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J Mol Biol. 2017 Nov 10;429(22):3546-3560. doi: 10.1016/j.jmb.2017.05.025. Epub 2017 Jun 3.

Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10.

Author information

1
Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, Ontario M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto, Ontario M5S3E1, Canada.
2
Cancer Epigenetics DPU, Oncology Research & Development, GlaxoSmithKline Inc, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.
3
Department of Aquaculture, Fisheries College, Guangdong Ocean University, Guangdong, 524088, China.
4
Department of Marine Biology, Fisheries College, Guangdong Ocean University, Guangdong, 524025, China.
5
Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G1L7, Canada.
6
Immuno-Oncology Combinations DPU, Oncology Research & Development, GlaxoSmithKline Inc, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.
7
Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5G1L7, Canada.
8
Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, Ontario M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto, Ontario M5S3E1, Canada. Electronic address: j.moffat@utoronto.ca.
9
Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, Ontario M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto, Ontario M5S3E1, Canada. Electronic address: sachdev.sidhu@utoronto.ca.

Abstract

Post-translational modification of the p53 signaling pathway plays an important role in cell cycle progression and stress-induced apoptosis. Indeed, a large body of work has shown that dysregulation of p53 and its E3 ligase MDM2 by the ubiquitin-proteasome system (UPS) promotes carcinogenesis and malignant transformation. Thus, drug discovery efforts have focused on the restoration of wild-type p53 activity or inactivation of oncogenic mutant p53 by targeted inhibition of UPS components, particularly key deubiquitinases (DUBs) of the ubiquitin-specific protease (USP) class. However, development of selective small-molecule USP inhibitors has been challenging, partly due to the highly conserved structural features of the catalytic sites across the class. To tackle this problem, we devised a protein engineering strategy for rational design of inhibitors for DUBs and other UPS proteins. We employed a phage-displayed ubiquitin variant (UbV) library to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2. We were able to identify UbVs that bound USP7 or USP10 with high affinity and inhibited deubiquitination activity. We solved the crystal structure of UbV.7.2 and rationalized the molecular basis for enhanced affinity and specificity for USP7. Finally, cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin, demonstrating the therapeutic potential of inhibiting USP7 by this approach.

KEYWORDS:

USP10; USP7; inhibitor design; p53

PMID:
28587923
DOI:
10.1016/j.jmb.2017.05.025
[Indexed for MEDLINE]

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