Discovery of Benzodiazepine-Based Inhibitors of the E2 Enzyme UBCH10 from a Cell-Based p21 Degradation Screen

p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often down-regulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.

Automated summary

p21Cip1 is a CDK inhibitor that can halt cell proliferation and tumor growth. However, its expression is often decreased in cancer cells due to the loss of transcriptional activators or increased protein degradation. In this study, a benzodiazepine series of molecules was identified as inhibitors of p21 degradation. Further analysis revealed that UBCH10, a ubiquitin-conjugating enzyme, serves as a cellular target of the benzodiazepine series. Moreover, an optimized benzodiazepine analogue was found to inhibit UBCH10 activity and prevent substrate proteolysis.