure using a method known to those skilled in the art.
  • the aforementioned label can also be chemically combined and appended to the prokaryotic proteins of the present disclosure.
  • a method known to those skilled in the art can be used.
  • a method of performing PCR using a primer having a mutation can be used.
  • the site into which the mutation is introduced may, for example, be a site selected from the group consisting of TM2, TM1, TM5 and/or TM8 of the prokaryotic homologous protein of the GLUT, preferably located in the extracellular region selected from the group consisting of TM2, TM1, TM5 and/or TM8 The location.
  • the host which expresses the prokaryotic protein of the present disclosure is not particularly limited as long as it can express the protein of the present disclosure in a certain yield, and for example, Escherichia coli (JM109 strain, BL21 (commercially available) can be mentioned. DE3) strain, W3110 strain, etc.), Bacillus subtilis. It is further preferred to use E. coli as a host.
  • the polynucleotide In the case of using a polynucleotide encoding a prokaryotic protein of the present disclosure to transform a host, the polynucleotide itself can be used, and it is more preferable to use an appropriate one in an expression vector (for example, a plasmid which is usually used for transformation of prokaryotic cells, etc.).
  • An expression vector for the polynucleotide of the present disclosure is inserted at a site.
  • the expression vector is not particularly limited as long as it can stably exist and replicate in the transformed host.
  • Escherichia coli as a host, a commercially available pET plasmid vector, pUC plasmid vector can be exemplified. , pTrc plasmid vector, pCDF plasmid vector, pBBR plasmid vector.
  • a method known to those skilled in the art for example, the method described in Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992
  • the transformant obtained by the above-described method can be screened by an appropriate method, for example, by screening with a drug resistance gene carried on an expression vector, thereby obtaining an expression capable.
  • the expression vector of the present disclosure In order to prepare the expression vector of the present disclosure from the transformant of the present disclosure, it can be prepared by extracting the expression vector of the present disclosure from the transformant of the present disclosure by a method suitable for the host used in the transformation. The extraction can be carried out using an alkaline extraction method or any commercial kit commonly used in the art.
  • the prokaryotic protein of the present disclosure is recovered from the obtained culture (including a host cell and a culture medium in which the expression vector of the present disclosure is transformed), whereby the prokaryotic protein of the present disclosure can be produced.
  • the above culture may be carried out using a medium and culture conditions suitable for the host used in the transformation.
  • the medium may also contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cysteamine, thioglycolate, and dithiothreitol as needed.
  • the culture medium may be a LB (Luria-Bertani) medium supplemented with a necessary nutrient source for the wild type or mutant XylE protein, and the culture temperature may be 10 ° C to 40 ° C. °C, preferably 20 ° C to 37 ° C, more preferably about 25 ° C, the pH of the medium is from pH 6.0 to pH 8.5, preferably around pH 7.0.
  • the vector of the present disclosure contains an inducible promoter
  • the inducer IPTG (isopropyl- ⁇ -D-thiogalactopyranoside, isopropyl- ⁇ -D-thiogalactoside) can be exemplified.
  • the host is Escherichia coli
  • the turbidity (absorbance at 600 nm) of the culture solution is measured, and when it is about 1.0 to 2.0, an appropriate amount of IPTG is added, and then the culture is continued.
  • the concentration of IPTG added may be appropriately selected from the range of 0.01 to 1.0 mmol/L, preferably in the range of 0.1 to 0.5 mmol/L.
  • the various conditions associated with IPTG induction may be carried out according to conditions well known in the art.
  • separation/purification can be carried out from the culture using a method suitable for the expression pattern of the prokaryotic protein of the present disclosure in the transformant. For example, after the cells are separated and collected by centrifugation, the cells are disrupted by adding an enzyme treatment agent, a surfactant, or the like, or using ultrasonic waves, a French press, or the like, and the protein of the present disclosure is extracted and then purified. For the broken cells, preliminary separation of the components can be carried out by centrifugation.
  • a method known in the art may be used.
  • purification using a centrifugation method is mentioned.
  • the centrifugation method there are ultracentrifugation method, differential centrifugation method, density gradient centrifugation method, etc., and the purification operation can also be carried out by combining these centrifugation methods.
  • purification using liquid chromatography can be mentioned.
  • the liquid chromatography there are ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, affinity chromatography, and the like, and purification operations can also be carried out by combining these chromatography methods.
  • the prokaryotic protein of the present disclosure is a membrane protein
  • a detergent for example, a dodecyl- ⁇ -D- Maltoside (DDM).
  • the protein of the present disclosure is crystallized by an evaporation-diffusion equilibrium crystallization method of a hanging drop at a suitable protein concentration.
  • the crystals of the prokaryotic proteins of the present disclosure can be obtained by optimization of crystallization conditions (including precipitants and their concentrations, pH buffers, salts, additives, detergents, crystallization time, etc.) by methods well known to those skilled in the art.
  • diffraction data is collected by an X-ray crystal diffraction apparatus (for example, SSRF BL17U harness station), integral calculation of diffraction data, molecular replacement of integration results, construction of a structural model, and structural correction are performed by software commonly used by those skilled in the art, thereby Analyze the structure of the protein.
  • an X-ray crystal diffraction apparatus for example, SSRF BL17U harness station
  • integral calculation of diffraction data, molecular replacement of integration results, construction of a structural model, and structural correction are performed by software commonly used by those skilled in the art, thereby Analyze the structure of the protein.
  • the screening method of the present disclosure is characterized in that a prokaryotic protein model simulating GLUT is used to screen for a binding agent that can bind to GLUT.
  • the screening methods of the present disclosure employ prokaryotic proteins that mimic specific GLUT conformers to screen for binding agents that can bind to the particular GLUT conformer.
