ASSAY AND REAGENTS FOR DETECTING INHIBITORS OF UBIQUITIN-DEPENDENT DEGRADATION OF CELL CYCLE REGULATORY PROTEINS

摘要

The present invention provides a systematic and practical approach for the identification of candidate agents able to inhibit ubiquitin-mediated degradation of a cell-cycle regulatory protein, such as cyclins. One aspect of the present invention relates to a method for identifying an inhibitor of ubiquitin-mediated proteolysis of a cell-cycle regulatory protein by (i) providing a ubiquitin-conjugating system that includes the regulatory protein and ubiquitin under conditions which promote the ubiquitination of the target protein, and (ii) measuring the level of ubiquitination of the subject protein brought about by the system in the presence and absence of a candidate agent. A decrease in the level of ubiquitin conjugation is indicative of an inhibitory activity for the candidate agent. The level of ubiquitination of the regulatory protein can be measured by determining the actual concentration of protein:ubiquitin conjugates formed; or inferred by detecting some other quality of the subject protein affected by ubiquitination, including the proteolytic degradation of the protein.