Introduction: Despite good survival rates for localized renal cell carcinoma a proportion of patients present with or will progress to metastatic disease, which portends worse prognosis and poor overall survival. Survival has been improved with the use of new targeted therapies and immunotherapy regimens; many patients, however, ultimately develop resistance. There is therefore a need for identification and development of new therapeutics. Clear cell renal cell carcinoma (ccRCC), the most common histologic subtype, generally harbors loss of the tumor suppressor von Hippel Lindau (VHL). The serine/threonine protein phosphatase-5 (PP5) plays a role in the regulation of numerous signaling pathways essential for cancer growth. Previously we have shown that PP5 is targeted for ubiquitination and degradation by a VHL-containing E3 ubiquitin ligase complex in a hypoxia-independent manner. Expression and activity of PP5 is consequently increased in ccRCC, and it plays a pro-survival role. Knock-down of PP5 expression or targeting its activity through inhibition of the kinase casein kinase-1d (CK1d) caused apoptosis in VHL-null ccRCC. The objective of this study was to develop a novel PP5 inhibitor for ccRCC. Methods: An in silico docking screen using the crystal structure of the PP5 active site and a large compound library was used to screen for candidate inhibitors. A series of candidates were then evaluated using both in vitro and cell-based assays for their ability to inhibit PP5 activity and cause apoptosis in ccRCC cells. In vitro PP5 activity was assessed using specific phospho-peptide substrates and in cells by immunoblotting to assess phosphorylation levels of known substrates. Two primary candidates were conjugated to biotin for PP5 pulldown experiments to assess specificity. Results: Our in silico screen identified approximately 200 candidate inhibitors. Further screening of about 20 of these compounds identified two compounds, P5 and P13, which inhibit PP5 activity both in vitro and in ccRCC cells and were further refined based on structure. Pulldown using biotin-conjugated drugs demonstrated good inhibitor specificity. Furthermore, treatment with these inhibitors leads to apoptosis specifically in VHL-null ccRCC cell lines. Conclusions: PP5 promotes cell survival in ccRCC and knockdown or inhibition of PP5 leads to cellular apoptosis. We have identified a small molecule which specifically binds to and inhibits PP5. This novel PP5 inhibitor causes apoptosis in VHL-null ccRCC cells and may serve as a new therapeutic strategy for treatment of advanced ccRCC. SOURCE OF Funding: This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R35GM139584 (M.M.) and DoD KC190038 (M.M.).
