The ubiquitin system regulates the majority of cellular processes in eukaryotic species. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumor suppressor and other proteins critical for tumor cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved<br/>with small molecule deubiquitinase inhibitors. Here, using nuclear magnetic resonance (NMR)-based screening and structure-based drug design, we describe the development of selective and orally bioavailable USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce hematopoietic and solid tumor cell death and enhance cytotoxicity with standard-of-care chemotherapeutic agents. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target an interface between the thumb, fingers, and palm regions of the USP7 catalytic<br/>domain, 12Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. Within the USP7 thumb region, GNE-6640 and GNE-6776 interact with acidic residues that mediate H-bond interactions with the ubiquitin Lys-48 side-chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties having free Lys-48 side-chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7<br/>binding via NMR, a study that substantiated our hypothesis. This preferential binding significantly protracted the depolymerization kinetics of Lys-48-linked ubiquitin chains relative to Lys-63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Fragment guided path to a selective inhibitor of USP7 and its implications for chain linkage specificity

255th National Meeting (2018)

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