  • the screening methods of the present disclosure employ a prokaryotic protein that mimics the outwardly opening GLUT conformer to screen for binding to an outwardly opening GLUT conformer.
  • the screening method of the present disclosure employs a first prokaryotic protein that mimics the GLUT and a second prokaryotic protein that mimics a specific GLUT conformer, and by analyzing differences in the binding of the candidate molecule to the two, the screening can be combined with A binding agent for a different conformational isomer of the GLUT conformer.
  • the screening method of the present disclosure employs a first prokaryotic protein that mimics the GLUT and a second prokaryotic protein that mimics the outwardly opening GLUT conformer, and the screening can be combined by analyzing the difference in binding of the candidate molecule to the two. A binding agent for the inwardly opening GLUT conformer.
  • the method of the present disclosure preferably employs XylE as the first prokaryotic protein. Further, it is preferred to use a mutant XylE having a large sterically hindered side chain amino acid residue mutation as the second prokaryotic protein. It is further preferred to employ a large sterically hindered side chain amino acid residue mutation in a region of the extracellular region of TM2 and a region selected from the group consisting of the extracellular region of TM1, the extracellular region of TM5, and the extracellular region of TM8. The mutant XylE acts as a second prokaryotic protein.
  • the number of amino acid residue mutations in the large sterically hindered side chain is preferably two or more, and more preferably two, three, four, five, six, seven, eight, nine, ten or 11 or more.
  • the large sterically hindered side chain amino acid is preferably selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine.
  • the amino acid of the composition group is more preferably an amino acid selected from the group consisting of tryptophan, tyrosine, and phenylalanine, and more preferably tryptophan.
  • prokaryotic protein simulating GLUT in addition to XylE, if other prokaryotic-derived homologous proteins using GLUT can achieve the object of the present disclosure according to the concept and technical flow of the present disclosure, it also belongs to the present disclosure. range.
  • the method of the present disclosure for screening a binding agent for GLUT preferably comprises the steps of 1) and/or 2) below:
  • the screening method of the present disclosure may further include the following steps of 3)
  • step 3 A step of determining the binding of the candidate molecule on the GLUT based on the detection results of step 1) and/or step 2). According to the binding condition judged in the step 3), it can be judged whether or not the candidate molecule is a binding agent for the GLUT.
  • the first prokaryotic protein of step 1) is preferably a prokaryotic protein model of GLUT.
  • the second prokaryotic protein of step 2) is preferably a protein comprising an amino acid sequence having a large sterically hindered side chain amino acid residue mutation in the amino acid sequence of the aforementioned first prokaryotic protein.
  • the second prokaryotic protein of step 2) is also preferably a prokaryotic protein that mimics the exo-opening type GLUT conformer.
  • the determination of the binding of the candidate molecule on the GLUT in step 3) is preferably such that if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule can bind to the GLUT, ie, the candidate The molecule is a binding agent to the GLUT.
  • the determination of the binding of the candidate molecule on the GLUT in step 3) is also preferably to determine which conformer of the GLUT can be bound by the candidate molecule by comparing the binding result of the first prokaryotic protein and the second prokaryotic protein with the candidate molecule. .
  • the protein which mimics the outward-opening type GLUT conformer is used as the second prokaryotic protein in the step 2), if the candidate molecule is detected in the step 2) Binding of the second prokaryotic protein, it is judged that the candidate molecule can bind to the outward-opening GLUT conformer; if only the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), no detection is detected in step 2) The binding of the candidate molecule to the second prokaryotic protein determines that the candidate molecule is capable of binding to the inwardly opening GLUT conformer.
  • the second prokaryotic protein mimicking the exo-opening type GLUT conformer is preferably a prokaryotic protein having a large sterically hindered side chain amino acid residue mutation.
  • the candidate molecule for the binding agent of GLUT may be any natural or artificial molecule, and may be any small molecule or macromolecule, such as a chemical molecule or a biomolecule, including a chemical small molecule, a chemical macromolecule, a biological macromolecule or the like.
  • a candidate molecule for a binding agent to GLUT can be an antibody molecule, and as an antibody molecule, can be an intact antibody, or an antibody fragment.
  • a fragment of an intact antibody refers to a region of a part of the aforementioned intact antibody, for example, a monoclonal antibody or a polyclonal antibody, and examples thereof include Fab, Fab', F(ab') 2, Fv (antibody variable region), and single Chain antibodies (H chain, L chain, H chain V region and L chain V region, etc.), scFv, diabody (scFv dimer), dsFv (disulfide stabilized V region), at least partially complementary Peptides, Nanobodies, etc. of the complementarity determining region (CDR).
  • CDR complementarity determining region
  • a candidate molecule for a binding agent to GLUT may be a molecule known to have some pharmaceutically or biological activity, or a molecule that has not been demonstrated to have any pharmaceutical or biological activity. Any commercially available small molecule or macromolecular library can be used as a library of candidate molecules for binding agents to GLUT. Those skilled in the art can also construct a library comprising any small molecule or macromolecule as needed, and screen the binding agent for GLUT using the screening method of the present disclosure.
  • steps 1) and 2) is not particularly limited as long as the binding result of the first prokaryotic protein to the candidate molecule and the binding result of the second prokaryotic protein to the candidate molecule can be separately obtained, and steps 1) and 2) are also This can be done, for example, synchronously or simultaneously.
  • step 1) may include the following sub-steps:
  • the above step 2) may include the following sub-steps:
  • steps 1-1) and 1-2) is not particularly limited as long as the result of the binding of the first prokaryotic protein to the candidate molecule and the binding force of the two are obtained, and steps 1-1) and steps are not particularly limited. 1-2) can also be carried out, for example, simultaneously or simultaneously.
