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WO1994024269A1 - Sequence adn codant une synthase d'oxyde nitrique - Google Patents

Sequence adn codant une synthase d'oxyde nitrique Download PDF

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Publication number
WO1994024269A1
WO1994024269A1 PCT/DK1994/000146 DK9400146W WO9424269A1 WO 1994024269 A1 WO1994024269 A1 WO 1994024269A1 DK 9400146 W DK9400146 W DK 9400146W WO 9424269 A1 WO9424269 A1 WO 9424269A1
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WIPO (PCT)
Prior art keywords
inos
dna
nitric oxide
dna sequence
sequence
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PCT/DK1994/000146
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English (en)
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Allan Ertmann Karlsen
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Novo Nordisk A/S
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Publication of WO1994024269A1 publication Critical patent/WO1994024269A1/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)

Definitions

  • the present invention relates to a DNA construct comprising a DNA sequence encoding a nitric oxide synthase, a method of producing the nitric oxide synthase, a method of using the nitric oxide synthase to screen for inhibitors of nitric oxide synthase, and a test kit for use in the method.
  • IL-1 ⁇ acts on ⁇ -cells via IL-1 receptors, but the signal transduction mechanism is unknown.
  • Important post-receptor events associated with the inhibitory action of IL-1/3 on ⁇ -cells include a rapid increase in cytosolic Na + , protease activation, de novo protein synthesis, impaired mitrochondrial glucose oxidation and the induction of intracellular free oxygen and nitric oxide radicals (T. Mandrup-Poulsen et al.,
  • Nitric oxide is synthesized by the enzyme nitric oxide synthase (NOS) which converts L-arginine to citrulline and NO.
  • NOS nitric oxide synthase
  • Initial chracterization of NO synthases from different cell types suggests that two distinct forms exist: a constitutively expressed Ca 2+ /calmodulin-dependent form and a cytokine-inducible, calmodulin-independent form.
  • Constitutive production of nanomolar amounts of NO by endothelial cells appears to be vital to the regulation of homeostasis. Additionally, constitutive production of NO is critical for signal transduction in the central nervous system.
  • NOS NOS
  • monocytes monocytes
  • liver etc. The inducible form of NOS (iNOS) found in macrophages, monocytes, liver etc., produces micromolar amounts of NO which are likely to contribute to local tissue damage and systemic hypertension which accompanies septic shock as well as other inflammatory disorders. These two forms exhibit differences in regulation of expression, cofactor dependence, tissue distribution and subcellular localization.
  • pancreatic islet cells can be induced by cytokines to produce nitric oxide synthase suggest that a specific form of nitric oxide synthase involved in ⁇ -cell destruction is found in islets. The inhibition of this nitric oxide synthase may therefore be desirable to prevent or at least delay the onset of IDDM. It is the object of the present invention to prepare a pancreatic islet cell cytokine inducible nitric oxide synthase to be used in the screening for substances which act as inhibitors of the nitric oxide synthase.
  • the present invention relates to a DNA construct comprising a DNA sequence encoding a pancreatic islet cell inducible nitric oxide synthase (iNOS).
  • iNOS pancreatic islet cell inducible nitric oxide synthase
  • the present invention relates to a method of isolating inhibitors of pancreatic islet cell inducible nitric oxide synthase, the method comprising incubating the iNOS encoded by said DNA sequence with a substance suspected of being an iNOS inhibitor in the presence of a suitable substrate for iNOS, and detecting any effect of said substance on the interaction of the iNOS with said substrate.
  • the term "inhibitor” is intended to indicate a substance which inhibits the catalytic activity of the enzyme to convert L-arginine to citrulline and nitric oxide. It is the object of the present invention to isolate an inhibitor which is capable of specifically inhibiting the iNOS produced by pancreatic islets so that on administration, it will not interfere with the various essential functions of NO synthase elsewhere in the body.
