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WO1993018156A1 - Synthase de l'oxyde d'azote endothelial - Google Patents

Synthase de l'oxyde d'azote endothelial Download PDF

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Publication number
WO1993018156A1
WO1993018156A1 PCT/US1993/001951 US9301951W WO9318156A1 WO 1993018156 A1 WO1993018156 A1 WO 1993018156A1 US 9301951 W US9301951 W US 9301951W WO 9318156 A1 WO9318156 A1 WO 9318156A1
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WIPO (PCT)
Prior art keywords
nitric oxide
mammal
oxide synthase
preparation
endothelial nitric
Prior art date
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PCT/US1993/001951
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English (en)
Inventor
Kenneth D. Bloch
Stefan P. Janssens
Donald B. Bloch
Original Assignee
The General Hospital Corporation
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Publication date
Application filed by The General Hospital Corporation filed Critical The General Hospital Corporation
Priority to AU37891/93A priority Critical patent/AU3789193A/en
Publication of WO1993018156A1 publication Critical patent/WO1993018156A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C12N9/0075—Nitric-oxide synthase (1.14.13.39)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13039—Nitric-oxide synthase (NADPH dependent) (1.14.13.39)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • NO is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS) and diffuses into subjacent smooth muscle cells where it stimulates guanylate cyclase and induces vasodilatation.
  • NOS nitric oxide synthase
  • the invention features a substantially pure preparation of a nucleic acid sequence which encodes endothelial cell nitric oxide synthase (ECNOS) , preferably human ECNOS.
  • ECNOS endothelial cell nitric oxide synthase
  • the ECNOS nucleic acid sequence is essentially identical to Seq ID No: 1; and the ECNOS nucleic acid sequence encodes an amino acid sequence essentially the same as the amino acid sequence given in Seq ID No: 2.
  • Preferred embodiments of the invention include: a vector which includes a nucleic acid sequence encoding ECNOS, preferably human ECNOS, e.g., a nucleic acid sequence encoding an amino acid sequence which is essentially that of Seq ID No: 2; a cell which includes the vector; a cell which includes a nucleic acid sequence encoding ECNOS integrated into the genome of the cell (wherein the ECNOS nucleic acid is derived from a different species than the cell) ; a cell which expresses the ECNOS encoding nucleic acid; an essentially homogeneous population of cells each of which includes the vector; an essentially homogeneous population of cells each of which includes a sequence encoding ECNOS (wherein the ECNOS nucleic acid is derived from a different organism than the cell) integrated into the genome of the cell.
  • ECNOS preferably human ECNOS, e.g., a nucleic acid sequence encoding an amino acid sequence which is essentially that of
  • the invention includes a substantially pure preparation of ECNOS, preferably human ECNOS, e.g., an ECNOS with a sequence essentially identical to Seq ID No: 2.
  • the invention also includes a therapeutic composition including ECNOS or an enzymatically active fragment thereof, in a pharmaceutically-acceptable carrier.
  • the therapeutic composition is essentially free of other proteins of eukaryotic origin.
  • the invention features a method for manufacture of ECNOS including: providing a cell which includes a cloned sequence encoding ECNOS, preferably human ECNOS; culturing the cell in a medium so as to express the sequence; and purifying ECNOS from the cell or the medium.
  • the invention also includes a preparation of ECNOS made by this process.
  • the invention features a purified preparation of an antibody preferably a monoclonal antibody, which specifically binds ECNOS, and an affinity column including the antibody.
  • the invention features a method of catalyzing the formation of nitric oxide including contacting a substrate, e.g., a guanidino nitrogen, e.g., the terminal guanidino nitrogen of L-arginine, with a substantially purified preparation of ECNOS.
  • a substrate e.g., a guanidino nitrogen, e.g., the terminal guanidino nitrogen of L-arginine
  • the invention features a method of treating a mammal e.g., a human, having vascular or circulatory disorder, e.g., systemic or pulmonary hypertension, accelerated-atherosclerosis associated with angioplasty, or coronary artery spasm (Prinzmetal's angina) , including administering to the mammal an effective amount of ECNOS.