  • steps 2-1) and 2-2) is not particularly limited as long as the result of the binding of the second prokaryotic protein to the candidate molecule and the binding force of the two are obtained, and step 2-1) and the step are not particularly limited. 2-2) can also be carried out, for example, simultaneously or simultaneously.
  • a method of detecting the binding of the prokaryotic protein of the present disclosure to the candidate molecule there is no particular limitation as long as it is a method capable of measuring the magnitude of binding and/or binding force.
  • the detection is carried out in a solution environment.
  • Specific examples of the method include an isothermal calorimetric method, a microcalorimetric method, a surface plasmon resonance method, and the like, and an indirect detection of binding by detecting the influence of the candidate molecule on the transport activity of the prokaryotic protein of the present disclosure. Methods such as liposome transport assays.
  • the detection of binding can be carried out by using a liposome transport assay, and the magnitude of the binding force can be determined by isothermal calorimetry or microcalorimetry.
  • the detection methods used in steps 1) and 2) may be the same or different.
  • the detection methods employed in any two of steps 1-1), 1-2), 2-1) and 2-2) may be the same or different.
  • the method of the present disclosure determines whether a candidate molecule binds to and/or binds to a first prokaryotic protein or a second prokaryotic protein using isothermal calorimetry and/or microcalorimetry.
  • the first prokaryotic protein is a protein comprising the amino acid sequence of SEQ ID NO: 4, and the second prokaryotic protein is one or more of the XylEs described in the aforementioned " Prokaryotic Protein Model of GLUT " section. mutant.
  • the methods of the present disclosure further comprise the step of detecting the effect of the candidate molecule on the transport activity of the prokaryotic protein mimicking the GLUT by a liposome transport assay. Based on the results of the liposome transport assay, the effect of the candidate molecule on the transport activity of the GLUT can be judged. For example, if the candidate molecule inhibits the transport activity of the prokaryotic protein model of the GLUT, it is judged that the candidate molecule can inhibit the transport activity of the GLUT.
  • the prokaryotic protein of the mimetic GLUT utilized in the liposome transport assay may be the same as or different from the first prokaryotic protein in step 1).
  • the sequence between the steps of the liposome transport experiment and any of the steps 1), 2) and 3) is not particularly limited, and may also be, for example, in steps 1), 2) and 3) Any of the steps are performed synchronously or simultaneously.
  • Non-Patent Document 9 For Isothermal Titration Calorimetry (ITC), reference can be made to Non-Patent Document 9 in a manner well known to those skilled in the art.
  • the protein having a concentration between 10 and 1000 ⁇ mol/L is added to the reaction cell of the isothermal calorimeter at a reaction temperature of 15 to 30 ° C, and the concentration is 0.1 mmol/
  • the substrate or candidate molecule of L ⁇ 100 mmol / L was titrated to determine the binding force data between the two.
  • MST Microscale Thermophoresis
  • protein is used at a concentration between 500 and 50,000 nmol/L, and a 1:1 gradient is applied to a substrate or candidate molecule having a starting concentration between 10 and 10000 ⁇ mol/L.
  • the substrate was mixed with a gradient diluted substrate or candidate molecule, and then the binding force was measured using a micro thermophoresis.
  • liposomes carrying the prokaryotic proteins of the present disclosure are first prepared using a nitrogen-dried polar lipid of E. coli, followed by addition to the proteoliposome at 25 °C. Specific concentration of candidate binder molecules, after incubation, add 1 ⁇ Ci of 3 H-labeled substrate to 100 ⁇ l of KPM (50 mmol/L potassium phosphate buffer pH 6.5, 2 mmol/L magnesium chloride), then add 2 ⁇ l to the solution.
  • KPM 50 mmol/L potassium phosphate buffer pH 6.5, 2 mmol/L magnesium chloride
  • the proteoliposome after 30 s of reaction, the solution containing the proteoliposome was filtered through a 0.22 ⁇ m filter, then the filter was rinsed with 2 ml of KPM solution, and finally the filter was added to 500 ⁇ l of scintillation fluid and incubated overnight. After reading with the counter.
  • the screening methods of the present disclosure can be used to screen for binding agents to GLUT, such as binding agents that inhibit the transport activity of GLUT.
  • the binding agent that inhibits the transport activity of GLUT may also be referred to as an inhibitor of the transport activity of GLUT, or a GLUT inhibitor.
  • the screening methods of the present disclosure can be used to screen for drugs against GLUT, such as drugs for treating cancer, which are cancers associated with overexpression of GLUT.
  • the cancer includes, but is not limited to, lymphoma, colorectal cancer, hepatocellular carcinoma, head and neck cancer, stomach cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, pancreatic cancer, sarcoma, laryngeal cancer, esophageal cancer, brain cancer, breast cancer. , choriocarcinoma, ovarian cancer, endometrial cancer, retinoblastoma, rhabdomyosarcoma, glioma, cervical cancer, gallbladder cancer, oral cancer, squamous cell carcinoma, bladder cancer, multiple myeloma, melanoma, Testicular seminoma and the like. Screening of the GLUT inhibitor or drug can be carried out by methods and procedures similar to those described above for screening for binding agents to GLUT.
  • the kit for screening a binding agent for GLUT of the present disclosure is not particularly limited as long as it contains a prokaryotic protein that mimics GLUT.
  • the kit of the present disclosure preferably comprises a prokaryotic protein that mimics a specific conformer of the GLUT.
  • the kit of the present disclosure comprises a prokaryotic protein that mimics an outwardly open GLUT conformer.
