Interaction Between the VHL Tumour Suppressor and Hypoxia Inducible Factor, and Assay Methods Relating Thereto

1 . An assay for a modulator of VHL-HIF α subunit interaction, which comprises:

a) bringing into contact a VHL protein, a HIF α subunit protein and a putative modulator compound under conditions where the VHL protein and the HIF α subunit protein, in the absence of modulator, are capable of forming a complex; and

b) measuring the degree of inhibition of complex formation caused by said modulator compound.

2 . An assay according to claim 1 in the form of a two-hybrid assay.

3 . An assay according to claim 1 in the form of an immunoprecipitation.

4 . An assay according to claim 1 wherein at least one of said proteins is labelled with a detectable label.

5 . An assay according to any one of the preceding claims wherein at least one of said proteins is in the form of a fusion protein.

6 . An assay according to any one of the preceding claims wherein the ubiquitylation of the HIF α subunit is determined.

7 . An assay a modulator of VHL-HIF α subunit interaction, which comprises:

a) bringing into contact a VHL protein, a HIF α subunit protein and a putative modulator compound under conditions where the VHL protein and the HIF α subunit protein, in the absence of modulator, are capable of forming a complex;

b) providing an HIF response element to which the HIF α subunit protein is capable of binding and transcriptionally activating; and

c) measuring the degree of modulation of binding of the α subunit to, or transcriptional activation of, the response element caused by said modulator compound.

8 . An assay according to claim 6 wherein said response element is operably linked to a reporter gene.

9 . An assay according to any one of the preceding claims wherein said VHL protein is human VHL (Genbank accession number AF010238) or a fragment thereof comprising at least residues 63-156.

10 . An assay according to any one of the preceding claims wherein said HIF α subunit protein is human HIF α subunit protein (Genbank accession number U22431) or a fragment thereof comprising at least residues 549-572.

11 . An isolated polypeptide which consists of from 5 to 50 amino acids whose sequence is found in region 549-652, particularly 549-572 of human HIF-1α (Genbank accession number U22431).

12 . An isolated polypeptide which consists of from 5 to 50 amino acids, said polypeptide being capable of forming a complex with VHL, and characterised by the presence of a sequence selected from:

LAPYIPMD;

SEQ ID NO: 1

LAPYISMD;

SEQ ID NO: 2

LLPYIPMD;

SEQ ID NO: 3

LVPYIPMD;

SEQ ID NO: 4

IAPYIPMD;

SEQ ID NO: 5

IAPYIPME;

SEQ ID NO: 6

and

LVPYISMD.

SEQ ID NO: 7

13 . A polypeptide according to claim 12 which comprises the sequence:

DLDLEMLAPYIPMDDDFQL;

(SEQ ID NO: 8)

and

variants thereof in which there are from 1 to 4 substitutions.

14 . A polypeptide according to claim 12 which comprises the sequence:

PFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSP;

(SEQ ID NO: 9)

and

variants thereof in which there are from 1 to 4 substitutions.

15 . A polypeptide comprising the polypeptide of any one of claims 11 to 14 fused to a membrane translocation sequence.

16 . A polypeptide according to any one of claims 11 to 14 for use in a method of inhibiting the interaction of a HIF α subunit with VHL.

17 . A method of promoting angiogenesis and/or cellular survival in a cell exposed to a hypoxic environment, said method comprising blocking the interaction between VHL and a HIF α subunit.

18 . An assay for a modulator which promotes VHL-HIF α subunit interaction, which assay comprises:

a) bringing into contact a HIF α subunit protein and a putative modulator compound in the presence or absence of a VHL protein,

b) providing a VHL protein where said protein is absent in step (a); and

c) determining whether the VHL-HIF α subunit interaction has been promoted by the presence of the modulator.