  • vascular or circulatory disorder e.g., systemic or pulmonary hypertension, accelerated-atherosclerosis associated with angioplasty, or coronary artery spasm (Prinzmetal's angina)
  • the invention features a method of determining whether a mammal, e.g., a human, is at risk for a circulatory disorder, e.g., systemic or pulmonary hypertension, accelerated-atherosclerosis associated with angioplasty, or coronary artery spasm (Prinzmetal's angina) , including determining the structure of the mammal's ECNOS gene. This can be done by determining the nucleic acid sequence of all or part of the mammal's ECNOS gene, or by restriction fragment length polymorphism analysis. A lesion, e.g., a chromosomal rearrangement, e.g., a deletion, in the mammal's ECNOS gene being predictive of risk for the disorder.
  • a circulatory disorder e.g., systemic or pulmonary hypertension, accelerated-atherosclerosis associated with angioplasty, or coronary artery spasm (Prinzmetal's angina)
  • the invention features a method determining whether a mammal, e.g., a human, is at risk for a circulatory disorder, e.g., systemic or pulmonary hypertension, accelerated-atherosclerosis associated with angioplasty, or coronary artery spasm (Prinzmetal's angina) , including determining the level and or pattern of expression of the mammal's ECNOS gene, lower than wild type expression or an altered temporal or spatial pattern of expression being indicative of risk for the disorder.
  • the level of expression includes measuring the level of ECNOS mRNA in a sample taken from the mammal.
  • the invention features a method of relaxing a smooth muscle in a mammal, e.g., a human, including administering to the mammal an effective amount of ECNOS.
  • the invention features a method of increasing the level of endothelial nitric oxide synthase in mammal, e.g., a human, including introducing a nucleic acid sequence encoding endothelial nitric oxide synthase into the mammal and expressing the sequence.
  • the invention features a method of activating guanylyl cyclase including contacting guanylyl cyclase with a substantially pure preparation of ECNOS.
  • the invention features a method of inhibiting or reversing platelet aggregation in a mammal, e.g., a human, including administering to the mammal a platelet inhibiting amount of ENOS.
  • the invention features a method of inhibiting platelet aggregation in a sample comprising contacting the sample with a platelet inhibiting amount of the preparation of ECNOS.
  • the invention features a method of evaluating a compound, e.g., for anti-inflammatory activities, including contacting the compound with a preparation of ECNOS and determining the ability of the compound to inhibit the production of NO by the preparation.
  • the determination is made by monitoring any of the ability of the preparation to change the aggregation properties of platelets, or the ability of the preparation to induce vasodilation.
  • the invention features a method of evaluating a compound for the ability to inhibit ECNOS including contacting the compound with a preparation of ECNOS and determining the ability of the compound to inhibit the production of NO by the preparation. In preferred embodiments the determination is made by monitoring any of the ability to produce NO, the ability of the preparation to change the aggregation properties of platelets, or the ability of the preparation to induce vasodilation.
  • the invention features a method of evaluating a compound for the ability to stimulate ECNOS activity including contacting the compound with a preparation of ECNOS and determining the ability of the compound to promote the production of NO by the preparation. In preferred embodiments the determination is made by monitoring any of the ability to produce NO, the ability of the preparation to change the aggregation properties of platelets, or the ability of the preparation to induce vasodilation.
  • Compounds which stimulate NO production can be used, e.g., to lower blood pressure.
  • the invention features a method of evaluating a compound for ECNOS-agonist activity including determining the ability of the compound to promote the production of NO, e.g., by contacting the compound with a sample containing ECNOS. In preferred embodiments the determination is made by monitoring any of the ability to produce NO, the ability of the preparation to change the aggregation properties of platelets, or the ability of the preparation to induce vasodilation.
  • the invention features a method of inhibiting smooth muscle cell proliferation in a mammal, e.g., a human, including administering an effective amount of ENOS to the mammal.
  • the invention includes, a method of inhibiting smooth muscle cell proliferation after angioplasty in a mammal, e.g., a human, including performing angioplasty on the mammal, and administering to the mammal an effective amount of ENOS.
  • the ENOS can be administered before, during, or after angioplasty.
