Chemical Approaches to Intervening in Ubiquitin Specific Protease 7 (USP7) Function for Oncology and Immune Oncology Therapies

Ubiquitin specific protease 7 (USP7), the most widely studied among the nearly 100 deubiquitinating enzymes, supports cancer by positively affecting tumor growth and negatively affecting the patient’s immune response to tumors. Great interest exists, therefore, in developing USP7 inhibitors for clinical evaluation. While the proteasome inhibitor field has enjoyed clinical success, very few clinically appropriate effectors of deubiquitinating (protease) or ubiquitinating (ligase) enzymes have appeared. The ubiquitin protease/ligase field is moving from the initial discovery of potent, selective modulators with cell proof of concept and <i>in vivo</i> activity to the optimization of these molecules to impart drug-like properties or the discovery of new inhibitor scaffolds by improved screening or rational design. This Perspective focuses on the current status of USP7 inhibitors from various organizations active in developing these compounds for the clinic and suggests undertakings that are both achievable and necessary to lead to successful clinical outcomes for USP7 inhibitors in cancer treatment

Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60

<div><p>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 <i>in vivo</i> 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.</p></div

P217564 regulates the degradation of USP7 substrates and shows durable inhibition of USP7.

<p>(A) HCT116, PC-3, and Jurkat cells were treated with DMSO or P217564 at indicated concentrations for 24 hours; crude cell extracts were subjected to SDS-PAGE electrophoresis, followed by IB with indicated antibodies. (B) P217564 produces long-lasting USP7 inhibition in cells. Jurkat cells were treated with 10μM P217564 for 2 hours, the compound was washed out, and the USP7 activity was measured at various times up to 48 hours using the USP7 IP/activity assay. Cellular levels of USP7 substrates including UHRF1, DNMT1, and HDM2 were detected by Western blot. Anti-Actin blot was used as loading control.</p

Selective USP7 inhibitor P217564 regulates the stability of USP7.

<p>(A) P217564 decreases the USP7 level in a dose-dependent manner. Jurkat cells were treated with DMSO or P217564 at indicated concentrations for 24 hours; crude cell extracts were subjected to SDS-PAGE electrophoresis, followed by IB with indicated antibodies. *n.s indicates non-specific band. Relative quantitation of band intensity is given. (B) P217564 decreases the half-life of USP7. HCT116 cells were treated with DMSO or P217564 in the presence of 50ug/mL cycloheximide (CHX), and the levels of USP7 and HDM2 were analyzed using western blotting at indicated time points. The IB of Actin was used as the loading control.</p

DUB-based UbiTest to capture P217564-induced ubiquitination of USP7 substrates and analyze their polyubiquitin chain topology.

<p>(A) Schematic representation of DUB-based UbiTest. (B) Jurkat cells were treated with or without P217564 in the presence or absence of bortezomib (BTZ) for 2 hours. Total ubiquitinated proteins were then isolated from crude cell extracts using tandem ubiquitin binding entity (TUBE). All pull-down products were eluted, digested with USP2, and then subjected to IB analysis with indicated antibodies (left). The increased signal of individual substrate post USP2 digestion represents the amount of ubiquitination of each substrate. The relative change of the ubiquitination level of each substrate post compound treatment was quantified (right). (C) Samples were digested with USP2, a broad spectrum DUB, or with K48 (OUTB1) or K63 (AMSH) chain type specific DUBs, and followed by IB analysis of substrates with indicated antibodies.</p

P217564 inactivates USP7, resulting in increased ubiquitination and degradation of Tip60 and Foxp3 in Treg cells.

<p>(A) Treg cells were isolated from mice and treated with 10 μM P217564 for 2 hours; crude cell extracts were incubated with Ub-VME probe, followed by IB against USP7 to analyze the formation of USP7-Ub-VME complexes. (B) Mouse Treg cells were treated with DMSO or 10 μM P217564 for 2 hours; crude cell extracts were subjected to SDS-PAGE electrophoresis, followed by IB with indicated antibodies. Relative quantitation of band intensity is shown. (C) P217564-induced ubiquitination of Tip60, Foxp3 and USP7 in Treg cells was analyzed using the DUB-based UbiTest described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189744#pone.0189744.g006" target="_blank">Fig 6</a>.</p

Covalent mode of inhibition of USP7 inhibitor.

<p>(A) Purified wild-type USP7 core (USP7core WT) is able to form a complex with Ub-VME; P217564 prevents the formation of the USP7 core-Ub-VME complex. Purified active site mutant USP7 core-C223A cannot react with Ub-VME. (B) P217564 binds to USP7 covalently at its active site cysteine. Purified USP7core WT or C223A was incubated with either DMSO or P217564, and then subjected to LC-MS analysis to detect the formation of compound adduct on the USP7 core protein. (C) LC-MS/MS analysis to identify the conjugation site of P217564 on USP7.</p

NMR chemical shift perturbation analysis of P217564 binding to USP7core and mapping of the inhibitor-binding site.

<p>(A) Inhibitor-induced chemical shift perturbations (Δω, Hz) plotted for each residue of USP7 core. Resonances that were detected in the free protein only but disappeared in the complex were arbitrarily set to 80 Hz. (B) USP7 core solvent accessible surface colored according to NMR chemical shift perturbation from (A) (pdb ID: 1NB8). The degree of magenta represents increased chemical shift changes induced by P217564 binding. USP7 core residues for which resonance assignments are missing in ¹H-¹⁵N HSQC spectra are shown in light gold. Catalytic cysteine residue C223 is shown in red. The structure is shown in two orientations. The active site and the ubiquitin-binding “fingers” region are labeled.</p

P217564 regulates the degradation of USP7 substrates and shows durable inhibition of USP7.

<p>(A) HCT116, PC-3, and Jurkat cells were treated with DMSO or P217564 at indicated concentrations for 24 hours; crude cell extracts were subjected to SDS-PAGE electrophoresis, followed by IB with indicated antibodies. (B) P217564 produces long-lasting USP7 inhibition in cells. Jurkat cells were treated with 10μM P217564 for 2 hours, the compound was washed out, and the USP7 activity was measured at various times up to 48 hours using the USP7 IP/activity assay. Cellular levels of USP7 substrates including UHRF1, DNMT1, and HDM2 were detected by Western blot. Anti-Actin blot was used as loading control.</p

P217564 selectively inhibits USP7 activity in cultured cells.

<p>(A) Jurkat cells were treated with DMSO or P217564 as indicated for 2 hours; USP7 was isolated by immunoprecipitation (IP) and its activity quantified by UB-EK_L-based activity assay (middle). Simultaneously, equal amounts of crude cell extracts were used to determine total DUB activity using the UB-EK_L assay (Right). (B) Schematic of Ub-VME reaction. (C) Jurkat cells were treated with DMSO or P217564; crude cell lysates were incubated with the Ub-VME probe and then immunoblotted against USP7 to determine formation of USP7-Ub-VME complexes. (D) Jurkat cells were treated with DMSO or P217564; crude cell extracts were labeled with HA-Ub-VME probe, followed by immunoblotting (IB) with indicated antibodies.</p