  • the kit of the present disclosure preferably comprises a first prokaryotic protein that mimics the GLUT and a second prokaryotic protein that mimics a particular conformer of the GLUT.
  • the second prokaryotic protein mimics an outward open-type GLUT conformer.
  • the first prokaryotic protein and the second prokaryotic protein comprised by the kit of the present disclosure may be a prokaryotic protein simulating a GLUT and a prokaryotic protein mimicking a specific conformer of the GLUT, respectively, as described in any part of the disclosure, wherein the second prokaryotic protein Prokaryotic proteins that mimic the outwardly open conformational conformation of the GLUT can be described in any part of the disclosure.
  • the first prokaryotic protein contained in the kit of the present disclosure is preferably a protein comprising the amino acid sequence of SEQ ID NO: 4.
  • the second prokaryotic protein contained in the kit is preferably a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in the amino acid sequence of the first prokaryotic protein.
  • a mutant protein having a large sterically hindered side chain amino acid residue mutation mimics an outwardly opening GLUT conformer.
  • the kit of the present disclosure comprises a large space in the extracellular region portion of TM2 and a region selected from the group consisting of the extracellular region portion of TM1, the extracellular region portion of TM5, and the extracellular region portion of TM8
  • a mutant of the first prokaryotic protein mutated by the amino acid residue of the side chain is used as the second prokaryotic protein.
  • the number of amino acid residue mutations in the large sterically hindered side chain is preferably two or more, and more preferably two, three, four, five, six, seven, eight, nine, ten or eleven More than one.
  • the large sterically hindered side chain amino acid is preferably selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine.
  • the amino acid of the composition group is more preferably an amino acid selected from the group consisting of tryptophan, tyrosine, and phenylalanine, and more preferably tryptophan.
  • the second prokaryotic protein comprised by the kit of the present disclosure is preferably one or more of the XylE mutants described in the " Prokaryotic Protein Model of GLUT " section above.
  • prokaryotic protein of the mimetic GLUT included in the kit of the present disclosure, in addition to using XylE, if other prokaryotic-derived homologous proteins of GLUT are used, the present disclosure can also realize the present disclosure. The purpose is also within the scope of the present disclosure.
  • kits of the present disclosure can be used to screen for binding agents to GLUT, such as binding agents that inhibit the transport activity of GLUT.
  • the inhibition of GLUT rotation The active binding agent may also be referred to as an inhibitor of the transport activity of GLUT, or a GLUT inhibitor.
  • the kits of the present disclosure can be used to screen for drugs against GLUT, such as drugs for treating cancer, which are cancers associated with overexpression of GLUT.
  • the cancer includes, but is not limited to, lymphoma, colorectal cancer, hepatocellular carcinoma, head and neck cancer, stomach cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, pancreatic cancer, sarcoma, laryngeal cancer, esophageal cancer, brain cancer, breast cancer.
  • choriocarcinoma ovarian cancer, endometrial cancer, retinoblastoma, rhabdomyosarcoma, glioma, cervical cancer, gallbladder cancer, oral cancer, squamous cell carcinoma, bladder cancer, multiple myeloma, melanoma, Testicular seminoma and the like.
  • the kit of the present disclosure may comprise, in addition to the prokaryotic proteins of the present disclosure, reagents, tools and/or devices for detecting binding of prokaryotic proteins of the present disclosure to candidate molecules.
  • the kit of the present disclosure comprises reagents, tools and/or devices for detecting binding of a prokaryotic protein of the present disclosure to a candidate molecule by microcalorimetry.
  • one or more control samples may be included in the kit, and the control sample may be a positive control or a negative control sample.
  • kits containing the reagents therein and preferably suitably aliquoted are typically included.
  • the components of the kit may be packaged in an aqueous medium or in lyophilized form.
  • the kit may also contain one or more excipients, diluents and/or carriers.
  • excipients, diluents, and/or carriers include water, buffers, physiological saline.
  • the kit may also include instructions for using the kit components as well as any other reagents not included in the kit. Additionally, the kit is not limited to the particular items identified above and may comprise any reagent for manipulating or characterizing the binding of the prokaryotic proteins of the present disclosure to the candidate molecule.
  • the method of the present disclosure for immobilizing a conformer of a prokaryotic homologous protein of GLUT is characterized in that the conformation of the prokaryotic homologous protein is immobilized by introducing a mutation of an amino acid residue into a prokaryotic homologous protein of GLUT.
  • the conformation of the prokaryotic homologous protein is immobilized by introducing a large sterically hindered side chain amino acid residue mutation into the prokaryotic homologous protein of GLUT.
  • the conformation of the prokaryotic homologous protein is fixed to an outward open conformation by introducing a large sterically hindered side chain amino acid residue mutation into the prokaryotic homologous protein of GLUT.
  • the method of immobilizing the prokaryotic homologous protein conformation of GLUT of the present disclosure preferably introduces two or more, preferably two, three, four, five, six, seven, eight on the homologous prokaryotic protein.
  • One, nine, ten or more large sterically hindered side chain amino acid residue mutations Preferably, at least one mutation in the amino acid residue mutation of the two or more large sterically hindered side chains is located in the extracellular region of the prokaryotic protein of TM2, and at least one other mutation is located in the extranuclear protein selected from the extracellular domain of TM1.
  • the method of the present disclosure for immobilizing the conformational isomer of a prokaryotic homologous protein of GLUT comprises the steps of:
  • a large sterically hindered side chain amino acid residue mutation is introduced in a region of the prokaryotic homologous protein selected from the group consisting of the extracellular region portion of TM1, the extracellular region portion of TM5, and the extracellular region portion of TM8.