  • the invention features a method of evaluating a treatment, e.g., the administration of a compound, e.g., the administration an arginine analog, for the ability to specifically or preferentially inhibit a first isotype of nitric oxide synthase, e.g., macrophage inducible NO synthase, without substantially inhibiting a second isotype of nitric oxide synthase, e.g., endothelial NO synthase, including contacting the compound with the first isotype and determining if the first isotype is inhibited and contacting the second isotype and determining if the second isotype is substantially inhibited.
  • a treatment e.g., the administration of a compound, e.g., the administration an arginine analog
  • substantially pure describes a preparation which is at least 60% by weight the compound of interest, e.g., a protein, or a polypeptide, e.g., hECNOS.
  • the preparation is at least 75%, more preferably at least 90%,.and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • substantially pure nucleic acid refers to preparation of RNA or DNA molecules, e.g., genomic DNA, cDNA, or episomal DNA.
  • a fragment of a larger nucleic acid e.g., genomic fragments, fragments of cDNAs, fragments of RNAs, fragments of an episome or plasmid
  • substantially pure nucleic acid refers to a nucleic acid sequence that is not associated with the sequences that flank it in a naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that are adjacent to the fragment, e.g., the sequences adjacent to the fragment in its normal site in the genome.
  • substantially unfragmented nucleic acid molecules e.g., RNA molecules, e.g., mRNA or tRNA molecules, episomal molecules, organellar DNA, e.g., chloroplast or itochondrial DNA, or essentially whole genomic molecules, e.g., viral genomes, or chromosomes
  • substantially pure nucleic acid refers to a nucleic acid preparation that is less than 50% by weight other components, e.g., proteins, lipids, or other nucleic acids, that naturally accompany the nucleic acid.
  • substantially pure nucleic acid refers to a preparation in which at least 50% by weight of the nucleic acid is the nucleic acid of interest.
  • homologous refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules, or two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half, e.g., 5 of 10, of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous the two sequences share 90% homology.
  • the DNA sequences 3 ⁇ TTGCC5 and 3'TATGGC'5 share 50% homology.
  • substantially homologous is meant largely but not wholly homologous.
  • the invention is useful for: investigating the role of ECNOS in endothelial regulation of vascular tone; investigating the molecular basis of a variety of vascular disorders, e.g., systemic or pulmonary hypertension, accelerated-atherosclerosis associated with angioplasty, or coronary artery spasm (Prinzmetal's angina) ; inducing the relaxation of smooth muscle; supplying a transgenic source of ECNOS to a mammal, e.g., a mammal with a vascular disorder or a mutant ECNOS locus; screening for inhibitors of ECNOS, for use, e.g., as anti-inflammatory agents; determining if a mammal is at risk for a vascular disorder, e.g., systemic or pulmonary hypertension, accelerated-atherosclerosis associated with angioplasty, or coronary artery spasm (Prinzmetal's angina) by determining the structure or level of expression of the ECNOS gene in the mammal;
  • Drawings Fig. 1 is the sequence of hECNOS (Seq ID No: 1) ..
  • Fig. 2 is an alignment of the amino acid sequences of ECNOS (Seq ID No: 2 ) with brain NOS (Seq ID No: 3).
  • Fig. 3 is a RNA blot hybridization showing ECNOS expression in spleen, kidney, lung, and HUVEC.
  • Fig. 4 is a photograph showing the localization of NADPH diaphorase activity in bovine pulmonary artery endothelial cells (panel A) , in NIH/3T3 cells expressing the human endothelial NOS cDNA (panel B) , and in untransfected NIH/3T3 cells (panel C) .
  • Fig. 5 is a graph of the catalytic activity of ECNOS in control and transfected cells. Overview
  • a human cDNA encoding vascular endothelial nitric oxide synthase which also confers NADPH diaphorase activity on transfected NIH/3T3 cells has been isolated.
  • Endothelial cell-produced NO acts as an endogenous nitrovasodilator by activating soluble guanylyl cyclase, Moncada et al., Pharmacol. Rev. 43, 109-142 (1991), in vascular smooth muscle cells.
  • a similar cGMP-dependent inhibition of platelet aggregation and adhesion accounts for the potent antithrombotic action of NO, Radomski et al., Br. J. Pharmacol. 92, 639-646 (1987).
