Assay and Reagents for Detecting Inhibitors of Ubiquitin-dependent Degredation o

Summary Issued US patent 5,726,025 :

SUMMARY OF THE INVENTION

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 in cells from vertebrate organism, especially mammals, preferably humans. For instance, the assays permit identification of agents which inhibit ubiquitination of the cyclin proteins, e.g., B-type 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, a mitotic destruction complex (e.g., comprising CDC27 and CDC16), 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. In certain embodiments, the present assay comprises an in vivo ubiquitin-conjugating system, such as a cell able to conduct the regulatory protein through at least a portion of a ubiquitin-mediated proteolytic pathway. In other embodiments, the present assay comprises an in vitro ubiquitin-conjugating system comprising a reconstituted protein mixture in which at least the ability to transfer ubiquitin to the regulatory protein is constituted.

Applications For Further Information Please Contact the Director of Business Development Michal Preminger Email: [email protected] Telephone: (617) 432-0920

Inventor(s): Kirschner, Marc W.

Type of Offer: Licensing



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