Assays for modulators of asparaginyl hydroxylase

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase

Reexamination Certificate

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Reexamination Certificate

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07544480

ABSTRACT:
A method of identifying an agent which modulates hydroxylation of hypoxia inducible factor (HIF), comprises contacting a HIF asparagine hydroxylase and a test substance in the presence of a substrate of the hydroxylase under conditions in which asparagine in the substrate is hydroxylated in the absence of the test substance; and determining hydroxylation of the substrate. Preferably the substrate is a HIF polypeptide comprising HIF-1α, a fragment thereof comprising Asn 803 of HIF-1α or a peptide analogue of HIF-1α or fragment thereof comprising an asparagine equivalent to Asn 803 of HIF-1α and wherein hydroxylation of Asn 803 or of a said equivalent asparagine is determined.

REFERENCES:
patent: 2006/0040265 (2006-02-01), Flamme et al.
patent: WO 00/74725 (2000-12-01), None
patent: WO 03/025013 (2003-03-01), None
Lando et al. Asparagine Hydroxylation of the HIF Transactivation Domain: A Hypoxic Switch; Science, vol. 295 (2002) pp. 858-861.
Seffernick et al. Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different; Journal of Bacteriology, vol. 183, No. 8 (2001) pp. 2405-2410.
Wells, J.A. Additivity of Mutational Effects in Proteins; Biochemistry, vol. 29, No. 37 (1990) pp. 8509-8517.
Lando, D. et al., “Asparagine Hydroxylation of HIF Transactivation Domain: A Hypoxic Switch”, Science, Vo. 295, No. 5556, pp. 858-861, (Feb. 1, 2002).
Lando, D. et al., “FIH-1 is an Asparaginyl Hydroxylase Enzyme that Regulates the Transcriptional Activity of Hpoxia-Inducible Factor”, Genes & Development, vol. 16, No. 12, pp. 1466-1471, (Jun. 15, 2002).
Hewitson, K. S. et al., “Hypoxia-Inducible Factor (HIF) Asparagine Hydroxylase is Identical to Factor Inhibiting HIF (FIH) and is Related to the Cupin Structural Family” The Journal of Biological Chemistry, vol. 27, No. 29, pp. 26351-26355, (Jul. 19, 2002).
McNeill, L. A. et al., “Hypoxia-Inducible Factor Asparaginyl Hydroxylase (FIH-1) Catalyses Hydroxylation at the β-Carbon of Asparagine-803”, Biochemical Journal, Vo. 367, No. 3, pp. 571-575, (2002).
Mahon, P. C. et al., “FIH-1: A Novel Protein that Interacts with HIF-1α and VHL to Mediate Repression of HIF-1 Transcriptional Activity”.
Freedman, S. et al., “Structural Basis for Recruitment of CBP/p300 by Hypoxia-Inducible Factor-1α”, Proc. Natl. Acad. Sci. USA, vol. 99(8), Apr. 2002, pp. 5367-5372.
U.K. Patent Office Search Report for Application No. GB 0211920.4.
Lando, D. et al., “Asparagine Hydroxylation of HIF Transactivation Domain: A Hypoxic Switch”, Science, Vo. 295, No. 5556, pp. 858-861, (Feb. 1, 2002).
Lando, D. et al., “FIH-1 is an Asparaginyl Hydroxylase Enzyme that Regulates the Transcriptional Activity of Hpoxia-Inducible Factor”, Genes & Development, vol. 16, No. 12, pp. 1466-1471, (Jun. 15, 2002).
Hewitson, K. S. et al., “Hypoxia-Inducible Factor (HIF) Asparagine Hydroxylase is Identical to Factor Inhibiting HIF (FIH) and is Related to the Cupin Structural Family”, The Journal of Biological Chemistry, vol. 27, No. 29, pp. 26351-26355, (Jul. 19, 2002).
McNeill, L. A. et al., “Hypoxia-Inducible Factor Asparaginyl Hydroxylase (FIH-1) Catalyses Hydroxylation at the β-Carbon of Asparagine-803”, Biochemical Journal, Vo. 367, No. 3, pp. 571-575, (2002).
Mahon, P. C. et al., “FIH-1: A Novel Protein that Interacts with HIF-1α and VHL to Mediate Repression of HIF-1 Transcriptional Activity”, (2001).
Freedman, S. et al., “Structural Basis for Recruitment of CBP/p300 by Hypoxia-Inducible Factor-1α”, Proc. Natl. Acad. Sci. USA, vol. 99(8), Apr. 2002, pp. 5367-5372.
U.K. Patent Office Search Report for Application No. GB 0211920.4, (Dec. 14, 2000).

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