Assay for identifying a modulator of HIF hydroxylase
View Patent ↗A novel class of hydroxylases is described having the amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, and variants and fragments thereof having HIF hydroxylation activity. The polypeptides of the invention have in particular prolyl hydroxylase activity. An assay method monitors the interaction of the HIF hydroxylase with a substrate. Modulators of HIF hydroxylase are provided for use in the treatment of a condition associated with increased or decreased HIF levels or activity or for the treatment of a condition where it is desirable to modulate HIF levels or activity.
1. An assay method for identifying an agent which modulates the interaction of a hypoxia inducible factor (HIF) hydroxylase with a substrate of the hydroxylase, the method comprising:
contacting a HIF hydroxylase and a substrate of the hydroxylase under conditions in which the hydroxylase interacts with the substrate, in the presence or absence of a test substance; and
determining the interaction, or lack of interaction of, the hydroxylase and the substrate, wherein the interaction of the hydroxylase with the substrate in the presence or absence of the test substance is determined by measuring the hydroxylase activity of the hydroxylase;
wherein the HIF hydroxylase is chosen from:
(a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, FLJ21620 (BAB15101) or Clorf12 (NP071334);
(b) a variant thereof having at least 60% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having HIF hydroxylase activity; or
(c) a fragment of either (a) or (b) having HIF hydroxylase activity.
2. An assay method according to claim 1 , wherein the hydroxylase and the test substance are contacted in the presence of 2-oxoglutarate and the hydroxylase activity is determined by measuring the turnover of said 2-oxoglutarate to succinate and CO 2 .
3. An assay method according to claim 1 , wherein the hydroxylase activity is determined by measuring the hydroxylation of one or more proline residues in the substrate.
4. The assay method of claim 1 , wherein the HIF hydroxylase is a human HIF hydroxylase.