  • steps 1) and 2) is not particularly limited as long as the above-mentioned mutation can be introduced, and steps 1) and 2) can also be carried out, for example, simultaneously or simultaneously.
  • the mutation of the large sterically hindered side chain amino acid residue may be, for example, a mutation produced by Gly58, Ala62, and Leu65 located in the extracellular region of TM2; Ala29 and Ser32 located in the extracellular region of TM1.
  • Glu36 located in the extracellular region of TM5; Leu315, Thr318, Ile319, Gly322 located in the extracellular region of TM8.
  • the amino acid number therein corresponds to the number of the amino acid in the sequence of XylE shown in SEQ ID NO: 4.
  • prokaryotic homologous proteins of other GLUTs other than XylE one of skill in the art can readily confirm the corresponding position of the above site on the prokaryotic homologous protein using any known and commonly used amino acid sequence alignment algorithm or program.
  • the large sterically hindered side chain amino acid is preferably selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine.
  • the group is further preferably selected from the group consisting of tryptophan, tyrosine and phenylalanine, and most preferably tryptophan.
  • At least one mutation is located at the 58th, 62nd, and 65th
  • the site of the group consisting of at least one other mutation is located in a group selected from the group consisting of the 29th, 32nd, 36th, 176th, 315th, 318th, 319th, and 322th positions. Site.
  • At least one mutation is selected from the group consisting of Gly58Trp, Ala62Trp and Leu65Trp
  • at least one other mutation is selected from the group consisting of Ala29Trp, Ser32Trp, Glu36Trp, Leu176Trp, Leu315Trp, Thr318Trp, Ile319Trp, Gly322Trp.
  • the amino acid number therein corresponds to the number of the amino acid in the sequence of XylE shown in SEQ ID NO: 4. Those skilled in the art can readily determine the corresponding positions of these sites on prokaryotic homologous proteins of other GLUTs other than XylE as above.
  • the methods of the present disclosure may further comprise introducing mutations at other sites as long as the mutation does not affect the fixation of the conformation of the prokaryotic homologous protein of GLUT.
  • physical properties and chemical properties which are generated outside the conserved sites of prokaryotic proteins or a similar substitution between two amino acids may be mentioned.
  • one, two, three, four, five, six, seven, eight, nine or ten or more generated outside the conserved site of the prokaryotic protein may be mentioned.
  • Addition and/or deletion of amino acids any one of the two, two, three, four, five, six, seven, eight, nine or more amino acids may be Neighbor or not adjacent.
  • a method known in the art can be used.
  • a primer having a nucleotide mutation can be used, and a polynucleotide encoding a desired prokaryotic protein (such as cDNA) can be used as a template for PCR; or a target mutant prokaryotic protein can be used.
  • the amino acid sequence is converted into a nucleotide sequence, and a polynucleotide comprising the nucleotide sequence is artificially synthesized.
  • the host transformed with the expression vector is cultured under appropriate conditions to express and obtain a conformation-fixed mutation.
  • Type protein The specific operation can be carried out by those skilled in the art using conventional methods in the art, as described above.
  • the 3'-end reverse primer CGGATCCTCGAGTTACAGCGTAGCAGTTTGTTGTG amplifies the full-length DNA encoding the XylE protein, and the DNA sequence was confirmed by sequencing to be the sequence shown in SEQ ID NO: 3.
  • the DNA encoding the XylE protein was cloned into the pET15b vector (Novagen) by molecular cloning means, and the vector was transformed into Escherichia coli BL21 (DE3) strain, and XylE protein was expressed by using an expression system of Escherichia coli BL21 (DE3) strain, which has The amino acid sequence is shown in SEQ ID NO: 4.
  • an initial culture of Escherichia coli BL21 (DE3) transformed with XylE plasmid was added to 1 L of LB medium, cultured at 37 ° C for 4 h on a shaker at 220 rpm, and induced by adding 250 ⁇ mol/L IPTG. Incubate for 4 h at 37 ° C in a shaker at 220 rpm.
  • BL21(DE3) cells expressing the XylE protein were collected by centrifugation, and after disrupting the cells using ultrasonic waves, the pure cell membrane fraction was separated by velocity gradient centrifugation.
  • the XylE protein in the cell membrane is extracted by using the detergent dodecyl- ⁇ -D-maltoside (DDM), specifically, 1-2% of DDM is added to the cell membrane solution crushed by the homogenizer, Incubate for 1-2 h in a 4 ° C cold room.
  • DDM detergent dodecyl- ⁇ -D-maltoside
  • the XylE protein was then purified using a combination of affinity chromatography (for polyhistidine tags) and size exclusion chromatography (using a dextran 200 column). Affinity chromatography was carried out using a Ni-NTA column, and the supernatant of the membrane protein extract was added to the column.
  • a rinse solution (20 mmol/L imidazole, 25 mmol/L Tris buffer pH 8.0) was used. Rinsing with 150 mmol/L sodium chloride, 0.02% DDM), then eluting the protein with an eluent (250 mmol/L imidazole, 25 mmol/L Tris buffer pH 8.0, 150 mmol/L sodium chloride, 0.02% DDM) . The protein was then concentrated to 2 ml with a 50 KD concentrating tube, and subjected to molecular exclusion chromatography using a dextran 200 column. The buffer conditions were (25 mmol/L MES buffer pH 6.5, 150 mmol/L sodium chloride, 0.056%). Cymal-6).
  • the purified XylE protein was verified using size exclusion chromatography (see above). A single peak is shown in the resulting chromatogram, shown as a in Figure 3.
  • the purified XylE protein was verified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using SDS-PAGE of 16% denaturing gel.
