Identification of cyano indenopyrazine derivatives as a novel series of potent ubiquitin-specific protease inhibitors

C220 Regulated protein turnover is primarily controlled by the ubiquitin-proteasome system. The only marketed drug related to the ubiquitin-proteasome system, bortezomib, is acting as a proteasome inhibitor and has been approved for the treatment of some hematological cancers. Targeting the upstream ubiquitin conjugation/deconjugation system carries out promises of therapeutics with increased specificity and selectivity. Ubiquitin-specific proteases (USP) are involved in the deubiquitination of specific target substrates regulating their stability, subcellular localization and/or activation status. USP represent a drugable target class due to their thiol-protease catalytic core which is amenable to pharmacological inhibition by small molecules. A genome-wide RNAi screen of the catalytically active human USP in cancer-relevant cellular models and phenotypic assays identified USP7 and USP8 as promising cancer targets. Advanced HTS-compatible, HTRF-based screening assays using optimized USP substrates including various ubiquitin derivatives (ubiquitin precursor, branched ubiquitin chains) as well as specific, physiological substrates were developed to screen a chemically diverse library. Primary hits were run through a lead optimization program to improve potency, selectivity, cellular activity and pharmacokinetic profile. We report here the identification of a series of cyano indenopyrazine derivatives as novel and potent USP inhibitors. One of our lead compounds, HBX 41,108, was found to reversibly inhibit USP7 catalytic activity with an IC50 of 0.4 µM and to exhibit dose-dependent antiproliferative activity with sub-micromolar GI50. Reminiscent to RNAi-mediated USP7 silencing in cancer cells, HBX 41,108 stabilizes and activates p53 in a non-genotoxic manner, leading to cell growth inhibition and p53-dependent apoptosis as demonstrated using p53 wild-type and null isogenic cell lines. This compound also sensitizes colorectal carcinoma cells to the p53-dependent cytotoxic effect of 5-fluorouracil. A related compound displaying exquisite USP8 selectivity in vitro (HBX 90,659) exhibited antiproliferative and pro-apoptotic activities with sub-micromolar GI50 in different cancer cell lines and induced a marked increase in total cell protein ubiquitination, recapitulating the phenotypes observed in USP8 knock-down cells. We report the development of unique, advanced ubiquitin-specific proteases screening assays and the identification of a novel chemical series of drug-like, small-molecule USP inhibitors. These will help further validate this novel class of molecular targets and may provide a structural basis for the development of new anticancer drugs.