Virtual Screening–Guided Identification of Candidate USP7-Inhibitory Scaffolds Exhibiting Antiproliferative Activity

Abstract

Background/Objectives: Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that regulates multiple oncogenic and tumor-suppressive pathways and has emerged as a promising target for anticancer drug discovery. However, most reported USP7 inhibitors belong to a limited number of structural classes, and no USP7-targeted therapy has yet reached clinical application. This study aimed to identify structurally diverse candidate USP7-inhibitory scaffolds with antiproliferative activity using an integrated computational and experimental screening strategy. Methods: A drug discovery workflow combining pharmacophore modelling, structure-based virtual screening, molecular docking, and in silico pharmacokinetic assessment was employed to identify candidate USP7 inhibitors. Selected compounds were evaluated for inhibition of recombinant USP7 and for antiproliferative activity in a panel of human cancer cell lines. Molecular docking analyses were performed to investigate predicted binding modes. Results: The primary screening campaign identified four active compounds (>50% inhibition at 10 µM) and twenty-one weakly active compounds (10–49% inhibition at 10 µM), including structurally distinct approved drugs and compounds from an in-house chemical library. The four active compounds were subsequently validated by dose–response assays against recombinant USP7 and exhibited micromolar inhibitory activity. These candidate USP7-inhibitory scaffolds also exhibited antiproliferative activity across cancer cell lines with different molecular backgrounds. Docking studies predicted binding within a pharmacologically relevant region of the USP7 catalytic cleft and revealed putative interactions with key residues involved in ligand recognition. Among the compounds evaluated, rafoxanide demonstrated the most favorable combination of predicted USP7 binding and antiproliferative activity. Conclusions: This integrated virtual screening and experimental validation approach enabled the identification of candidate USP7-inhibitory scaffolds with preliminary antiproliferative activity. The identified hits include approved drugs with previously unreported USP7 inhibitory activity, as well as underexplored scaffolds that expand the chemical space of candidate USP7-targeting molecules. These candidate scaffolds warrant further medicinal chemistry optimization, orthogonal validation, and mechanistic characterization to establish their potential as USP7-targeted anticancer agents.