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • the specific formula is: per ml of 60ml SDS gel: 24ml 40% acrylamide / fork acrylamide (37.5:1 by volume), 15ml 1.5M Tris buffer PH8.8, 300 ⁇ l 20% SDS (m / v), 420 ⁇ l 10 % ammonium persulfate, 45 ⁇ l TEMED, dilute to 60 ml with water.
  • the resulting electrophoresis results show a single band, shown as b in Figure 3. The above results confirmed that a high purity XylE protein was obtained.
  • the tryptophan mutation Gly58Trp was introduced into the extracellular region of TM2 of wild-type XylE using conventional PCR, and the color ammonia was introduced in the extracellular region of TM8.
  • the acid mutation Leu315Trp gave a mutant XylE (hereinafter referred to as XylE-X0).
  • PCR is used to first amplify a DNA sequence from the 5' start to the mutation site in the entire sequence, and then a DNA sequence from the 3' end to the mutation site in the entire sequence is amplified, and then the two fragments are mixed.
  • the 5' start-end forward primer and the 3' end sequence reverse primer were used to amplify the entire sequence.
  • the primers used to introduce the Gly58Trp mutation are:
  • the primers used to introduce the Leu315Trp mutation are:
  • the nucleotide residues of the mutation site are indicated in lower case letters in the primer sequence.
  • the 5' start-end forward primer and the 3' end sequence reverse primer were the same as in Example 1.
  • the full-length DNA sequence encoding the XylE-X0 mutant was confirmed to be the sequence of SEQ ID NO: 5, and the amino acid number of the above-mentioned mutation site corresponds to the sequence number of the amino acid in SEQ ID NO: 4.
  • the XylE-X0 mutant has an amino acid sequence as shown in SEQ ID NO: 6. Expression and purification of the XylE-X0 mutant were carried out in the same manner as in Example 1.
  • the purified XylE-X0 mutant protein was verified by size exclusion chromatography using the same procedure as in Example 1, and the resulting chromatogram showed a single peak, which is shown in a of Fig. 4. Further, the purified XylE-X0 mutant protein was verified by the same operation as in Example 1 using SDS-PAGE, and the obtained electrophoresis results showed a single band, which is shown in b of Fig. 4. The above results confirmed that a high purity XylE-X0 mutant protein was obtained.
  • the concentration of the purified XylE-X0 mutant protein was adjusted to about 5 mg/ml, and then crystallization was carried out by an evaporation-diffusion equilibrium crystallization method of a hanging drop.
  • a crystal having a higher diffraction quality was obtained by using crystallization conditions of 0.1 M NaCl, 0.1 M Li 2 SO 4 , 0.1 M MES pH 6.5, 30% PEG 400 (v/v).
  • SSRF Synchrotron Radiation Center
  • the environment in which the protein is placed is a solution environment or a lipid membrane environment.
  • the inventors performed the following liposome-based transport assays and polyethylene glycol labeling experiments.
  • the assembly of protein liposomes was first carried out by dissolving polar lipids of Escherichia coli with chloroform and methanol solution (3:1 by volume) and blowing with nitrogen. After drying, the dried lipid was resuspended to 10-25 mg/ml using KPM solution (50 mmol/L potassium phosphate buffer pH 6.5, 2 mmol/L magnesium chloride), and repeatedly frozen and thawed by liquid nitrogen for 5 to 10 times, and then used.
  • KPM solution 50 mmol/L potassium phosphate buffer pH 6.5, 2 mmol/L magnesium chloride
  • the 0.4 ⁇ m pore size filter was filtered back and forth 15 to 25 times, then 0.5% to 1.3% of the detergent OG was added to the solution, and incubated in a cold room at 4 ° C for half an hour, and then 0.8% to 1.5% according to the lipid concentration. (mass ratio) protein was added, and then incubated in a cold room at 4 ° C for one hour, then added to Biobead three times per 1 g of detergent to add Biobead (Avanti Polar Lipids, Inc.) to remove detergent.
  • Biobead was added, it was incubated in a cold room at 4 ° C for one hour, then repeatedly frozen and thawed by liquid nitrogen for 5 to 10 times, and then filtered back and forth 15 to 25 times with a 0.4 ⁇ m pore size filter. After ultracentrifugation, the protein lipid was removed. The plastid is resuspended to 50-120 mg/ml with the predetermined solution to replace the liposome to a sugar-free solution. In order to complete the preparation of proteoliposomes.
  • the liposome transport experiments were then carried out as follows: All reactions were carried out at 25 °C by first adding a specific concentration of candidate binder molecules to the proteoliposome, incubating for half an hour, and then adding 1 ⁇ Ci of 3 in 100 ⁇ l of KPM solution. H-labeled xylose, then add 2 ⁇ l of proteoliposome to the solution. After 30 s of reaction, the solution mixed with proteoliposome was filtered through a 0.22 ⁇ m filter, then the filter was rinsed with 2 ml of KPM solution, and finally The filter was added to 500 ⁇ l of scintillation fluid and incubated overnight before reading with a counter. The results obtained are shown in a of Figure 7. The results of SDS-PAGE of the blank liposome, the XylE-loaded liposome, and the liposome carrying the XylE-X0 mutant by the same procedure as in Example 1 are shown in b of Fig. 7.
  • a in Figure 7 shows that the XylE-X0 mutant introduced with two large sterically hindered side chain amino acid residue mutations completely lost the activity of the transport substrate, thus demonstrating that the mutant protein is still immobilized in the lipid membrane environment. The process of transporting the substrate cannot be completed by conformation in the outward opening.
  • b in Figure 7 shows that the amount of protein in the wild-type XylE and XylE-X0 mutants contained in the liposomes was consistent in the transport experiments.
