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Discovery and characterization of highly potent and selective allosteric USP7 inhibitors

Abstract

Given the importance of ubiquitin-specific protease 7 (USP7) in oncogenic pathways, identification of USP7 inhibitors has attracted considerable interest. Despite substantial efforts, however, the development of validated deubiquitinase (DUB) inhibitors that exhibit drug-like properties and a well-defined mechanism of action has proven particularly challenging. In this article, we describe the identification, optimization and detailed characterization of highly potent (IC50 < 10 nM), selective USP7 inhibitors together with their less active, enantiomeric counterparts. We also disclose, for the first time, co-crystal structures of a human DUB enzyme complexed with small-molecule inhibitors, which reveal a previously undisclosed allosteric binding site. Finally, we report the identification of cancer cell lines hypersensitive to USP7 inhibition (EC50 < 30 nM) and demonstrate equal or superior activity in these cell models compared to clinically relevant MDM2 antagonists. Overall, these findings demonstrate the tractability and druggability of DUBs, and provide important tools for additional target validation studies.

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Figure 1: High-resolution X-ray co-crystal structure of USP7 in complex with 2 (PDB ID 5N9R).
Figure 2: High-resolution X-ray crystal structure of USP7 in complex with 5 (PDB ID 5N9T).
Figure 3: Selectivity profile of 4 against a panel of DUBs.
Figure 4: Target engagement and selectivity profile of 4 against selected USPs in cells.
Figure 5: Treatment of cells with 4 caused nongenotoxic stabilization of p53 and decreased levels of MDM2.

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Acknowledgements

The authors thank collaborators at Ubiquigent (Dundee, UK), Boston Biochem (Boston, US) and Beactica (Uppsala, Sweden) for their contributions, as well as Crelux GmbH (Martinsried, Germany) for solving the crystal structures and C. Scott and J. Burrows (Queen's University, Belfast) for helpful discussions and for providing plasmids, respectively. This study was supported by the Almac Group, the European Regional Development Fund and Invest Northern Ireland (Grant RD1010668).

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T.H. conceived the concept and directed the research. C.R.O'D. and G.G. helped develop the concept and designed and supervised medicinal chemistry and biology experiments. M.D.H., E.A., J.F. and H.M. carried out the design, synthesis and characterization of compounds. A.D. performed surface plasmon resonance experiments. C.H., K.M., E.O., E.C., A.D. and N.P. carried out compound screening, target validation and biochemical and cellular profiling studies. O.B. carried out computational modeling and structural analysis. T.H. and G.G. wrote the manuscript with input from other authors.

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Correspondence to Timothy Harrison.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–16 (PDF 3747 kb)

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Gavory, G., O'Dowd, C., Helm, M. et al. Discovery and characterization of highly potent and selective allosteric USP7 inhibitors. Nat Chem Biol 14, 118–125 (2018). https://doi.org/10.1038/nchembio.2528

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