  • the present invention relates to a test kit for isolating inhibitors of pancreatic islet cell inducible nitric oxide synthase, the kit comprising in separate containers (a) iNOS encoded by the DNA sequence according to any of claims 1-6, and
  • the DNA construct of the invention encoding the iNOS may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the iNOS by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).
  • the DNA sequence encoding the iNOS is preferably of mammalian origin, i.e. derived from a mammalian pancreatic islet genomic DNA or cDNA library.
  • the DNA sequence may be of rodent origin, e.g. rat or mouse origin, or of human origin.
  • the DNA construct of the invention encoding the iNOS may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • the DNA construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire DNA construct, in accordance with standard techniques.
  • the DNA construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
  • the DNA construct of the invention comprises the following partial DNA sequence
  • CCATGGAGCA CCCAAAGTAC GAATGGTTCC AAA (SEQ ID NO:1) or a homologue thereof encoding a protein with iNOS activity.
  • the partial DNA sequence shown above has been isolated from a rat islet cDNA library as described below. The sequence has been found to be significantly homologous to the previously published (C.R. Lyons et al., J. Biol. Chem. 267, 1992, pp. 6370-6374) mouse macrophage sequence (cf. Fig. 1A and 1B).
  • mouse macrophage iNOS is inducible by lipopolysaccharide and interferon- ⁇
  • iNOS produced in pancreatic islets is inducible by interleukin-1 (IL-1) (rat) or a mixture of IL-1, tumour necrosis factor ⁇ (TNF- ⁇ ) and interferon- ⁇ (IFN- ⁇ ) (human), indicating that significant differences may exist between the mouse macrophage iNOS and the rat islet iNOS.
  • IL-1 interleukin-1
  • TNF- ⁇ tumour necrosis factor ⁇
  • IFN- ⁇ interferon- ⁇
  • the term "homologue” is intended to indicate a natural variant of the DNA sequence encoding rat islet iNOS, such as a variant produced in pancreatic islets of another species, in particular in human pancreatic islets, or a variant produced by modification of the DNA sequence shown above.
  • suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid sequence of the iNOS but which may correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure.
  • any protein produced from such a homologous DNA sequence should exhibit an iNOS activity (e.g. with respect to substrate specificity) similar to that of the native iNOS.
  • the invention relates to the full-length rat islet iNOS shown in the Sequence Listing as SEQ ID NO: 6, or a suitable modification thereof, as defined above.
  • the present invention relates to a DNA sequence encoding human islet iNOS, comprising the partial DNA sequences shown in the Sequence Listing as SEQ ID NO: 9 and SEQ ID NO: 10, or a suitable modification thereof, as defined above.
  • the present invention relates to a recombinant expression vector comprising a DNA construct of the invention.
  • the recombinant expression vector into which the DNA construct of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • the DNA sequence encoding the iNOS should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA encoding the iNOS in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al . , Science 222 ( 1983 ) , 809 - 814 ) or the adeno-virus 2 major late promoter.
  • the DNA sequence encoding the iNOS may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) terminators.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • fungal cells may be used as host cells of the invention.
  • suitable yeasts cells include cells of Saccharomyces spp. or Schizo-saccharomyces spp., in particular strains of Saccharomyces cerevisiae.
  • Other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277.
  • the iNOS may then be produced by a method which comprises culturing a cell as described above in a suitable nutrient medium under conditions which are conducive to the expression of the iNOS and recovering the resulting iNOS from the culture.
  • the medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the inhibitory activity of a suspected iNOS inhibitor may be determined by measuring the amount of L-arginine or citrulline after incubation, compared to a control which does not contain any suspected iNOS inhibitor.
