Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223) by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.
The ubiquitin-specific protease 7 (USP7) has emerged as an attractive therapeutic target owing to its critical roles in several cancer signaling pathways as well as its essential role in maintaining Foxp3+ T-regulatory cell (Treg) functions. Pharmacological inhibition of USP7 using Progenra’s small molecule inhibitors resulted in direct anti-tumor activity as well as the unleashing of anti-tumor immunity by suppressing Treg functions. However, the precise mechanism of action these compounds was not well characterized. Using a combination of NMR spectroscopy, mass spectrometry, and single amino acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic cleft of USP7 and covalently modify its active site cysteine (Cys223). Pharmacokinetic studies revealed sustained irreversible USP7 inhibition after short term inhibitor treatment as well as subsequent changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition has allowed us to incorporate rational design strategies into the lead optimization to obtain improved molecules that will serve as the basis of a new class of anti-cancer immunotherapy agents. Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Charles Grove, Phuong Nguyen, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Wayne Hancock, Irina Bezsonova, Suresh Kumar. Characterization of selective covalent inhibitors of USP7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2017-5336
The ubiquitin-specific protease 7 (USP7) has emerged as an attractive oncology/immune-oncology target owing to its critical roles in several cancer-related signaling pathways as well as its essential role in maintaining functions of Foxp3+ T-regulatory cells (Tregs), the key players in tumor immune evasion. Progenra has developed a series of compounds that inhibit purified USP7 selectively and attenuate USP7 activity in cells and in vivo; these inhibitors exert antitumor activity directly and also facilitate immune-mediated antitumor activity by suppressing Treg functions. However, the precise mechanism of action of these compounds remains unclear. In this study, using a combination of NMR spectroscopy, mass spectrometry, and single amino-acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic pocket of USP7 and modify its active site cysteine (Cys223) by forming a covalent adduct. Consistent with the covalent binding mechanism, pharmacokinetic studies revealed long-lasting, irreversible USP7 inhibition after a short pulse treatment with inhibitor, accompanied by changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage-specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition will permit the rational design of improved inhibitors as a new class of anticancer agent. Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Lee Chen, Brigid Cunnion, David Sterner, Charles Grove, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Irina Bezsonova, Wayne W. Hancock, Suresh Kumar. Characterization of selective active-site targeted covalent inhibitors of usp7 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B193.
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