WO2004047852A1 - Composition pharmaceutique pour maitriser la stabilite de hif-1$g(a) - Google Patents

Composition pharmaceutique pour maitriser la stabilite de hif-1$g(a) Download PDF

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WO2004047852A1
WO2004047852A1 PCT/KR2003/002577 KR0302577W WO2004047852A1 WO 2004047852 A1 WO2004047852 A1 WO 2004047852A1 KR 0302577 W KR0302577 W KR 0302577W WO 2004047852 A1 WO2004047852 A1 WO 2004047852A1
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ardl
hif
protein
pharmaceutical composition
compound
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PCT/KR2003/002577
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Kyu-Won Kim
Joo-Won Jeong
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Kyu-Won Kim
Joo-Won Jeong
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Priority to AU2003284772A priority Critical patent/AU2003284772A1/en
Publication of WO2004047852A1 publication Critical patent/WO2004047852A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91051—Acyltransferases other than aminoacyltransferases (general) (2.3.1)
    • G01N2333/91057—Acyltransferases other than aminoacyltransferases (general) (2.3.1) with definite EC number (2.3.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention in general, relates to the use of arrest-defective protein 1 (ARDl) to modulate the stability of hypoxia-inducible factor- l ⁇ (HIF- l ⁇ ). More particularly, the present invention relates to a pharmaceutical composition for promoting the degradation of HIF-l ⁇ protein, which includes ARDl, a stimulator of ARDl or a nucleic acid molecule to encode ARDl, a method of screening a compound stimulating expression of ARDl, and a yeast two-hybrid system for screening ARDl.
  • ARDl arrest-defective protein 1
  • HIF- l ⁇ hypoxia-inducible factor- l ⁇
  • the present invention is concerned with a pharmaceutical composition for inhibiting the degradation of HIF-1 ⁇ , which includes an inhibitor of ARDl, an antibody specific to ARDl or an antisense to hybridize with a polynucleotide encoding ARDl, and a method of screening a compound inhibiting expression of ARDl.
  • HIF-1 hypoxia-inducible factor 1
  • HIF-1 stimulates the transcription of hypoxia-responsive genes including erythropoietin and NEGF (vascular endothelial growth factor), of which translational products function to increase oxygen availability by promoting erythropoiesis and angiogenesis or activating genes associated with glucose transport and metabolism (Semenza, G.L., Annu. Rev. Cell Dev. Biol. 15, 551-578, 1999).
  • HIF-1 is composed of two subunits: the hypoxia-regulated ⁇ subunit and the hypoxia-insensitive ⁇ subunit (Wang, G.L., et al., Proc. Natl. Acad. Sci. USA 92, 5510-5514, 1995).
  • HIF-1 ⁇ subunit Under normoxic conditions, the HIF-1 ⁇ subunit is rapidly degraded via an ubiquitin-proteasome pathway mediated by the von Hippel-Lindau tumor suppressor gene product (pNHL) (Salceda, S. et al., J. Biol. Chem. 272, 22642-22647, 1997; Huang, L.E., et al., Proc. ⁇ atl. Acad. Sci. USA 95, 7987-7992,
  • pNHL von Hippel-Lindau tumor suppressor gene product
  • HIF-l ⁇ The interaction between HIF-l ⁇ and pNHL under normoxia conditions is initiated by the posttranslational hydroxylation of specific proline residues (Pro402 and Pro564) in a polypeptide segment of HIF-1 ⁇ , known as the oxygen-dependent degradation (ODD) domain, (Ivan, M., et al.,
  • HIF-PHs HIF- prolyl hydroxylases
  • p53 directly interacts with HIF-1 ⁇ and limits the hypoxia-induced expression of HIF-1 ⁇ by stimulating Mdm2 -mediated ubiquination and proteasomal degradation under hypoxic conditions (Ohh, M., et al., Nat. Cell Biol. 2, 423-427, 2000; and Ravi, R., et al., Genes Dev. 14, 34-44, 2000).
  • the degradation of HIF-1 ⁇ by p53 in a hypoxia condition is inhibited by direct interaction with the Jun activation domain binding protein- 1 (Jabl) and the ODD domain by blocking the interaction with p53(Bae, M.K., et al., J. Biol. Chem. 277, 9-12, 2002).
  • HIF-1 activation Phosphorylation and dephosphorylation in specific signaling pathways can result in HIF-1 activation (Richard, D.E., et al., J. Biol. Chem. 274, 22642-22647, 1999).
  • the ⁇ 42/p44 MAPK signaling induces the posttranslational phosphorylation of HIF-1 ⁇ and up-regulates the transcriptional activity of HIF-1 (Richard, D.E., et al., J. Biol. Chem. 274, 32631-32637, 1999).
  • HIF-l the stability and activity of HIF-l are modulated by multiple proteins and several translational modifications.
  • the modulation of HIF- l ⁇ by interaction with other proteins is known to play an important role in its stabilization and activation.
  • the posttranslational prolyl hydroxylation targets HIF-l ⁇ to pNHL, and the pNHL then mediates the ubiquitination and proteasomal degradation of HIF-1 ⁇ .
  • studies associated with posttranslational modification of HIF-1 ⁇ were limited to hydroxylation, ubiquitination and phosphorylation.
  • Acetylation is a type of posttranslational modifications of proteins, such as histone, which is well-known to undergo posttranslational acetylation (Kouzarides, T., EMBO J. 19, 1176-1179, 2000).
  • histone acetyltransferases which include PCAF/GC ⁇ 5, TAF250, p300/CBP, SRC1 and MOZ (Kouzarides, T., Curr. Opin. Genet. Dev. 9, 40-48, 1999), and these enzymes are mainly present in the nucleus.
  • acetyltransferases have been identified to modify other proteins in addition to histone, including general transcriptional factors, such as E2F1, MyoD, GATA-1 and p53 (Kouzarides, T., EMBO J. 19, 1176-1179, 2000; Ogryzok, N.N., et al., Cell 94, 35-
  • the posttranslational acetylation can regulate the DNA-binding activity of general transcriptional factors and their interaction with other proteins, such as costimulator proteins and transcriptional regulators (Bannister, A.J. and Miska, E.A., Cell. Mol. Life Sci. 57, 1184-1192, 2000; and Barlev, N.A., et al.,
  • Nonhistone proteins such as general transcriptional factors and DNA- binding proteins, are identified as substrates for PCAF and/or p300/CBP
  • transcriptional factors include p53 (Gu and Roeder, Cell 90, 595- 606, 1997), GATA-1 (Boyes, J., et al, Nature 396, 594-598, 1998; and Hung, H.L., et al, Mol. Cell. Biol. 19, 3496-3505, 1999), EKLF (Zhang, W. and Bieker, J.J.,
  • EKLF and TCF has been unclear (Bannister, A.J. and Miska, E.A., Cell. Mol. Life Sci. 57, 1184-1192, 2000).
  • ARDl was known to form a homodimer or a heterodimer with NAT1, where the complexes have N-terminal acetyltransferase activity (Park, E.G. and Szostal, J.W., EMBO J. 11, 2087-2093, 1992). Thereafter, in mammalian cells, a human ARDl homologue was detected in all tissues examined (Tribioli, C, et. al., Hum. Mol. Genetics 3, 1061-1067, 1994). However, functions and substrates of the human ARDl homologue have not been established.
  • the present inventors screened proteins interacting with the ODD domain of HIF-1 using a yeast two-hybrid system. DNA sequencing and database searches resulted in the finding that selected clones correspond to a 700-bp mouse cDNA of a mouse homologue for ARDl N-acetyltransferase. That is, the present inventors found that ARDl regulates both induction and degradation of HIF-l ⁇ by binding to HIF-l ⁇ , and identified first that the ARDl protein participates in the control of the cellular levels of HIF-1 in such a way to stimulate degradation of HIF-l ⁇ by acetylation.