  • Fig. 1 shows the sequence of a human ECNOS (Seq ID No: 1) .
  • Oligonucleotides corresponding to amino acids in domains shared by cytochrome P-450 reductase and the recently-identified brain NOS, Bredt et al. Nature 351, 714-718 (1991) were synthesized to amplify a partial cDNA encoding a bovine endothelial cell NOS.
  • This partial cDNA was used to isolate a cDNA was used to isolate a cDNA encoding a human vascular endothelial NOS.
  • the translated human protein is 1203 amino acids in length and shares approximately 50% percent of its amino acid sequence with brain NOS.
  • oligonucleotide primers based upon the sequence of a specific domain of brain NOS, Bredt et al. (1991) .
  • cDNA prepared from bovine aortic endothelial cell RNA were synthesized.
  • the primer sequences corresponded to the NADPH-ribose and NADPH-adenine cofactor binding sites. These sites are highly conserved between brain NOS and cytochrome P450 reductase Bredt et al., (1991).
  • PCR products approximately 350-bp in length were purified by agarose-gel electrophoresis and cloned into pGEM7zf (Promega) .
  • the nucleotide sequences of several partial cDNA clones were determined; one of these showed significant amino-acid homology with portions of brain NOS.
  • a restriction fragment prepared from this bovine cDNA was used to screen a ⁇ GTIO cDNA library prepared from lipopolysaccharide-stimulated human umbilical vein endothelial cells, Staunton et al. , Cell 52, 925-933 (1988).
  • cDNA inserts from a hybridizing bacteriophage were subcloned in pUC19. Nucleotide sequence determination of the longest cDNA was performed by the dideoxynucleotide chain termination method using nested deletions. Both sense and antisense DNA strands were sequenced.
  • One of the amplified partial cDNAs was approximately 350-bp in length and encoded a peptide which shared significant amino acid homology with segments of brain NO synthase and a ⁇ GTIO cDNA library prepared from lipopolysaccharide-stimulated human umbilical vein endothelial cells, Staunton et al., (1988) , was screened with this partial cDNA, and hybridizing bacteriophage were isolated. A clone with a 4099-bp insert was thus obtained. It contained a 3609-bp open reading from encoding a 1203-amino acid protein. The first methionine lies within a Kozak consensus sequence for initiation of translation, Kozak, Cell 44, 283-292 (1986) .
  • this endothelial cell protein shares only 52 percent of its amino acid sequence with brain NOS though striking homology is evident at sequences corresponding to flavin mononucleotide (FMN) , flavin adenine dinucleotide (FAD) , and NADPH binding regions, see Fig. 2.
  • FIG. 2 shows the amino acid sequence alignment of endothelial NOS (Seq ID No: 2) with brain NOS (Seq ID No: 3) .
  • the amino acid sequence of human endothelial NOS is shown in upper-case single letter code.
  • the sequence of rat brain NOS is shown in lower case. Residue numbers are indicated on the left with the final residue number portrayed on the bottom right.
  • Endothelial NOS shares 52% of its amino acid sequence with the brain enzyme. Sequences which are highly conserved between the two proteins and which probably represent contact sites for binding of enzyme cofactors, Bredt et al., (1991), are enclosed in boxes, including FMN, FAD-pyrophosphate and-isoalloxazine groups, and NADPH-adenine and-ribose groups.) This degree of homology may account for the recognition of NOS-immunoreactivity in endothelial cells by an antiserum raised against brain NOS, Bredt et al., (1990).
  • the encoded peptide is predicted to be about 133 kDa in size which is smaller than brain NO synthase (predicted molecular weight 160 kDa) and closer to the 135-kDa type III endothelial cell constitutive NO synthase, recently characterized by Pollock et al. , Pollock et al. Proc. Natl. Acad. Sci. U.S.A. 88, 10480-10484 (1991).
  • RNA blot hybridization confirmed that the gene encoding this endothelial NO synthase was expressed in human umbilical vein endothelial cells (HUVEC) as well as human lung, kidney, and spleen, see Fig. 3.
