Ernst Larissa N, Stahlecker Jason, Mier Finn, Serafim Ricardo A M, Wydra Valentin R, Masberg Benedikt, Jaag Simon J, Knappe Cornelius, Lämmerhofer Michael, Stehle Thilo, Gehringer Matthias, Boeckler Frank M
Department of Pharmacy and Biochemistry, Laboratory for Molecular Design & Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Tübingen, Germany.
Department of Medicinal Chemistry, Faculty of Medicine, Institute for Biomedical Engineering, Eberhard Karls Universität Tübingen, Tübingen, Germany.
Arch Pharm (Weinheim). 2025 Aug;358(8):e70053. doi: 10.1002/ardp.70053.
The cysteine protease ubiquitin-specific protease 7 (USP7), also known as herpes-associated ubiquitin-specific protease (HAUSP), has gained increasing attention in recent years due to its proven overexpression in several cancer types and its role in tumorigenesis. Herein, after a design based on molecular docking experiments, we report the synthesis of a series of mildly electrophilic compounds that covalently modify the catalytic cysteine 223 in USP7 through a nucleophilic aromatic substitution (SAr) reaction. The compounds were first evaluated using differential scanning fluorimetry (DSF) to describe their influence on the melting temperature of native and mutant USP7 variants. Furthermore, the possible formation of a covalent bond was analyzed using intact protein mass spectrometry (MS). For promising derivatives, IC values were determined in an enzyme activity assay to confirm an inhibitory effect on USP7. Finally, a co-crystal structure revealed that the prototype compound (7a) arylates the catalytic cysteine in the apo form of USP7 via an unconventional binding mode near the catalytic triad. The synthesis and biological evaluation of this compound series provides valuable structure-activity relationships (SAR) and reveals an interesting and unprecedented binding mode, thus providing a basis for improving USP7 inhibitors.
半胱氨酸蛋白酶泛素特异性蛋白酶7（USP7），也被称为疱疹相关泛素特异性蛋白酶（HAUSP），近年来因其在多种癌症类型中被证实的过表达及其在肿瘤发生中的作用而受到越来越多的关注。在此，基于分子对接实验进行设计后，我们报告了一系列温和亲电化合物的合成，这些化合物通过亲核芳香取代（SAr）反应共价修饰USP7中的催化半胱氨酸223。首先使用差示扫描荧光法（DSF）评估这些化合物对天然和突变型USP7变体熔解温度的影响。此外，使用完整蛋白质质谱（MS）分析共价键的可能形成。对于有前景的衍生物，在酶活性测定中确定IC值以确认对USP7的抑制作用。最后，共晶体结构表明原型化合物（7a）通过在催化三联体附近的非常规结合模式使USP7的无配体形式中的催化半胱氨酸芳基化。该化合物系列的合成和生物学评价提供了有价值的构效关系（SAR），并揭示了一种有趣且前所未有的结合模式，从而为改进USP7抑制剂提供了基础。
