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 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.
Claims29
1-55. (canceled)
A pharmaceutical composition comprising
- a substance that has been identified as inhibiting the activity of a HIF hydroxylase in mediating the hydroxylation of one or more proline residues of a HIF-α protein by means of an assay method in which
- a HIF prolyl hydroxylase and a substrate of the hydroxylase are contacted under conditions in which the hydroxylase interacts with the substrate, in the presence or absence of a test substance;
- and
- the interaction, or lack of interaction of, the hydroxylase and the substrate is determined by measuring the hydroxylase activity of the hydroxylase;
- wherein the HIF prolyl hydroxylase is a fragment of SEQ ID NO:
- 4 that
- contains the sequence HXD[X]nH, X being any amino acid and n being any number between 1 and 200,
- contains a β-barrel jelly roll structure,
- retains HIF prolyl hydroxylase activity, and
- hydroxylates the proline residue of a motif LXXLXP contained in the substrate, where X is any amino acid;
- and
- a pharmaceutically acceptable excipient.
The pharmaceutical composition of 70, wherein the ring is pyrolidine or an unsaturated derivative thereof.
The pharmaceutical composition of 67, wherein R1 in the substance formula is an acid group.
The pharmaceutical composition of 67, 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.