  • Fig. 3 shows the identification of endothelial NOS mRNA by RNA blot hybridization. Expression of the endothelial NOS gene was detected in cultured human umbilical vein endothelial cells and human spleen, lung, and kidney. The mRNA is approximately 4300 nucleotides long. The migration of ribosomal RNA is indicated (28S and 18S) ) .
  • This tissue distribution of expression differs markedly from that of the brain NOS gene, Bredt et al. (1991) .
  • the mRNA encoding the human endothelial gene was approximately 4300-nucleotides in length and much smaller than brain NOS mRNA.
  • the presence of abundant mRNA in unstimulated endothelial cells and its homology to the constitutively expressed brain NOS further suggests that the endothelial cell cDNA encodes a constitutively expressed NOS, Radomski et al., Proc. Natl. Acad. Sci. U.S.A. 87, 10043-10047 (1990).
  • the endothelial cell cDNA was ligated 3' to the SV40 promoter in the pSPORT expression vector and transfected into NIH/3T3 cells (described below) .
  • Bovine pulmonary artery endothelial cells incubated in the presence of NADPH and nitro blue tetrazolium showed small amounts of blue cytoplasmic staining consistent with low levels of NADPH diaphorase activity, Fig. 4A.
  • Control NIH/3T3 cells contained no detectable blue staining, Fig. 4C.
  • transfected cells with high levels of the endothelial cell mRNA showed abundant blue cytoplasmic staining, Fig. 4B.
  • the presence of high levels of NADPH diaphorase activity in transfected cells demonstrated that the expressed cDNA encoded a protein which was functional enzyme with properties similar to brain NOS.
  • the human endothelial NOS cDNA was cloned into the EcoRl site of pSPORT (Bethesda Research Laboratories) .
  • the expression plasmid together with a plasmid encoding neomycin resistance were transfected into NIH/3T3 cells by the calcium-phosphate method, Sambrook Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1989) , hereby incorporated by reference. Following selection in the antibiotic G418, resistant clones containing endothelial NOS mRNA were identified.
  • NADPH diaphorase staining was performed by incubating cells with 1 mH NADPH, 0.2 mM nitro blue tetrazolium, 0.1 M Tris-HCl (pH 7.2), and 0.2% Triton X- 100 for 30 min at 37°C, Dawson et al., Proc. Natl. Acad. Sci. U.S.A. 88:7797-7801 (1991). Cells were photographed at the same magnification under bright-field illumination. A blue cytoplasmic staining pattern is indicative of NADPH diaphorase activity. ECNOS transfected NIH 3T3 release NO To test whether transfected cells release NO, a co-incubation assay, Marsden et al. , Am.
  • RFL-6 cells exposed to coverslips coated with transfected cells contained significantly increased concentrations of cGMP.
  • transfected cells and RFL-6 cells were co-incubated in the presence of the NOS inhibitor, N G -nitro-L-arginine methyl ester (L-NAME) , Rees et al. , Br. J. Pharmacol, 101, 746-752 (1990).
  • the inhibitor significantly decreased the ability of transfected cells to stimulate cGMP production in RFL-6 cells.
  • Fig. 5 shows the functional expression of endothelial NOS enzyme activity.
  • NOS catalytic activity in transfected 3T3 cells was assessed by measuring the cGMP response to nitric oxide in a co- incubation assay using reporter rat lung fibroblasts (RFL-6 cells) .
  • Cellular cGMP content was measured following co-incubation of RFL-6 cells with untransfected NIH/3T3 cells (CONTROL 3T3) and with transfected cells expressing the human endothelial NOS CDNA (hECNOS-TRANSFECTED 3T3) in the absence (UNTREATED) and presence of N G -nitro-L-arginine methyl ester (L-NAME- TREATED) .
  • Data are expressed as means + standard error of six determinations from representative experiment.
  • Rat fetal fibroblasts (RFL-6 cells; American Type Culture Collection #CCL191) were cultured in Ham's F12 medium supplemented with 10% fetal bovine, serum and glutamine in 12 well tissue culture plates. Control and transfected 3T3 cells were grown on coverslips (approximately 10 5 control cells per coverslip and 6xl0 4 transfected cells per coverslip in the representative experiment shown) . RFL-6 cells were pretreated for 10 minutes with 200 U/ml superoxide dismutase and 0.1 mM 3-isobutyl-l-methylxanthine.