  • a substatially unchanged amount of L-arginine in the incubation mixture is indicative of the presence of an inhibitor, as is a decreased amount or absence of citrulline.
  • the formation of nitric oxide (NO) resulting from the incubation may be determined, decreased NO formation indicating the presence of an iNOS inhibitor.
  • the formation of NO may be measured indirectly by adding an indicator.
  • an indicator may, for instance, be guanylate cyclase which is strongly activated by NO to produce cyclic GMP from GTP.
  • the formation of NO is determined by measuring the amount of cyclic GMP formed on incubation (formation of cyclic GMP may for instance be measured as described in J.A. Corbett et al., Biochem. J. 287, 1992, pp. 229-235).
  • NO formation may also be determined by measuring the amount of nitrite formed on incubation (NO is converted to nitrite in the presence of free oxygen) (e.g. as described by L.C. Green et al., Anal. Biochem. 126, 1982, pp. 131-138).
  • Rat islets were isolated from newborn rats after collagenase digestion of rat pancreases (as described by J. Brunstedt et al., in Methods in Diabetes Research, vol. 1 (Laboratory Methods, Part C), J. Lamer and S.J. Pohl (eds.), Wiley & Sons, New York, 1984, pp. 254-288). After isolation, the islets were precultured for 3-7 days in RPMI 1640 + 10% fetal calf serum at 37°C.
  • the islets were then incubated with 150 U/ml recombinant IL-1 ⁇ (prepared by Novo Nordisk A/S) for 48 hours in a medium containing 150 islets/300 ⁇ l of RPMI 1640 + 0.5% normal human serum. Control islets were cultured similarly in a medium which did not contain any IL-1.
  • the culture media were collected and analysed for nitrite production by mixing 150 ml of medium with an equal volume of Griess reagent (1 part of 0.1% naphthylethylene diamine dihydrochloride, 1 part of 1% sulfanilamide in 5% H 3 PO 4 ) and incubated for 10 minutes at room temperature (L.C. Green et al., Anal. Biochem. 126, 1982, pp. 131-138).
  • the absorbance at 550 nm was determined on an immuno reader (NIPPON InterMed KK, Tokyo, Japan) against a sodium nitrite standard curve.
  • the resulting clones were sequenced by the method described by Tabor and Richardson, Proc. Natl. Acad. Sci. USA 84, 1987, pp. 4767-4771 by means of the Sequenase kit (available from US Biochemical Corp.).
  • the resulting sequence showed a high degree of homology to the previously published mouse macrophage iNOS sequence (cf. Fig. 1A and IB, wherein RATBCL indicates the partial rat islet iNOS sequence of the invention, and MUSMAC indicates the corresponding mouse macrophage iNOS sequence).
  • Islets were isolated from 3-6 days old Wistar rats (M ⁇ llegaard, Lille Skensved, Denmark) following collagenase digestion of the pancreas as described by Brunstedt J, Nielsen JH, Lernmark A, and The Hagedorn Study Group (1984) Isolation of islets from mice and rats. In: Lamer J, Pohl SL, ed. Methods in diabetes research, (Laboratory methods, part C). New York: Wiley & Sons, 254-288. vol 1).
  • a partial (1 kb) rat islet cDNA was first cloned by standard RT-PCR using the primer 5'CCAAGCTTGCCGCCACCATGGCTTGCCCCTGG (SEQ ID NO: 3) in conjunction with degenerate primers 3'TG(GA)AACCA(CT)TC(GA)TA(CT)T(TG) (GT)GG(GA)TG(CT)TCCAT spanning the bases 256-1254 of the mouse macrophage iNOS cDNA (Xie Q, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, Nathan C (1992) Cloning and characterization of inducible nitric oxide synthase from mouse macrophages.
  • the cloned cDNA were sequenced using the automatic laser fluorescence DNA sequencer (ALF ##) and the sequences were analyzed using the software package from the University of Wisconsin Genetic Computer Group (Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12:387-396).
  • ALF automatic laser fluorescence DNA sequencer
  • the coding region was subcloned into the Hindlll and Notl sites of the pcDNA3 vector (Invitrogen) and expressed under control of the CMV promoter in the human embryonic kidney cell line 293 (publicly available from the American Type Culture collection as ATCC CRL 1573) following standard calciumphosphate transfection.
  • CTCTAGACCT CAACAAAGCT CTCAGCAGCA TCCACGCCAA GAACGTGTTC ACCATGAGGC 2220