  • the present invention based on the finding that ARDl serves as a negative regulator versus HIF-1 ⁇ , relates to a pharmaceutical composition for promoting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of ARDl.
  • the present invention relates to a pharmaceutical composition for promoting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of stimulator of ARD 1.
  • the present invention relates to a pharmaceutical composition for promoting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of nucleic acid molecule encoding ARDl.
  • the present invention relates to a method of screening a compound stimulating expression of ARDl, including culturing a cell expressing the ARDl protein, contacting the cultured cell with a compound to be tested, and comparing an expression level of the ARDl protein in the cell contacted with the test compound with that in a control not contacted with the compound.
  • the present invention relates to a pharmaceutical composition for inhibiting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of inhibitor of ARDl.
  • the present invention relates to a pharmaceutical composition for inhibiting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of antibody specific to ARDl .
  • the present invention relates to a pharmaceutical composition for inhibiting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of antisense to hybridize with a polynucleotide encoding ARDl.
  • the present invention relates to a method of screening a compound inhibiting expression of ARDl, including culturing a cell expressing the ARDl, contacting the cultured cell with a compound to be tested, and comparing an expression level of the ARDl in the cell contacted with the test compound with that in a control not contacted with the compound.
  • the present invention relates to a yeast two-hybrid system for screening a protein interacting with HIF-l ⁇ , including a bait vector to encode a GAL4 DNA -binding domain fused to the HIF-1 ⁇ , a prey vector to encode a GAL4 transactivation domain fused to a candidate protein for interacting with the HIF-l ⁇ , and a his " yeast strain having LacZ and HIS genes and a GAL4 binding region in a transcriptional regulatory region of the genes.
  • the present invention relates to a method of screening a compound stimulating binding of ARDl to HIF-1 ⁇ , including transfecting a his " yeast strain, having LacZ and HIS genes and a GAL4 binding region in a transcriptional regulatory region of the genes, with both a bait vector to encode a GAL4 DNA-bi ⁇ ding domain fused to the HIF-1 ⁇ and a prey vector to encode a GAL4 transactivation domain fused to the ARDl, in the presence of a compound to be tested; and comparing histidine auxotrophy and ⁇ -galactosidase expression in the resulting transfectant with those in a control transfected with the bait and prey vectors in the absence of the test compound.
  • the present invention relates to a method of screening a compound inhibiting binding of ARDl to HIF-l ⁇ , including transfecting a his " yeast strain, having LacZ and HIS genes and a GAL4 binding region in a transcriptional regulatory region of the genes, with both a bait vector to encode a GAL4 DNA-binding domain fused to the HIF-1 ⁇ and a prey vector to encode a GAL4 transactivation domain fused to the ARD 1 , in the presence of a compound to be tested; and comparing histidine auxotrophy and ⁇ -galactosidase expression in the resulting transfectant with those in a control transfected with the bait and prey vectors in the absence of the test compound.
  • FIG. 1A is a schematic representation of structures of full-length HIF-l ⁇ and its deletion derivatives, wherein gray boxes represent the ODD domain (1 : full-length HIF-l ⁇ , 1 to 827 amino acid residues; 2: 1 to 603 amino acid residues;
  • FIG. IB shows a result of GST-pull-down assay for HIF-1 ⁇ and its deletion derivatives, which have been translated by programmed reticulocyte lysates in the presence of [ 35 S]methionine (Nos. 1 to 6 are same as in FIG. 1 A);
  • FIG. IC shows a result of Western blot analysis and its quantitative analysis, indicating that ARDl interacts with HIF-l ⁇ in vivo (IP: immunoprecipitation; N: normoxia, 21% O 2 ; and H: hypoxia, 1% O 2 );
  • FIG. 2 A shows a result of luciferase assay, indicating that ARDl inhibits transcriptional activation mediated by HIF-1 ⁇
  • FIG. 2B shows results of RT-PCR, Western blot analysis and quantitative analysis for NEGF mR ⁇ A levels, indicating that ARDl inhibits NEGF expression
  • FIG. 3A shows results of Western blot analysis, RT-PCR and quantitative analysis for HIF-1 ⁇ protein levels, indicating that ARDl decreases HIF-1 ⁇ stability;
  • FIG. 3B shows a result of immunoblot analysis, indicating that the HIF-l ⁇ protein level decreased by ARDl is restored to the level of hypoxia- treated cells in the presence of a specific inhibitor of the 26S proteasome, MG132;
  • FIG. 3C shows a result of autoradiograph image analysis after pulse-chase experiments, demonstrating the inhibitory effect of ARDl versus HIF-1 ⁇ stability
  • FIG. 3D shows a result of Western blot analysis, indicating that an antisense ARDl inhibits degradation of HIF-l ⁇ under normoxia, and a result of RT-PCR, indicating that endogenous ARDl expression is effectively suppressed by the antisense ARDl
  • FIG. 3E shows results of Western blot analysis and its quantitative analysis, indicating that ARDl increases interaction of HIF-l ⁇ with pNHL and thus can serve as a negative regulator of HIF-l ⁇ stability
  • FIG. 4A shows results of immunoblot analysis and its quantitative analysis, indicating that degradation of HIF-l ⁇ is stimulated by a deacetylase inhibitor
  • FIG. 4B shows results of Western blot analysis and quantitative analysis for acetylated HIF-l ⁇ , indicating that acetylation of HIF-l ⁇ is increased by ARDl;
  • FIG. 4C shows results of Western blot analysis and quantitative analysis for acetylated HIF-1 ⁇ , displaying acetylated HIF-l ⁇ protein levels under normoxia and hypoxia
  • FIG. 4D shows results of autoradiography of SDS-PAGE, Western blot analysis and Coomassie brilliant blue staining, indicating that HIF-l ⁇ serves as a direct substrate for ARDl, and that ARDl functions as a HIF-l ⁇ acetyltransferase by direct interaction with the ODD domain
  • FIG. 5A shows results of Western blot analysis and Coomassie brilliant blue staining, indicating that ARDl specifically acetylates Lys532 in the ODD domain of HIF-1 ⁇ ;
  • FIG. 5B shows a result of MALDI-TOF MS analysis, demonstrating that Lys532 in HIF-l ⁇ is a target site of ARDl;
  • FIG. 5C shows an amino acid sequence alignment of human and mouse
  • FIG. 6 A shows a result of Western blot analysis, indicating that HIF-l ⁇ - K532R mutant is more stable than wild-type HIF-l ⁇ under normoxia
  • FIG. 6B shows results of Western blot analysis and quantitative analysis for interaction of HIF-1 ⁇ with pNHL, indicating that the interaction of pNHL with
  • HIF-l ⁇ -K532R mutant is much weaker than that with wild-type HIF-1 ⁇ ;
  • FIG. 6C shows results of autoradiography of SDS-PAGE and quantitative analysis for interaction of HIF-1 ⁇ with pNHL, indicating that acetylation of Lys532 in HIF-1 ⁇ stimulates degradation of HIF-l ⁇ by- increasing pVHL- mediated ubiquitination;
  • FIG. 6D shows results of autoradiography of SDS-PAGE and quantitative analysis for ubiquitination of HIF-l ⁇ , indicating that acetylation by ARDl stimulates ubiquitination of HIF-l ⁇ ;
  • FIG. 6E shows a result of autoradiography of SDS-PAGE, demonstrating that Lys532 is not an ubiquitination site but an acetylation site
  • FIG. 7 A shows a result of Northern blot analysis, indicating that ARDl expresses in all human tissues including brain, heart, skeletal muscle and liver;
  • FIG. 7B shows a result of RT-PCR, indicating that ARDl expression is inhibited under hypoxia
  • FIG. 7C shows results of Northern blot analysis, acetylation assay
  • FIG. 7D shows a result of Western blot analysis, indicating that ARDl may act on HIF-l ⁇ in the cytosol
  • FIG. 8 shows a nucleotide sequence alignment of mouse, human and yeast
  • FIG. 9 shows a nucleotide sequence alignment of mouse and human ARDl proteins
  • FIG. 10 shows a construct of a fuil-length ARDl expression vector pCMV- Tag- ARDl according to the present invention
  • FIG. 11 shows a construct of a pET-ARDl vector used for in vitro translation and bacterial induction of ARDl
  • FIG. 12 shows a construct of a GST- ARD vector used for in vitro translation and bacterial induction of ARDl
  • FIG. 13 shows a construct of an antisense ARDl vector.