  • Coverslips were then juxtaposed to the reporter cell monolayer for 30 min. To inhibit NOS activity, identical wells were treated with 0.5 mM L-NAME for ten minutes — prior to addition of coverslips.
  • Cellular cGMP was extracted in 0.1 M HC1, Beasley et al. , J. Clin. Invest. 87, 602-608 (1991) , and was quantitated by radioimmunoassay after acetylation (Biomedical Technologies Inc.).
  • the peptides of the invention may be administered to a mammal, particularly a human, in one of the traditional modes (e.g., orally, parenterally, transdermally, or transmucosally) , in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels and liposomes or by transgenic modes.
  • the invention includes any protein which is 55% or more homologous to hECNOS (Seq ID No: 2) .
  • the protein is at least 65% or more, more preferably 80% or more, and most preferably 95% or more, homologous to hECNOS.
  • chimeric polypeptides that include an hECNOS.
  • the invention also includes analogs of naturally occurring hECNOS.
  • Analogs can differ from naturally occurring hECNOS by amino acid sequence dif erences or by modifications that do not affect sequence, or by both.
  • Analogs of the invention will generally exhibit at least 70%, more preferably 80%, more preferably 90%, and most preferably 95% or even 99%, homology with all or part of a naturally occurring hECNOS sequence.
  • the length of comparison sequences will generally be at least about 8 amino acid residues, usually at least 20 amino acid residues, more usually at least 24 amino acid residues, typically at least 28 amino acid residues, and preferably more than 35 amino acid residues.
  • Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation derived from cells that normally provide such processing, e.g., mammalian glycosylation enzymes. Also embraced are versions of the same primary amino acid sequence that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • modifications of glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation derived from cells
  • Analogs can differ from naturally occurring hECNOS by alterations of their primary sequence. These include genetic variants, both natural and induced. Induced mutants may be derived by various techniques, including random mutagenesis of the encoding nucleic acids using irradiation or exposure to ethanemethylsulfate (EMS) , or may incorporate Changes produced by site-specific mutagenesis or other techniques of molecular biology. See. Sambrook, Fritsch and Maniatis (1989) , Molecular
  • the invention also includes biologically active fragments of the polypeptides.
  • fragment as applied to a polypeptide, will ordinarily be at least about 10 contiguous amino acids, typically at least about 20 contiguous amino acids, more typically at least about 30 contiguous amino acids, usually at least about 40 contiguous amino acids, preferably at least about 50 contiguous amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length.
  • Fragments of hECNOS can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of hECNOS can be assessed by methods known to those skilled in the art.
  • hECNOS polypeptides containing amino acids that are normally removed during protein processing, including additional amino acids that are not required for the biological activity of the polypeptide, or including additional amino acids that result from alternative mRNA splicing or alternative protein processing events.
  • a hECNOS polypeptide, fragment, or analog is biologically active if it exhibits a biological activity of a naturally occurring hECNOS, e.g., the ability to catalyze the synthesis of NO from L-arginine.
  • Nucleic acids and proteins of the invention can be used to screen treatments, e.g., the administration of compounds, e.g. , the administration of an arginine analog, for isotype-selective NO synthase inhibition.
  • compounds e.g. , the administration of an arginine analog
  • isotype-selective NO synthase inhibition There are numerous isotypes of NO synthase in the body and it can be desirable to inhibit one isotype but not another.
  • macrophage inducible NO synthase is active in sepsis. It would be desirable to inhibit inducible macrophage NO synthase in sepsis patients (and thus increase their blood pressure) without substantially inhibiting endothelial NO synthase.
  • a treatment e.g., a compound
  • a treatment e.g., the administration of a compound, specifically or preferentially inhibits a first isotype if, at a predetermined concentration or dose, it results in a substantial effect on a parameter determined by the activity of the first isotype, e.g., macrophage NO synthase dependent changes in blood pressure in sepsis patients, but does not result in a substantial effect on a parameter determined by the action of the second isotype, e.g., endothelial NO synthase dependent changes in the vascular system.