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Abstract

Une séquence ADN codant une synthèse d'oxyde nitrique inductible (iNOS) de cellule d'îlot pancréatique, est utilisée pour préparer une iNOS d'îlot pancréatique recombinante. L'iNOS peut être utilisée dans un test d'identification des inhibiteurs de l'iNOS pancréatique en présence d'un substrat.
PCT/DK1994/000146 1993-04-16 1994-04-11 Sequence adn codant une synthase d'oxyde nitrique WO1994024269A1 (fr)

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DK0433/93 1993-04-16

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4411402A1 (de) * 1994-03-31 1995-10-05 Juergen Schrader DNA-Expressionsvektoren zur Verwendung in der gentherapeutischen Behandlung von Gefäßerkrankungen
WO1996040715A1 (fr) * 1995-06-07 1996-12-19 University Of Nebraska Board Of Regents Oligonucleotides therapeutiques ciblant les genes mdr1 et mrp chez l'homme
US5744340A (en) * 1995-06-09 1998-04-28 Schering Corporation Expression of human inducible nitric oxide synthase
US5830461A (en) * 1992-11-25 1998-11-03 University Of Pittsburgh Of The Commonwealth System Of Higher Education Methods for promoting wound healing and treating transplant-associated vasculopathy

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 154, Dialog Accession No. 08193824, CORBETT JA et al., "Does Nitric Oxide Mediate Autoimmune Destruction of Beta-Cells? Possible Therapeutic Interventions in IDDM"; & DIABETES (UNITED STATES), Aug 1992, 41 (8), p. 897-903. *
DIALOG INFORMATION SERVICES, File 154, Medline, Dialog Accession No. 08072618, Medline Accession No. 92210618, LYONS CR et al., "Molecular Cloning and Functional Expression of an Inducible Nitric Oxide Synthase from a Murine Macrophage Cell Line"; & J BIOL CHEM (UNITED STATES), 25-03-1992, 267 (9), p. 6370-4. *
DIALOG INFORMATION SERVICES, File 154, Medline, Dialog Accession No. 08481721, Medline Accession No. 93191721, NUNOKAWA Y et al.: "Cloning of Inducible Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells"; & BIOCHEM BIOPHYS RES COMMUN (UNITED STATES), 26 feb 1993, 191 (1), p 89-94. *
PROC. NATL. ACAD. SCI., Volume 86, July 1989, RICHARD G. KNOWLES et al., "Formation of Nitric Oxide from L-Arginine in the Central Nervous System: A Transduction Mechanism for Stimulation of the Soluble Guanylate Cyclase", page 5159 - page 5162. *
SCIENCE, Volume 255, 1992, HARALD H.H.W. SCHMIDT et al., "Insulin Secretion from Pancreatic B Cells Caused by L-Arginine-Derived Nitrogen Oxides", page 721 - page 723. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5830461A (en) * 1992-11-25 1998-11-03 University Of Pittsburgh Of The Commonwealth System Of Higher Education Methods for promoting wound healing and treating transplant-associated vasculopathy
US6103230A (en) * 1992-11-25 2000-08-15 University Of Pittsburgh Of The Commonwealth System Of Higher Education Methods for promoting wound healing and treating transplant-associated vasculopathy
DE4411402A1 (de) * 1994-03-31 1995-10-05 Juergen Schrader DNA-Expressionsvektoren zur Verwendung in der gentherapeutischen Behandlung von Gefäßerkrankungen
WO1995027070A1 (fr) * 1994-03-31 1995-10-12 Schrader Juergen Vecteurs d'expression a adn pour l'utilisation dans le traitement par therapie genique de maladies vasculaires
US6146887A (en) * 1994-03-31 2000-11-14 Jurgen Schrader DNA expression vectors for use in the gene therapeutic treatment of vascular disorders
US6149936A (en) * 1994-03-31 2000-11-21 Joseph Schrader DNA expression vectors for the use in the gene therapeutic treatment of vascular disorders
WO1996040715A1 (fr) * 1995-06-07 1996-12-19 University Of Nebraska Board Of Regents Oligonucleotides therapeutiques ciblant les genes mdr1 et mrp chez l'homme
US5744340A (en) * 1995-06-09 1998-04-28 Schering Corporation Expression of human inducible nitric oxide synthase

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