Assay for inhibitors of CIP/KIP protein degradation
AssigneeUniversite de Montreal
InventorsAnne Marinier, Sylvain Meloche, Benoit Pelletier
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Abstract
An assay and system compatible with high throughput screening (HTS) that is capable of identifying inhibitors, such as small-molecule inhibitors, of the degradation of the Cdk inhibitor p21, are described. The assay is based on the use of fusion protein comprising (i) a p2 polypeptide; and (i) a reporter protein linked to the C-terminal of said p21 polypeptide, wherein the fusion protein has a half-life that is similar to that of the p21 polypeptide. Inhibitors identified by this assay may be useful to inhibit the proliferation of tumor cells, and thus for the treatment of cancers.
Claims20
A method for determining whether a test compound inhibits the degradation of a Cip/Kip protein by the Ubiquitin-Proteasome system and may be useful for treating cancer, said method comprising
- (a) contacting said test compound with a cell expressing a fusion protein in the presence of an eukaryotic protein synthesis inhibitor, said fusion protein comprising (i) a Cip/Kip polypeptide;
- and (ii) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide, and
- (b) measuring a readout signal from the reporter protein,
- wherein a higher readout signal from the reporter protein in the presence of said test compound, relative to the readout signal in the absence of said test compound, is indicative that said test compound inhibits the degradation of a Cip/Kip protein by the Ubiquitin-Proteasome system and may be useful for treating cancer.
A system for determining whether a test compound inhibits the degradation of a Cip/Kip protein by the Ubiquitin-Proteasomes system and may be useful for treating cancer, said system comprising:
- a cell expressing a fusion protein, said fusion protein comprising (i) a Cip/Kip polypeptide;
- and (ii) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide;
- an eukaryotic protein synthesis inhibitor;
- and
- a detection system to measure a readout signal from the reporter protein.
Dependencies are inferred from the claim text.