  • the treatment results in a greater decrease in NO production of the first isotype than in NO production by the second isotype.

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Abstract

Préparation pratiquement pure d'un acide nucléique renfermant une séquence codant la synthase de l'oxyde d'azote endothélial.
PCT/US1993/001951 1992-03-05 1993-03-05 Synthase de l'oxyde d'azote endothelial WO1993018156A1 (fr)

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AU37891/93A AU3789193A (en) 1992-03-05 1993-03-05 Endothelial nitric oxide synthase

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5498539A (en) * 1992-07-02 1996-03-12 Daiichi Pharmaceutical Co., Ltd. Bovine endothelial nitric oxide synthase nucleic acids
EP0702518A4 (fr) * 1993-06-11 1998-07-01 Univ Leland Stanford Junior Traitement des maladies vasculaires degeneratives par modulation de l'activite ou de la production d'oxyde nitrique endogene
WO2000037653A1 (fr) * 1998-12-22 2000-06-29 Board Of Regents, The University Of Texas System Base structurelle de la fonction pterine dans l'oxyde nitrique synthase
US6146887A (en) * 1994-03-31 2000-11-14 Jurgen Schrader DNA expression vectors for use in the gene therapeutic treatment of vascular disorders
US6150500A (en) * 1996-07-12 2000-11-21 Salerno; John C. Activators of endothelial nitric oxide synthase
US6337321B1 (en) 1993-06-11 2002-01-08 The Board Of Trustees Of The Leland Stanford Junior University Enhancement of vascular function by modulation of endogenous nitric oxide production or activity
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US6989164B2 (en) 2000-12-22 2006-01-24 The Daily Wellness Company Method and composition for improving male fertility health
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US6337321B1 (en) 1993-06-11 2002-01-08 The Board Of Trustees Of The Leland Stanford Junior University Enhancement of vascular function by modulation of endogenous nitric oxide production or activity
EP0702518A4 (fr) * 1993-06-11 1998-07-01 Univ Leland Stanford Junior Traitement des maladies vasculaires degeneratives par modulation de l'activite ou de la production d'oxyde nitrique endogene
US7452916B2 (en) 1993-06-11 2008-11-18 The Board Of Trustees Of The Leland Stanford Junior University Enhancement of vascular function by modulation of endogenous nitric oxide production or activity
US6646006B2 (en) 1993-06-11 2003-11-11 The Board Of Trustees Of The Leland Stanford Junior University Enhancement of vascular function by modulation of endogenous nitric oxide production or activity
US6642208B2 (en) 1993-06-11 2003-11-04 The Board Of Trustees Of The Leland Stanford Junior University Enhancement of vascular function by modulation of endogenous nitric oxide production or activity
US6149936A (en) * 1994-03-31 2000-11-21 Joseph Schrader DNA expression vectors for the use in the gene therapeutic treatment of vascular disorders
US6146887A (en) * 1994-03-31 2000-11-14 Jurgen Schrader DNA expression vectors for use in the gene therapeutic treatment of vascular disorders
EP0875581A4 (fr) * 1995-11-13 2004-03-24 Shionogi & Co Diagnostic des maladies associees aux spasmes coronariens
US6150500A (en) * 1996-07-12 2000-11-21 Salerno; John C. Activators of endothelial nitric oxide synthase
US6750197B1 (en) 1996-07-12 2004-06-15 Rensselaer Polytechnic Institute Activators of endothelial nitric oxide synthase
US7229649B2 (en) 1998-04-03 2007-06-12 The Daily Wellness Company Method and composition for improving sexual fitness
WO2000037653A1 (fr) * 1998-12-22 2000-06-29 Board Of Regents, The University Of Texas System Base structurelle de la fonction pterine dans l'oxyde nitrique synthase
US6989164B2 (en) 2000-12-22 2006-01-24 The Daily Wellness Company Method and composition for improving male fertility health
US7045151B2 (en) 2000-12-22 2006-05-16 The Daily Wellness Company Method and composition for improving fertility health in female and male animals and humans
US7557087B2 (en) 2004-03-01 2009-07-07 Lumen Therapeutics, Llc Compositions and methods for treating diseases

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