  • HIF-1 hyperoxia-inducible factor- 1
  • HIF-1 levels are very low under normal oxygen levels (normoxia), while being increased under hypoxia.
  • HIF-1 is composed of two subunits of HIF- l ⁇ and HIF-1 ⁇ .
  • normoxia HIF-l ⁇ is hydroxylated at specific proline residues, and pVHL ubiquitinates the prolyl hydroxylated HIF-1 ⁇ for proteasomal degradation.
  • HIF-1 ⁇ exists as a complex with HIF-1 ⁇ , wherein the ⁇ subunit in the complex form is not available for the VHL-mediated degradation.
  • HIF-1 serves as a transcriptional stimulator to improve cellular viability.
  • HIF-1 of which levels are elevated under hypoxia, increases expression levels of enzymes required for anaerobic glycolysis generating energy in the absence of oxygen, and stimulates expression of other hypoxia- responsive genes, including those for proteins (e.g., VEGF) promoting angiogenesis in oxygen-deprived regions, proteins (e.g., erythropoiethin) increasing oxygen delivery in the blood and proteins (e.g., IGF2) improving cellular viability under oxygen deprivation conditions.
  • proteins e.g., VEGF
  • proteins e.g., erythropoiethin
  • IGF2 proteins
  • HIF-1 activates transcription of genes encoding a variety of proteins, including enzymes involved in energy metabolism, which are exemplified by adenylate kinase-3, carbonic anhydrase-9, glucose transporter- 1 and -3 and enzymes carrying out the anaerobic glycolysis; proteins involved in angiogenesis, which are exemplified by ⁇ lB adrenergic receptor, endothelin-1, heme oxygenase-1 (HO-1), nitric oxide synthase-2 (NOS-2), plasminogen stimulator inhibitor- 1 (PAI-1), VEGF, and VEGF receptor FLT-1; proteins involved in erythropoiesis, which are exemplified by ceruloplasmin, erythropoietin, transferrin and transferrin receptor; and proteins associated with cell proliferation and viability, which are exemplified by adrenomedullin, cyclin G2, EPO, heme oxygenase-1,
  • HIF-1 serves as a transcriptional stimulator
  • hypoxia and hypoglycemia caused by blocking of blood vessels supplying oxygen and nutrients (e.g., glucose) to organs or tissues.
  • oxygen and nutrients e.g., glucose
  • cerebral infarction and myocardial infarction as well as cerebral ischemic stroke can be treated by increasing the expression of HIF-1 to enhance energy generation and angiogenesis (Wurzel, J. and Goldman, B.I., N. Engl. J. Med., 343(2): 148-9, 2000).
  • HIF-1 can be used as a therapeutic means for ulcers (Baatar, D. et al., Am. J. Pathol., 161(4):1449-57, 2002) and injuries (Richard, DE., et al., Biochem. Biophys., Res. Commun., 266(3):718-22, 1999).
  • tumor growth can be inhibited by reducing HIF-1 levels to suppress angiogenesis (Semenza, G.L., Trends Mol. Med., 8(4 Suppl):S62- 7, 2002).
  • HIF-1 can be used as a target for therapeutic strategies to inhibit angiogenesis in molecular levels for rheumatoid arthritis (Folkman, J., Nat.
  • ARDl serves as a negative regulator of the HIF-l ⁇ protein.
  • HIF-l ⁇ is acetylated, and this modification is essential for regulating the stability of HIF-1 ⁇ .
  • acetylated levels of HIF-1 ⁇ gradually decrease, while ARDl expression is reduced.
  • ARDl acetylates HIF-l ⁇ by transferring an acetyl group from Ac-Co A to Lys532 residue in the ODD domain of HIF-1 ⁇ .
  • the ODD domain contains sequences responsible for O 2 -dependent ubiquitination of HIF-1 ⁇ (Sutter, C. H. et al., Proc. Natl. Acad. Sci, USA, 97, 47748-47753, 2000).
  • HIF-1 ⁇ is ubiquitinated by pVHL, which is the E3 ubiquitin-protein ligase, and then subjected to proteosomal destruction (Salceda, S. and Caro, J., J. Biol. Chem., 272, 22642-
  • the present inventors found that a K532R mutant of HIF- l ⁇ is not acetylated by ARDl while the mutant protein is stabilized and decreases in the interaction with pVHL under normoxia. Also, aceylation of the ODD domain by ARDl increased the interaction between HIF-l ⁇ and pVHL and stimulated ubiquitination of HIF-l ⁇ .
  • the present inventors found that the HIF-l ⁇ -K532R mutant is ubiquitinated at the same level as wild-type HIF-l while its binding to pVHL is not affected by ARDl. Based on these results, the present inventors suggest that the Lys532 residue of HIF-l ⁇ is the target site for acetylation by ARDl, not an ubiquitination site. Further, the present inventors found that acetylation of the Lys532 residue strongly increases the interaction of HIF-l ⁇ with pVHL and induces pVHL-mediated ubiquitination.
  • ARDl acetylates Lys532 in the ODD domain of HIF-l ⁇ by direct interaction with the ODD domain and plays a central role in HIF-1 ⁇ stability by accelerating HIF-1 ⁇ interaction with pVHL.
  • the present invention provides a pharmaceutical composition for promoting degradation of HIF-l ⁇ , including a pharmaceutically effective amount of ARD 1.
  • ARDl refers to the amino acid sequences of substantially purified ARDl obtained from all tissues or organs of mammals, for example, murine, rabbits, porcine, bovine, horses, and preferably, humans, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • a substantially purified polypepetide having a HIF-1 ⁇ acetyltransferase activity and comprising the amino acid sequence of SEQ ID NO: 1, or its variants or fragments may be used.
  • mouse ARDl (SEQ ID NO: 1), which is one of selected clones binding to HIF-1 ⁇ , has 51% amino acid homology to ARDl acetyltransferase of Saccharomyces cerevisiae (Tribioli, C, et al., Hum. Mol. Genetics 3, .1061-1067, 1994).
  • a substantially purified human ARDl protein having a HIF-l ⁇ acetyltransferase activity and comprising the amino acid sequence of SEQ ID NO: 2, its variants or fragments may be used.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75 % free, and most preferably about 90%) free from other components with which they are naturally associated.
  • a "variant" of ARDl refers to an amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine).
  • amino acids may be grouped into nonpolar (hydrophobic) amino acids (e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, etc.), polar neutral amino acids (e.g., glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc.), positively charged (basic) amino acids (e.g., arginine, lysine, histidine, etc.) and negatively charged (acidic) amino acids (e.g., aspartic acid, glutamic acid, etc.).
  • nonpolar (hydrophobic) amino acids e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, etc.
  • polar neutral amino acids e.g., glycine, serine, threonine,
  • the variant may have "non-conservative" changes (e.g., replacement of glycine with tryptophan).
  • the variant may also include amino acid deletions or insertions, or both.
  • An amino acid variant can be made by inducing site-directed mutagenesis in a nucleotide sequence encoding ARDl using methods commonly used in the art such as cassette mutagenesis and expressing a DNA construct encoding the variant through cell culturing.
  • Such amino acid sequence variant has typically the biological activity functionally identical to its natural form.
