Assays, methods and means
AssigneeIsis Innovation Limited
InventorsPatrick Henry Maxwell, Christopher William Pugh, Peter John Ratcliffe, Christopher Joseph Schofield
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Abstract
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 IIIF hydroxylase with a substrate. Modulators of IIIF 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.
Claims45
1-55. (canceled)
A method of treatment of a condition where a modulation in HIF levels is desirable by administering a substance that inhibits the hydroxylation of one or more proline residues of a human HIF-α protein mediated by a HIF hydroxylase, wherein that substance has been identified as inhibiting such hydroxylation by means of assays comprising
- contacting a HIF prolyl 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 by measuring the hydroxylase activity of the hydroxylase;
- wherein the HIF prolyl 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.
The method of 83, wherein R2 in the substance formula is selected from the group consisting of —SH, —OH, —CO2H, —SO3H, —B(OH)2, —PO3H2, —NHOH, —CONHR3, —CONHOR3, —CONHR3, and —CONR3OR3 where R3 is a branched or straight chain alkyl group of 1 to 6 carbon atoms which can be functionalized.
Dependencies are inferred from the claim text.