A program to enable discovery of catalytic and/or allosteric inhibitors of the USP4/11/15 family of deubiquitinating enzymes

Post-translational modification of cellular proteins with ubiquitin serves as a signal in virtually all eukaryotic pathways. These signals are carefully regulated by ubiquitin conjugation and deconjugation enzymes, making them prime candidates as drug targets. In particular deubiquitinating enzymes (DUBs), especially those with a cysteine-protease catalytic center and extensive allosteric regulation, have significant potential for drug discovery. However, development of DUB inhibitors has proven difficult, due to difficulties in identification of high affinity binders from screens and lack of high-resolution crystal structures of complexes. The ubiquitin specific proteases, USP4, USP11 and USP15 play important roles in TGF-beta signalling, and have been associated with RNA splicing, DNA damage response or neuroinflammation and Parkinson’s disease. These DUBs share a common domain structure, with N-terminal regulatory domains and a catalytic domain with a long internal insert. Previously we have solved the crystal structure of the USP4 catalytic core domain and we could show biochemically that the regulatory domains are important to promote product release. Working with CD3 we identified promising hits in a high-throughput screen for inhibitors of USP15 that now should be characterized, both biochemically and structurally, so that they can be tested by cell biology collaborators. Astex developed structure-based fragment-based screening approaches, an exciting novel method for identifying lead compound without prior assumptions that requires well-diffracting crystals. Now we want to promote drug discovery against USP4/11/15 by three separate lines: • Characterize and optimize binding mode of compounds derived from high-throughput screening of USP15 using biochemical, structural and biophysical approaches. • Develop crystal-growth conditions that are suitable for fragment screening and compound optimization. • Study intrinsic allosteric regulation of the USP4/11/15 class of DUB enzymes biochemically and structurally and develop new assays that can target this aspect of DUB regulation. The results of this project will provide assays and insight for targeting these important DUBs to develop novel inhibitors with high affinity and specificity. Meanwhile they will also provide fundamental insight into their regulatory properties by structural and biochemical analysis of the full-length proteins.