  • An amino acid substitution is typically made by the replacement of one base, or may be made by the replacement of two or more bases as long as the resulting variant has the biological activity functionally identical to naturally occurring ARDl.
  • An insertion is typically made by the addition of a consecutive amino acid sequence of about 1 to 20 amino acids, or may be made with a longer sequence.
  • a deletion is typically in the range of about 1 to 30 amino acid residues, or may be in part made in a longer sequence such as the absence of one domain.
  • the variant may be a functional equivalent with the biological activity identical to naturally occurring ARDl, or, if desired, may be selected from variants with altered structural and/or chemical properties.
  • ARDl protein may contain posttranslational modifications including myristylations, phosphorylations, glycosylations and proteolytic cleavages.
  • the ARDl protein may be prepared by direct isolation from mammalian tissues.
  • the isolation and purification of the ARDl protein contained in culture fluids or cell extracts may be achieved by a number of methods generally known in the art, for example, methods using solubility, such as salt precipitation and solvent precipitation, methods using a difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods using a difference in electric charges such as ion exchange chromatography, methods using in a difference in hydrophilic properties such as phase-reversed high performance liquid chromatography, and methods using a difference in pi values such as isoelectric focusing electrophoresis.
  • ARDl may be prepared by polypeptide chemical synthetic methods generally known in the art.
  • a peptide may be synthesized using a known technique such as systematic liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemical synthesis (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, (1997); and A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, (1989)).
  • the preferred method for preparing polypeptides is to use solid phase synthesis.
  • ARDl may be synthesized by a known solid phase method repeating condensation reaction between protected amino acids, with which amino acid synthesis starts from the C-terminus of ARDl and progresses sequentially according to the amino acid sequence of ARDl. After the condensation reaction, a protecting group and a C-terminal amino acid-linked carrier may be removed by a known process such as acid decomposition or amino lysis.
  • a protecting group and a C-terminal amino acid-linked carrier may be removed by a known process such as acid decomposition or amino lysis.
  • ARDl may be prepared by recombinant DNA techniques, for example, by constructing an expression vector carrying a nucleic acid sequence encoding ARDl, expressing the expression vector in a suitable host cell and isolating the ARDl polypeptide from the culture fluid. Genetic engineering approaches for polypeptide preparation are well known in the. art (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Sambrook et al., supra; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif. (1991); and Hitzeman et al., J. Biol. Chem., 255,
  • ARDl gene may be isolated using many techniques known to those skilled in the art.
  • cDNA or expression libraries can be produced by a known method by reverse transcription using mRNA isolated from, for example, mammalian cells or tissues, such as PCR or southern blot analysis. The obtained DNA may be subcloned to a vector such as pBR322, pUC19 or T vector.
  • a DNA sequence encoding ARDl may be cloned to suitable expression vectors, for example, plasmids derived from E.
  • coli e.g., p ⁇ T3A, pBluescript or pUC19
  • plasmids derived from Bacillus suhtilis e.g., pUBHO, pTB5 or pC194
  • bacteriophages e.g., lamda phage
  • animal viruses e.g., retrovirus
  • insect viruses e.g., vaculovirus
  • the resulting recombinant vector is introduced into a suitable host using a standard technique for transformation and phage infection.
  • a standard technique for transformation and phage infection for example, in case of E. coli, calcium chloride precipitation is used (S. N. Cohen, Proc. Natl. Acad. Sci. USA 69:2110, 1972). Transformation of Bacillus species may be carried out according to a known method in the literature [S. Chang, et al., Molecular and General Genetics,
  • Transformation of yeasts may be performed by lithium acetate or spheroplast methods according to a known procedure in the literature [Parent, et al., Yeast, 1 :83-138, 1985]. Transformation of animal cells may be accomplished according to a method, for example, described in the literature [Virology, 52:456, 1973]. Transformation of insect cells by vaculovirus may be performed according to a method described in the literature [Biotechnology, 6:47, 1988]. The transformants are cultivated depending on the host cells used by a standard technique suitable for the cells. For example, in case of culturing E. coli, the cells are grown in LB medium at 30 to 40°C until reaching the stationary phase or saturation.
  • ARDl protein may be extracted, isolated and purified from the culture fluids of the transformants, for example, cultured cells or culture supernatants.
  • the isolation and purification of ARDl protein contained in the culture supernatants or cell extracts may be accomplished according to many methods known in the art, for example, salt precipitation and solvent precipitation, methods using a difference in molecular weights, such as dialysis, ultraf ⁇ ltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods using a difference in electric charges such as ion exchange chromatography, methods using in a difference in hydrophilic properties such as phase-reversed high performance liquid chromatography, and methods using a difference in pi values such as isoelectric focusing electrophoresis.
  • ARDl functions to regulate the both acetylation and degradation of HIF-1 ⁇ by binding to HIF-1 ⁇ .
  • HIF-1 ⁇ is acetylated by the ARDl protein and then degraded, and thereby is reduced in activity.
  • ARD 1 decreases, leading to a decrease in ARDl protein levels, while the acetylation of HIF-l ⁇ by ARDl is reduced, resulting in an increase in HIF-1 ⁇ protein levels.
  • the present invention provides a pharmaceutical composition for promoting degradation of HIF-l ⁇ , including a pharmaceutically effective amount of stimulator of ARD 1.
  • the term "stimulator”, as used herein, refers to a molecule enhancing or maintaining the HIF-l ⁇ acetyltransferase activity of ARDl when binding to the ARDl protein.
  • the stimulator may include proteins, nucleic acids, carbohydrates and all other molecules, which bind to ARDl and regulate the effect of the ARDl protein on HIF-1 ⁇ .
  • the effectiveness of the stimulator may be detected according to a screening procedure including contacting ARDl protein or its equivalents with a sample containing a compound to be tested and detecting an increase in the acetyltransferase activity of ARDl protein.
  • a compound stimulating ARDl expression can be screened by culturing ARDl -expressing cells, contacting the cells with a compound to be tested and comparing expression levels of ARDl in the cells with a control not contacted with the test compound.
  • the present invention provides a method of screening a compound promoting expression of ARDl, including culturing a cell expressing the ARDl, contacting the cultured cell with a compound to be tested, and comparing an expression level of the ARDl in the cell contacted with the test compound with that in a control not contacted with the compound.
  • HIF-l ⁇ may be stimulated by increasing transcription of ARD 1.
  • the present invention provides a pharmaceutical composition for promoting degradation of HIF-l ⁇ , including a pharmaceutically effective amount of nucleic acid molecule encoding ARDl.
  • nucleic acid sequence contained in the pharmaceutical composition of the present invention which encodes ARDl, may be naturally occurring or non-naturally occurring synthetic modified.
  • the nucleic acid sequence may be single-stranded or double-stranded, and may be DNA (genome, cDNA or synthetic) or RNA molecules.
  • the RNA molecules include HnRNA molecules corresponding to DNA molecules " containing introns and mRNA molecules not containing introns. Additional coding or non- coding sequences may be present within the nucleic acid sequence of the present invention.
  • the aforementioned ARDl -encoding nucleic acid sequence may be altered by substitutions, deletions or insertions of one or more nucleotides without inducing a significant change in biological functionality of the resulting protein. This alteration includes alterations of heterogenous to homogenous genes.
  • the nucleic acid sequence encoding ARDl protein of the present invention is provided by a recombinant expression vector, which is prepared by operably linking the nucleic acid sequence to a vector.
  • the vector carrying the nucleic acid molecule encoding ARDl includes plasmids, phages, cosmids and viral vectors.
  • the vector may be self-replicated or incorporated into the host's chromosomal DNA.
  • the ARDl -encoding nucleic acid molecule may be linked to an expression regulatory sequence, such as promoter/enhancer sequences, and other sequences required for transcription, translation or processing.
