Development of p97 inhibitors as potential anti-cancer therapeutics

Abstract

P97/VCP is an important AAA ATPase not only due to its intriguing diverse cellular functions but also because it has been implicated in mediating turnover of many proteins involved in tumorigenesis. Specific small mol. inhibitors of p97/VCP are important tools to investigate diverse functions of this essential AAA ATPase and to evaluate its potential to be a therapeutic target. To quant. assay specificity of p97/VCP inhibitors, we develop three cell- based reporter assays with distinct dependences on p97/VCP for their stability based on changes of steady-state levels and half-lives by knocking down p97/VCP or expression of dominant-neg. p97/VCP. We apply these assays to screen against 160 cellpermeable kinase inhibitors to identify an irreversible p97/VCP inhibitor with IC50 of 1.7 ×M. Biochem. characterization demonstrates a cysteine within the second ATP binding pocket is responsible for the obsd. inactivation. Furthermore, we demonstrate the utility of these reporters on assessing effect of various kinase inhibitors in the ubiquitin proteasome pathway and provide insight into functions of kinases in the ubiquitin proteasome system. Most importantly, we can now apply the system to address specificity issue at an earlier stage to develop specific p97 inhibitors.