  • the polyethylene glycol labeling experiment was carried out in accordance with the method described in Huawei Zhou et al., Structural basis of the alternating-access mechanism in a b ile acid transporter, Nature 505, 569-573, January 2014, doi: 10.1038/nature 12811.
  • the inventors screened for a cysteine single point mutant Ile171Cys that was only labeled with mPEG-Mal-5K in the inward open conformation using a Cys-less mutant of XylE.
  • a single point mutation of Ile171Cys was introduced on the mutant without cysteine residue to obtain a mutant for labeling as I171C & WT.
  • the Ile171Cys mutation and the Gly58Trp and Leu315Trp mutations were introduced on the cysteine-free residue mutant to obtain a marker mutant designated as I171C & G58W/L315W.
  • the two mutant proteins were labeled with mPEG-Mal-5K under intact cell membrane conditions and under disrupted cell membrane conditions (sonication), respectively.
  • the results of Western Blot after the polyethylene glycol labeling experiments of I171C & WT and I171C & G58W/L315W are shown in Figure 8.
  • the I171C & WT mutant protein which does not introduce a large sterically hindered side chain amino acid residue mutation, is capable of transitioning between the outward and inward opening conformations.
  • the polyethylene glycol label mPEG-Mal-5K
  • the polyethylene glycol label is able to pass the hydrophilic channel formed by the inward opening close to the cysteine site at position 171 of I171C & WT, thereby completing the labeling.
  • the binding ability of the wild-type XylE protein obtained in Example 1 and the XylE-X0 mutant protein obtained in Example 2 to its natural substrate xylose was determined by isothermal calori titration experiments, and the two were determined. Binding to the natural substrate glucose of the glucose transporter GLUT.
  • the XylE protein or its mutant at a concentration of 100 ⁇ mol/L is added to a reaction cell of an isothermal calorimeter (GE Healthcare) at a reaction temperature of 15 to 30 ° C at a concentration of 5 mmol/L to 10 mmol/L.
  • the substrate was titrated to determine the binding force data between the two. The measurement results are shown in Fig. 9.
  • thermodynamic binding parameters shown in Table 1 show that the Gibbs free energy change ( ⁇ G) of the binding process is the case when wild-type XylE binds to xylose or glucose and XylE-X0 mutant binds to xylose or glucose. Negative values confirm that the four processes are spontaneous processes.
  • the large sterically hindered side chain residue is introduced, the enthalpy change ( ⁇ H) of the binding process changes from a positive value to a negative value, and the entropy decreases ( ⁇ S).
  • the change of Gibbs free energy is mainly caused by enthalpy change. provide.
  • the entropy change is mainly caused by conformational changes.
  • the above results indicate that the introduction of large sterically hindered side chain residue mutations limits the conformational changes of transporters. This result is mutually confirmed with the results of Example 3 and Example 4.
  • the binding ability of the wild type XylE obtained in Example 1 or the XylE-X0 mutant protein obtained in Example 2 to xylose or glucose was measured using a micro thermophoresis method.
  • the substrate xylose was diluted 1:1 from a starting concentration of 300 ⁇ mol/L, and the substrate glucose was 1:1 from a starting concentration of 1000 ⁇ mol/L.
  • the protein was mixed with a gradient diluted substrate by gradient dilution, after which the binding force was measured using a micro thermophoresis instrument (NanoTemper Technologies). The measurement results are shown in Fig. 10.
  • Figure 10 shows that the micro thermophoresis method can effectively measure the binding force of the transporter protein to the substrate molecule, and the measurement result is highly consistent with the results measured by the isothermal calorimetry experiment. It was thus demonstrated that the binding ability of the candidate molecule to the prokaryotic homologous protein of GLUT can be determined using a micro thermophoresis method, and the method of binding agent screening or drug screening can be performed by this method.
  • the two small molecule inhibitors are phloretin and cytochalasin B (CCB), the molecular structure of which is shown in the following formula.
  • the liposome-based transport assay was carried out by the same method as in Example 4, and the results of inhibition of the transport activity of the two small molecule inhibitors against wild-type XylE are shown in Fig. 11. Both small molecule inhibitors have a significant inhibitory effect on the transport activity of wild-type XylE protein.
  • the micro thermophoresis experiment was carried out by the same method as in Example 6.
  • the dissociation constant (K d ) of phloretin and CCB and the wild type XylE and XylE-X0 mutants were measured by microcalorimetry, respectively, and the results are shown in Fig. 12.
  • the full-length DNA encoding the GlcPse protein was amplified using the 5' start-end forward primer: gatgcacatATGAAAGCGAACAAGTACCTG and the 3'-end reverse primer: cggatcctcgagttaTTCGGTACGCGCGCCAG, and the sequence shown by SEQ ID NO: 51 was confirmed by sequencing.
  • the DNA encoding the GlcPse protein was cloned into the pET15b vector (Novagen) by molecular cloning means, and the expression in the E. coli BL21 (DE3) system was the same as in Example 1 except that the IPTG induction concentration was 400 ⁇ mol/L.
  • the GlcPse protein of the amino acid sequence shown in SEQ ID NO: 52.
  • size exclusion chromatography was carried out using the following buffer: 25 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 0.02% DDM, and the rest of the procedure was the same as in Example 1.
  • SDS-PAGE was carried out in the same manner as in Example 1.
  • the purified GlcPse protein was verified using size exclusion chromatography (see above).
  • the resulting chromatogram shows a main peak between fractions 10 to 16, shown as a in Figure 13.
  • Fractions 12 to 14 obtained by size exclusion chromatography were analyzed by SDS-PAGE, and the obtained electrophoresis results showed a single band, which is shown in b of Fig. 13.