  • the regulatory sequences indicate constitutive expression of nucleotides, as well as containing tissue-specific regulatory and/or inducible sequences.
  • the design of the expression vector may be determined depending on host cells to be transfected and other factors such as desired expression levels.
  • the expression vectors expressing the ARDl protein contained in the present composition are viral vectors, for example, replication-deficient retrovirus, adenovirus and adenovirus-related virus.
  • the viral vectors should meet the following criteria: (1) a viral vector having a suitable host range should be selected, which is capable of infecting a desired cell; (2) the gene delivered to a cell is capable of being conserved in the cell for a suitable period and being expressed therein; and (3) the vector should be safe to the host.
  • viral vectors usable for gene delivery to cells may include murine leukemia virus (MLV), JC, SV40, polioma, Epstein-barr virus, papillomavirus, vaccinia, poliovirus, Herpes virus, Sindbis virus, Rentivirus and other human and animal viruses.
  • MLV murine leukemia virus
  • JC JC
  • SV40 polioma
  • Epstein-barr virus Epstein-barr virus
  • papillomavirus Epstein-barr virus
  • papillomavirus vaccinia
  • poliovirus Herpes virus
  • Sindbis virus vaccinia
  • Rentivirus Herpes virus
  • other human and animal viruses may include murine leukemia virus (MLV), JC, SV40, polioma, Epstein-barr virus, papillomavirus, vaccinia, poliovirus, Herpes virus, Sindbis virus, Rentivirus and other human and animal viruses.
  • the present pharmaceutical composition including the ARDl protein, ARDl stimulator or ARDl -encoding nucleic acid molecule, as described above, may be useful for treating cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy, etc., by promoting the degradation of HIF-1 ⁇ .
  • an inhibitor of the ARDl protein may be used for inhibiting the activity of ARDl having a HIF-l ⁇ acetyltransferase activity. Therefore, in yet another aspect, the present invention provides a pharmaceutical composition for inhibiting degradation of HIF-l ⁇ , including a pharmaceutically effective amount of inhibitor of ARDl.
  • inhibitor refers to a molecule reducing the amount of the HIF-1 ⁇ acetyltransferase activity of ARDl or the period for which the activity is maintained, when binding to the ARDl protein.
  • the inhibitor may include proteins, nucleic acids, carbohydrates and all other molecules, which bind to ARDl and regulate the effect of the ARDl protein on HIF-1 ⁇ .
  • the effectiveness of the inhibitor may be detected according to a screening procedure including contacting ARDl protein or its equivalents with a sample containing a compound to be tested and detecting an increase in the acetyltransferase activity of
  • a compound inhibiting ARDl expression can be screened by culturing ARDl -expressing cells, contacting the cells with a compound to be tested and comparing expression levels of ARDl in the cells with a control not contacted with the test compound. Therefore, in yet another aspect, the present invention provides a method of screening a compound inhibiting expression of ARDl, including culturing a cell expressing the ARDl protein, contacting the cultured cell with a compound to be tested, and comparing the expression level of the ARDl protein in the cell contacted with the test compound with that in a control not contacted with the compound.
  • the activity of ARDl having a HIF-1 ⁇ acetyltransferase activity may be inhibited by using an antibody specifically binding to the ARDl protein.
  • the present invention provides a pharmaceutical composition for inhibiting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of antibody specific to ARDl .
  • antibody refers to polyclonal or monoclonal antibodies that are capable of forming specific antigen-antibody binding with the ARDl protein, as well as to fragments thereof, such as Fab, F(ab') 2 and Fv fragments.
  • the monoclonal antibody to ARDl may be prepared using any technique providing the production of antibody molecules through continuous cell line culture, for example, but is not limited to, hybridoma techniques, human B-cell hybridoma techniques and EBV-hybridoma techniques (Kohler, G. et al., Nature 256:495-497, 1975; Kozbor, D. et al., J. Immunol. Methods 81 :31-42, 1985; Cote, R. J. et al., Proc. Natl. Acad. Sci. 80:2026-2030, 1983; and Cole, S. P. et al., Mol.
  • chimeric antibodies may be used, which is base on splicing of a mouse antibody gene to a human antibody gene (Morrison, S. L. et al., Proc. Natl. Acad. Sci. 81 :6851-6855, 1984; Neuberger, M. S., et al, Nature 312:604-608, 1984; and Takeda, S. et al., Nature 314:452-454, 1985).
  • ARDl activity may be also inhibited by underexpression of ARDl gene in cells.
  • an antisense of ARDl is introduced into cells, ARDl expression is reduced, which HIF-1 protein levels are elevated.
  • antisense refers to all compositions containing nucleotide sequences that are complementarity to, that is, hybridize with a specific DNA or RNA sequence.
  • antisense strand means a nucleic acid strand that is complementary to the "sense” strand.
  • Antisense molecules include peptide nucleic acids (PNAs) and may be produced by any method including synthesis or transcription.
  • complementary nucleotides Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation.
  • complementary refers to the natural binding of polynucleotides by base-pairing under acceptable conditions of salts and temperature.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A”.
  • antisense molecules are mostly functionable. In particular, when containing sequences adjacent to the promoter region of ARDl, the antisense molecules have higher activity.
  • antisense molecules may include a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 5, which was illustratively used as an antisense molecule in the present invention, and a nucleic acid molecule comprising an antisense nucleic acid sequence for the full-length ARDl represented by SEQ ID NO: 5.
  • the antisense molecules are obtainable by solid phase synthesis using phosphite triester chemistry by growing the nucleotide chain in 3 '-5' direction in that the respective nucleotide is coupled to the first nucleotide which is covalently attached to the solid phase, comprising the steps of cleaving 5'-DMT (dimethoxytrityl) protecting group of the previous nucleotide, adding the respective nucleotide for chain propagation, modifying the phosphite group subsequently cap unreacted 5'-hydroxyl groups and cleaving the oligonucleotide from the solid support, followed by working up the synthesis product.
  • the antisense molecules may be modified as phosphorothioate derivatives. Modifications of the antisense molecules are advantageous since they are not as fast destroyed by endogenous factors when applied as this is valid for naturally occurring nucleotide sequences. In a preferred aspect, the modifications include phosphorothioate modifications.
  • the antisense molecules of the present invention may be used as pharmaceutical compositions in the form of oligonucleotides themselves or a recombinant expression vector constructed by introducing the antisense molecule into a vector. Therefore, in yet another aspect, the present invention provides a pharmaceutical composition for inhibiting degradation of HIF-1 ⁇ , including a pharmaceutically effective amount of antisense to hybridize with a polynucleotide encoding ARDl .
  • the present pharmaceutical composition including the ARDl inhibitor, antibody specific to ARDl or antisense to hybridize with a polynucleotide encoding ARDl, as described above, may be useful for treating ischemic diseases or ulcers by inhibiting the acetylation of HIF-1 ⁇ by ARDl.
  • the ischemic diseases include cerebral ischemic stroke and myocardial infarction.
  • the present pharmaceutical composition may be administered via various administration routes, for example, parenterally, topically, intramucosally and intraperitoneally.
  • the effective amounts of the ingredients contained in the present pharmaceutical composition are example 200 ⁇ g/ml for the ARDl protein, 2 mg/ample for the stimulator, 1 ⁇ g for the recombinant expression vector, 2 mg/ample for the inhibitor, 200 ⁇ g/ml for the antibody, and 1 ⁇ g for the antisense molecule.
  • the dose range may be changed by many factors such as administration methods, target cells and expression levels.
  • the present composition may contain acceptable carriers including all associated physiological solutions.
  • the carriers include dispersive media, coating agents, antibacterial agents, antifungal agents, and isotonic absorption retarders.
  • the yeast two-hybrid system is a genetic system for detection of protein- protein interactions in vivo, for example, using a transcriptional activator such as GAL4 that is composed of a DNA-binding domain and an activation domain.
