Deubiquitinating enzymes (DUBs) are key regulators of the ubiquitin pathway that catalyze the removal of ubiquitin from target substrates. Hence, these enzymes affect a wide variety of cellular processes by influencing the localization, stability and function of their targets. Importantly, inhibition of DUB activity has emerged as a promising therapeutic strategy to treat several diseases including various cancers & Alzheimer’s. Despite this, a paucity of specific and potent inhibitors aimed at DUB pathways has hindered attempts to exploit them for therapeutic benefit.
To address this issue, we developed a strategy using ubiquitin (Ub) as a scaffold to engineer specific & potent DUB inhibitors, called Ub variants (UbV). We have generated massively diverse and combinatorial libraries, comprising >7x1010 unique UbVs. Using phage-display we have screened our library to identify inhibitors of ~8/95 human DUBs. Although effective, an inability to purify many disease-relevant DUBs has limited the applicability of our in vitro screening system. Thus, we have developed a method based on the yeast two-hybrid (Y2H) system to detect UbV-DUB interactions in vivo. In a pilot study using a smaller library consisting of 105 unique UbVs, we identified UbVs that bind the DUB USP2 specifically, thus validating our approach. We are currently adapting this system to allow for the high-throughput screening of massive UbV libraries in vivo. In conjunction with established in vitro screening methods, we will use our in vivo screening platform to discover novel inhibitors against the full panel of human DUBs. Together, this work will make seminal contributions towards furthering our understanding of DUB function and alleviate a formidable bottleneck that obstructs the development of more powerful therapeutics aimed at the ubiquitin system.