  • the above results confirmed that a high purity GlcPse protein was obtained.
  • GlcP-6 mutant The preparation of a mutant of GlcPse protein having Gly45Trp/Ile277Trp double tryptophan mutation (hereinafter referred to as GlcP-6 mutant) was carried out in the same manner as in Example 2.
  • the primers used to introduce the Gly45Trp mutation are:
  • the primers used to introduce the Ile277Trp mutation are:
  • the nucleotide residues of the mutation site are indicated in lower case letters in the primer sequence.
  • the 5' start-end forward primer and the 3'-end reverse primer were the same as in Comparative Example 1.
  • the full-length DNA sequence of the obtained GlcP-6 mutant was confirmed to be the sequence shown in SEQ ID NO: 53, and the amino acid number of the above-mentioned mutation site corresponds to the sequence. The number of the amino acid in No. 52.
  • the DNA encoding the GlcP-6 mutant was cloned into the pET15b vector (Novagen) by molecular cloning means, and the GlcP-6 mutant having the amino acid sequence of SEQ ID NO: 54 was expressed and purified by the same procedure as in Comparative Example 1. .
  • the purified GlcP-6 mutant was verified using the same size exclusion chromatography as in Comparative Example 1.
  • the resulting chromatogram is shown in a of Figure 14.
  • the chromatograms show that a large amount of GlcP-6 mutant protein is retained on the exclusion chromatography column and cannot be purified by size exclusion chromatography.
  • the fractions Nos. 7 to 17 obtained by size exclusion chromatography were analyzed by SDS-PAGE, and the obtained electrophoresis results are shown in b of Fig. 14.
  • the electrophoresis results showed that the GlcP-6 mutant had poor purification effect, low yield and obvious impurities.
  • the above results show that the GlcP-6 mutant has poor stability in a solution environment.
  • the binding of the GlcPse protein obtained in Comparative Example 1 and the GlcP-6 mutant obtained in Comparative Example 2 to glucose was measured by a micro thermophoresis method.
  • the operation of the micro thermophoresis experiment was the same as in Example 6 except that the buffer was 25 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 0.02% DDM, and the results are shown in Fig. 16.
  • Gly45 and Ile277 sites of GlcPse represented by SEQ ID NO: 52 correspond to the Gly58 and Leu315 sites of XylE shown in SEQ ID NO: 4, and can be easily confirmed by those skilled in the art. Such a correspondence.
  • the XylE mutant protein shown in Table 4 was constructed and prepared by the same method as in Example 2.
  • the binding ability of the XylE mutant protein in Table 4 to the GLUT inhibitor phloretin was determined by the same microcalorimetry as in Example 6, and the results are shown in Figures 17A-17C.
  • the binding experiments of Figures 17A-17C show that the XylE mutants with large sterically hindered side chain amino acid residue mutations prepared by combining the mutation sites of Table 1 and the mutation region 2 shown in Table 3 are capable of
  • the GLUT inhibitor binds to a method of screening a binding agent for GLUT of the present disclosure.
  • Wild-type XylE was used to simulate GLUT, and XylE-X0 mutant was used to simulate the outward-opening GLUT conformer, and small molecule compounds were screened.
  • Test library includes 25 small molecules containing molecules known to bind to GLUT. This library was used to test the effectiveness of the screening methods of the present disclosure for screening binding agents for GLUT.
  • the binding ability of the small molecule in the library to the wild-type XylE or XylE-X0 mutant was determined by the same microcalorimetry as in Example 6. Two of the screens were shown to show both binding to wild-type XylE and also to XylE-.
  • the small molecule compounds bound by the X0 mutant are phloridzin and Fasentin, respectively. The structure of the two is shown in the following equation.
  • a large amount of information for the drug screening process can be obtained as follows: 1. Inhibition of the transport activity of the drug molecule to the prokaryotic protein model of GLUT can be performed to evaluate whether the molecule can inhibit the GLUT protein. 2. For a molecule that can inhibit the transport activity of a prokaryotic protein model of GLUT, the dissociation constant obtained by binding assay can be used to evaluate the binding strength of the molecule to the GLUT protein; 3.
  • the protein model and the binding force of the mutant prokaryotic protein that mimics the specific conformer of the GLUT can be judged whether the molecule binds to the GLUT in the outward-facing conformation or to the GLUT in the inward-facing conformation.

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Abstract

La présente invention concerne une protéine homologue procaryote de GLUT, un procédé de préparation de la protéine homologue procaryote, un procédé de criblage d'un liant pour GLUT utilisant la protéine homologue procaryote, et un kit comprenant la protéine homologue procaryote destinée au criblage du liant pour GLUT.
PCT/CN2017/080108 2017-04-11 2017-04-11 Protéine procaryote destinée au criblage de liant pour le transporteur de glucose (glut) et son procédé de préparation et son utilisation Ceased WO2018187942A1 (fr)

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PCT/CN2017/080108 WO2018187942A1 (fr) 2017-04-11 2017-04-11 Protéine procaryote destinée au criblage de liant pour le transporteur de glucose (glut) et son procédé de préparation et son utilisation

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Title
DENG DONG ET AL.: "Structural basis and transport mechanism of major facility superfamily Transporter(MFS)", CHINESE SCIENCE BULLETIN, vol. 60, no. 8, 31 December 2015 (2015-12-31), pages 720 - 728 *
SUN, LINFENG ET AL.: "Crystal structure of a bacterial homologue of glucose transporters GLUT1 -4", NATURE, vol. 490, 18 October 2012 (2012-10-18), pages 361 - 368 *

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