  • the DNA-binding domain functions to position the transcriptional activator to a specific DNA sequence located at the upstream from a gene to be regulated, while the activation domain functions to bind to factors necessary for transcriptional initiation and thus activate transcription. Therefore, for initiating transcription, the two domains should be juxtaposed on the transcriptional regulatory region.
  • two plasmid systems are employed. One is constructed to have the GAL4 DNA-binding domain fused to a test protein X.
  • the other is constructed to have the GAL4 activation domain fused to another test protein Y or a genomic library.
  • These two plasmids are introduced into a yeast strain containing Laz gene having a GAL4 binding region in its transcriptional regulatory region.
  • the DNA-binding domain and the transactivating domain are juxtaposed on the transcriptional regulatory region, and thus initiate transcription of LacZ ( ⁇ -galactosidase) gene.
  • Media containing X-gal may be used for selecting colonies expressing LacZ.
  • yeast two-hybrid system allows for studying protein-protein interactions in DNA levels and in vivo.
  • the yeast two-hybrid system does not require purified proteins or antibodies, and is highly sensitive in comparison with co-immunoprecipitation and thus can detect weak or transient interactions.
  • the yeast two-hybrid system is a very strong and effective means for finding a new gene through searching genes physically interacting with known genes in genome scale, identifying action mechanisms of genes, finding domains interacting with a specific protein, or studying mutations affecting the interactions.
  • the yeast two-hybrid system cannot be used for some proteins, which are not stably expressed or not transported to the nucleus, and produces many false positive signals. Therefore, there is a need for development of effective screening methods.
  • the his " yeast strain in an embodiment of the present invention, the his " yeast strain
  • HF7C was co-transfected with a bait vector encoding HIF-l ⁇ fused to the GAL4 DNA-binding domain and prey vectors allowing expression of mouse embryonic and T cell lymphoma cDNA sequences and the GAL4 transactivation domain fused to the cDNA sequences.
  • the resulting yeast transformants were screened for histidine auxotrophy and ⁇ -galactosidase expression.
  • the present invention provides a yeast two-hybrid system for screening a protein interacting with HIF-1 ⁇ , including a bait vector to encode a GAL4 DNA-binding domain fused to the HIF-1 ⁇ , a prey vector to encode a GAL4 transactivation domain fused to a candidate protein for interacting with the HIF-1 ⁇ , and a his " yeast strain having LacZ and HIS genes and a GAL4 binding region in a transcriptional regulatory region of the genes.
  • the yeast two-hybrid system may be used for screening the interaction between HIF-1 ⁇ and ARDl, as well as for screening compounds stimulating or inhibiting the interaction between both proteins. Therefore, in yet another aspect, the present invention relates to a method of screening a compound stimulating or inhibiting binding of ARDl to HIF-l ⁇ and, including transfecting a his " yeast strain, having LacZ and HIS genes and a GAL4 binding region in a transcriptional regulatory region of the genes, with both a bait vector to encode a GAL4 DNA-binding domain fused to the HIF-l ⁇ and a prey vector to encode a GAL4 transactivation domain fused to the ARDl, in the presence of a compound to be tested; and comparing histidine auxotrophy and ⁇ - galactosidase expression in the resulting transfectant with those in a control transfected with the bait and prey vectors in the absence of the test compound.
  • Sodium butyrate(NaBu) and trichostatin A(TSA) were purchased from Sigma and MG132 from Calbochem.
  • HIF-l ⁇ antibody was kindly provided by Dr. J. W. Park(College of
  • PVHL, Flag, and GFP antibodies were purchased from Pharmigen, Sigma, and CLONTECH, respectively.
  • P53 and Ac-Lys antibodies were purchased from Upstate
  • Full length ARDl expression vector was constructed by PCR and subcloning into pCMV-Tag(Stratagene). Specifically, mouse cDNA library was used as template, and primers 5 -GAA TTC ATG AAC ATC CGC AAT-3 ' (SEQ ID NO: 7) and 5 -GTC GAC CTA GGA GGC AGA GTC-3' (SEQ ID NO: 8) (the bold correspond to EcoRI and Xhol sites, respectively) including EcoRI and Xhol restriction enzyme sites in both terminals were used.
  • PCR (1 cycle at 94 °C for 5min, 25 cycles at 94 °C for 30 sec, at 45 °C for 40 sec and at 72 °C for 30 sec, 1 cycle at 72 °C for 5min) produced 720bp.
  • PCR products were digested with EcoRI(l unit) and XhoI(l unit) for 3 hrs to obtain cDNA fragments of ARDl (nucleotide 1 - 708).
  • pCMV-Tag2B(Stratagene) vector was digested with EcoRI(l unit) and XhoI(l unit) for 3 hrs.
  • pCMV-Tag- ARD 1 vector(Fig 10) was constructed by reacting I ⁇ l of the digested vector and Ifd of the digested PCR products with 1 ⁇ l of 10 x ligase buffer and 1 ⁇ l of T4 DNA ligase at 20 °C for 2h.
  • PCR products(720bp) obtained in the above were digested with EcoRI(l unit) and XhoI(l unit) and the obtained ARDl cDNA fragments(nucleotide 1 - 308) were inserted in a reverse direction into pCDNA3.1/Myc-His(-)A vector(Invitrogen) with EcoRI(l unit) and XhoI(l unit) in the same manner of the construction of pCMV-Tag-ARDl.
  • pBOS-hHIF-l ⁇ , pBOS-hARNT and pSV40pro- EpoHRE-Luc vectors were kindly provided by Dr. Fujii-Kuriyama(Tohoku University. Japan)(Ema et al., Proc. Natl. Acad. Sci. USA 94, 4273-4278, 1997), and mutated EpoHRE-luc vector was provided by Dr. Eric L. Huang(NCI, National Institute of Health, Bethesda, Maryland, USA).
  • pGBT9-ODD, deletion mutant of HIF-1 ⁇ , and GFP-HIF-l ⁇ vector were prepared as previously described(Bae, M. K., et al., J. Biol. Chem. 277, 9- 12, 2002) .
  • His fusion proteins For the production of His fusion proteins, pET-ARDl and pET-ODD vectors were used and His- ARDl and His-ODD proteins were purified on a Talon metal affinity column(CLONETECH) according to the instruction of the manufacturer. All proteins were filtered using Sephadex G-25 column( Amersham Pharmacia
  • Example 1 Identification of ARDl for HIF-1 ⁇ - Interacting Protein.
  • yeast two-hybrid system was used to identify candidate proteins that interact with HIF-l ⁇ in vivo.
  • Yeast strains for two-hybrid experiments were obtained from CLONTECH as components of the MATCHMAKER two-hybrid
  • the his " yeast strain, HF7C was co- transformed with a bait vector, encoding the GAL4 DNA binding domain in- frame with HIF-1 ⁇ residues 401-603 (ODD domain) (Huang et al., Proc. Natl. Acad. Sci. USA. 95, 7987-7992, 1998) and prey vectors allowing expression of mouse embryonic and T-cell lymphoma cDNA sequences in-frame with GAL4 transactivation domain.
  • the bait did not show any self-activity of transcriptional-activation for the reporters.
  • HIF-l ⁇ constructs used in this assay bound GST alone.
  • HEK 293 cells we co-transfected the Flag-tagged ARDl expression vector and the GFP-HIF-l ⁇ expression vector into HEK 293 cells.
  • HEK293 cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotics in a humidified 5% CO 2 incubator. After 24h post-transfection, the cells were incubated under hypoxia for 6h and the cell extracts were prepared. Total cell lysates were immunoprecipitated with anti- FLAG antibody. The presence of GFP-HIF-l ⁇ in the immunopreciptiates was examined using anti-HIF-l ⁇ antibody (top of Fig IC). Arrowhead at left indicate nonspecific bands.
  • Immunoprecipitated materials and total cell extracts were analyzed by Western blots with anti-Flag antibody and anti-HIF-l ⁇ antibody, respectively (middle, bottom of Fig IC).
  • Preparation of protein extracts, immunoprecipitation, and Western blotting were performed as described previously (Tanimoto, K et al, EMBO. J. 19, 4298-4309, 2000)(Fifty micrograms of protein were blotted onto nitrocellulose filters following SDS-PAGE.
  • Anti- FLAG M2 (Kodak), anti-VHL (PharMingen) or anti-Arnt antibodies were used as primary antibodies, diluted 1 :500 in TBS containing 0.1 %> Tween-20 (TBS-T) and 1% non-fat milk for 1 h. After several washes, a 1 :1000 dilution of anti-mouse Ig- horseradish peroxidase conjugate (Amersham Life Science) in TBS-T buffer containing 1% non-fat milk was used as a secondary antibody and incubated with the sample for 1 h at room temperature. After extensive washing with TBS-T buffer, immunocomplexes were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech).
  • ARDl interacted with HIF-1 ⁇ under both normoxic and hypoxic conditions.
  • the interaction of ARDl with HIF-1 ⁇ under normoxia was set to 100% (right of Fig IC). However, this interaction was decreased when cells were exposed to 1% O 2j in spite of the higher GFP- HIF-l ⁇ expression level under this condition ( Figure IC).
  • Example 2 ARDl Inhibits Transcriptional Activation Mediated by HIF- l ⁇ .
  • HEK293 cells were cotransfected with pSV40pro- EpoHRE-Luc (1 ⁇ g) or mutated EpoHRE-Luc (1 ⁇ g), pBOS-hHIF-l ⁇ (0.1 ⁇ g), pBOS-hARNT (0.1 ⁇ g) or pEF-BOS alone (0.2 ⁇ g) and 2 ⁇ g of pCMV-ARDl or pCMV alone as indicated.
  • Transfected cells were incubated for 24 h at 21% O 2 , and then incubated at 21% ⁇ O 2 or 1% O 2 for an additional 16 h.
  • the luciferase and ⁇ -galactosidase enzyme assays were performed as described (Bae et al., J.
  • HIF-l ⁇ in a dose-dependent manner.
  • luciferase activity was not changed by either hypoxia or the ARDl protein.
  • HT1080 cells were transfected with pCMV-ARDl and selected using G418 (Invitrogen). HT1080 cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotics in a humidified 5% CO 2 incubator. HT1080 cells overexpressing the ARDl protein were designated as HT1080-ARD1 cells. For the hypoxic condition, cells were incubated at a CO level of 5% with 1% O 2 balanced with N 2 using a hypoxic chamber (Forma).
  • HRE hypoxia-response element
  • HT1080 cells were stably transfected with the indicated expression vector and left untreated or exposed to 1% O 2 for 16 h.
  • RT-PCR analysis top two Fig 2B was performed to detect gene expression using specific primer for VEGF and ⁇ -actin.
  • Anti-VEGF and anti- ⁇ -tubulin Western blots from total protein extracts bottom two Fig 2B were performed.
  • the VEGF mRNA expression level was measured in four independent experiments followed by densitometry.
  • the expression under normoxia was set to 100% (right Fig 2B).
  • the increased expressions of VEGF mRNA and protein by hypoxia were downregulated in HT1080-ARD1 cells.
  • ARD1 cells were exposed to 1%> or 21% O 2 for 2 h.
  • HIF-1 ⁇ protein levels and mRNA levels were examined by Western blot analysis (top two of Fig 3A) and
  • RT-PCR analysis bottom of Fig 3 A, respectively.
  • the relative HIF-1 ⁇ protein level from six independent assays was quantified by densitometry.
  • the HIF-1 ⁇ protein level under hypoxia was set to 100% (right of Fig 3 A).
  • the overexpressed ARDl downregulated the protein level of HIF-l ⁇ induced by hypoxia; but it did not affect mRNA level of HIF-1 ⁇ , suggesting that the reduction of the HIF-l ⁇ protein level by ARDl was not due to the transcriptional reduction of the HIF-l ⁇ gene. This suggests that the stability of the HIF-l ⁇ protein is reduced by ARDl protein.
  • HIF-l ⁇ protein in HT1080 and HT1080-ARD1 cells exposed to 1% O 2 for 2 h followed by the addition of 5 ⁇ M MG132 for 1 h as indicated. Cell lysates were harvested and subjected to immunoblot analysis of HIF-l ⁇ .
  • HEK293 cells were transfected with the antisense ARDl expression vector and left untreated or exposed to 1% O 2 for 2 h.
  • HIF-1 ⁇ protein levels by Western blot were examined (top two of Fig 4D) and diminished ARDl mRNA by the antisense ARDl expression vector was checked using RT-PCR (bottom two of Fig 3D).
  • the downregulation of the HIF-1 ⁇ protein level by ARDl under normoxic conditions was undetectable because HIF-l ⁇ is not present in this condition.
  • HIF-l ⁇ stability under normoxia was significantly increased by transfection of the antisense ARDl expression vector.
  • HIF-1 ⁇ stability under hypoxia was increased by the antisense ARDl expression vector.
  • Endogenous pVHL was immunoprecipitated from 5 M MG132 treated HT1080 and HT1080-ARD1 cell extracts with anti-pVHL antibody and coprecipitated HIF-l ⁇ was detected by Western blot analysis. Immunoprecipitated pVHL was visualized by Western blot analysis with anti-pVHL antibody for loading control (lower pane Fig 3E). The relative interaction from three independent experiments was quantified by densitometry.
  • Example 4 ARDl Acetylates the ODD Domain of HIF-l ⁇ .
  • HIF-1 ⁇ can be acetylated by the acetyltransferase activity of ARDl
  • HT1080 cells were exposed to 1% O 2 for 2 h after the addition of 10 mM NaBu or 100 ng/ml TSA for 2 h as indicated. Cell lysates were harvested and subjected to immunoblot analysis of HIF-1 ⁇ .
  • the relative HIF-1 ⁇ protein level from four independent assays was quantified by densitometry.
  • the HIF-1 ⁇ protein level under hypoxia was set to 100% (right Fig 4A).
  • sodium butyrate dramatically decreased the hypoxia-induced HIF- l ⁇ protein level.
  • a histone deacetylase inhibitor, TSA also decreased the expression of HIF- 1 ⁇ protein.
  • 1 ⁇ is stimulated by protein-acetylation.
  • HIF-l ⁇ HT1080 cells were exposed to 5 ⁇ M MG132 or 1% O 2 for 2 h as indicated and cell lysates were immunoprecipitated with anti-Ac-Lys antibody. Cell lysates and immunoprecipitates were subjected to Western blot analysis with anti-HIF-l ⁇ antibody.
  • HIF-l ⁇ could serve as a direct substrate for ARDl and ARDl protein could function as a HIF-1 ⁇ acetyltransferase by direct interaction with the ODD domain.
  • ARDl protein could function as a HIF-1 ⁇ acetyltransferase by direct interaction with the ODD domain.
  • substrates [1 ⁇ g of glutathione-S-transferase (GST)-p53 or histone] were added and incubated with 50 nCi [ 14 C]acetyl-coenzyme A in 30 ⁇ l of reaction buffer (50 mM Tris-HCl pH 8.0, 10% glycerol, 1 mM DTT, 100 ⁇ M EDTA, 1 mM phenylmethyl sulfonyl fluoride) for another 45 min at 37 °C. Acetylation was analyzed by SDS-PAGE followed by autoradiography, or by a phosphoimager.).
  • reaction buffer 50 mM Tris-HCl pH 8.0, 10% glycerol, 1 mM DTT, 100 ⁇ M EDTA, 1 mM phenylmethyl sulfonyl fluoride
  • Example 5 ARDl Acetylates the Lys532 in HIF-l ⁇ Protein.
  • A In general, the position- specific acetylation of a target protein is mediated by distinct acetyltransferases (Gu et al., Cell 90, 595-606, 1997).
  • Site- directed mutagenesies of 6 lysine resiues to arginine in both pET-ODD expression vector and Flag -tagged HIF-1 ⁇ expression vector were performed using the Quick Change site-directed mutagenesis kit (Stratagene).
  • the 6 mutated ODD proteins were purified and an in vitro acetyltransferase assay was executed.
  • Purified recombinant ODD and mutated ODD domain proteins were incubated with acetyl- CoA and ARDl protein for acetylation reaction.
  • In vitro acetylated proteins were subjected to Western blot analysis with anti-Ac-Lys antibody.
  • the in vitro acetylated ODD protein by ARDl protein was purified by immunoprecipitation with Ac-Lys antibody, digested with V8 protease, and then the resulting peptides were analyzed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
  • MALDI-TOF MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • Example 6 ARDl as a HIF-l ⁇ Acetyltransferase Stimulates HIF-1 ⁇ Degradation.
  • Lys532 is the target site for acetylation by ARDl protein, and ARDl decreased the stability of HIF-1 ⁇ .
  • N-TAD N-terminal transactivation domain
  • Lys532 by ARDl is actively involved in the degradation of HIF-l ⁇ under normoxic conditions.
  • Flag-tagged wild-type or mutant HIF- l ⁇ (substitution of Lys532 to Arg) was transiently expressed in HEK293 cells and incubated under normoxic or hypoxic conditions for 6 h.
  • Cell extracts were prepared and subjected to immunoblot analysis with anti-Flag antibody (top two of Fig 6A).
  • HIF-l ⁇ transfected HEK293 cells were pulse labeled with [ S]-methionine and chased in an unlabeled medium for the indicated times under normoxia.
  • Cell lysates were immunoprecipitated with anti-Flag antibody.
  • the intensities of Flag-HIF-l ⁇ signals from three independent experiments were quantified and plotted.
  • Figure 6A the transfected HIF-l ⁇ -K532R protein was stable in normoxia, whereas wild-type HIF-l ⁇ was degraded under the same condition.
  • HIF-1 ⁇ -K532R protein To determine the half-life of the HIF-1 ⁇ -K532R protein, we performed pulse-chase experiments using transiently transfected HEK293 cells under normoxia. After transfection of HEK293 cells with the indicated vectors, 5 ⁇ M MG132 was added for 2 h and total cell extracts were isolated. Endogenous pVHL was immunoprecipitated and coprecipitated HIF-1 ⁇ was detected by Western blot analysis. The relative interaction from three independent experiments was quantified by densitometry. The interaction of wild-type HIF-1 ⁇ with pVHL was set to 100% (bottom of Fig 6B).
  • HIF-l ⁇ -K532R protein is more stable than wild- type HIF-1 ⁇ protein under normoxia ( Figure 6A). Moreover, the interaction of pVHL with HIF-1 ⁇ -K532R protein was much weaker than that with wild-type protein ( Figure 6B). These results suggested that HIF- l ⁇ -K532R protein is stable under normoxic conditions when pVHL interaction is reduced.
  • Example 7 ARDl Expression is Decreased under Hypoxic Conditions.
  • A. The EST data from UniGene Cluster Mm. 5934 indicated that mouse ARDl was expressed in almost every cell type.
  • To confirm the expression of ARDl mRNA we performed Northern blot analysis of RNAs from various human tissues. The expression of hARDl mRNA was detected by Northern blot analysis using human multiple-tissue blot. A human ⁇ -actin probe was used as a loading control. As shown in Figure 7 A, ARDl was detected in all tissues tested, including brain, heart, skeletal muscle, and liver, suggesting that ARDl is broadly expressed.
  • B. To examine the expression level of ARDl during hypoxia, we performed reverse transcriptase PCR.
  • HT1080 cells were incubated for 2 h under hypoxic conditions or treated with 100 M cobalt chloride and 100 M 2, 2'- dipyridyl.
  • RT-PCR analysis was performed using specific primers for ARDl and ⁇ -actin.
  • the ARDl mRNA level was decreased in cells exposed to 1% O , and was also decreased when cells were treated with hypoxia mimicking agents, cobalt chloride and 2, 2'-dipyridyl ( Figure 7B).
  • ARDl mRNA level decreased upon exposure to hypoxia for 2 and 4 hrs.
  • the acetylation of HIF-1 ⁇ was also decreased in proportion to the increase of hypoxic exposure, whereas the protein level of HIF-l ⁇ was increased.
  • ARDl protein was detected only in the cytosolic fraction, not in the nucleus ( Figure 7D), indicating that ARDl may act on HIF-1 ⁇ in the cytosol.
  • composition comprising ARDl protein(Arrest-defective Protein 1), nucleic acid encoding ARDl or stimulator of ARDl is useful for treating cancer, rheumatoid arthritis, psoriasis or diabetic retinopathy by promoting degradation of HIF-l ⁇ and composition comprising inhibitor of ARDl, antibody specific to ARDl, or antisense to hybridize with a polynucleotide encoding ARDl is useful for treating an ischemic disease, especially a cerebral ischemic stroke or a myocardial infarction, or an ulcer by inhibiting degradation of HIF-l ⁇ .

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Abstract

L'invention concerne une composition pharmaceutique utilisée pour promouvoir la dégradation du facteur sensible à l'hypoxie HIF-1α, laquelle comprend la protéine ARD1, un stimulateur de ARD1 ou une molécule d'acide nucléique codant la protéine ARD1. Elle concerne également un procédé de criblage d'un composé stimulant l'expression de ARD1, ainsi qu'un système à deux hybrides de levure pour le criblage d'une protéine interagissant avec le HIF-1α. L'invention concerne en outre une composition pharmaceutique utilisée pour inhiber la dégradation du HIF-1α, laquelle comprend un inhibiteur de ARD1, un anticorps spécifique à ARD1 s'hybridant avec un polynucléotide codant la protéine ARD1, ainsi qu'une procédé de criblage d'un composé inhibant l'expression de ARD1.
PCT/KR2003/002577 2002-11-26 2003-11-26 Composition pharmaceutique pour maitriser la stabilite de hif-1$g(a) WO2004047852A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101514230B (zh) * 2008-02-18 2011-08-10 北京市肿瘤防治研究所 Ard1的单克隆抗体及其应用

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5795724A (en) * 1997-09-12 1998-08-18 Incyte Pharmaceuticals, Inc. Human N-acetyl transferase

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US5795724A (en) * 1997-09-12 1998-08-18 Incyte Pharmaceuticals, Inc. Human N-acetyl transferase

Non-Patent Citations (4)

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GL SEMENZAI: "HIF-1 and mechanisms of hypoxia sensing", CURRENT OPINION CELL BIOLOGY, vol. 13, 2001, pages 167 - 171, XP002416408, DOI: doi:10.1016/S0955-0674(00)00194-0 *
KW KIM ET AL: "Angiogenesis", KOREA SOCIETY OF MEDICAL BIOCHEMISTRY AND MOLECULAR BIOLOGY NEWS, vol. 9, no. 3, 30 September 2002 (2002-09-30), pages 9 - 12 *
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PH MAXWELL: "Oxygen homeostasis and cancer: insights from a rare disease", CLINICAL MEDICINE, vol. 2, no. 4, August 2002 (2002-08-01), pages 356 - 362, XP002681349, DOI: doi:10.7861/clinmedicine.2-4-356 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101514230B (zh) * 2008-02-18 2011-08-10 北京市肿瘤防治研究所 Ard1的单克隆抗体及其应用

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