Ernst Larissa N, Jaag Simon J, Wydra Valentin R, Masberg Benedikt, Knappe Cornelius, Gerstenecker Stefan, Serafim Ricardo A M, Liang Xiaojun Julia, Seidler Nico J, Lämmerhofer Michael, Gehringer Matthias, Boeckler Frank M
Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Laboratory for Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany.
Department of Pharmacy and Biochemistry, Eberhard Karls Universität, Pharmaceutical (Bio-)Analysis, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany.
Drug Des Devel Ther. 2025 Mar 25;19:2253-2284. doi: 10.2147/DDDT.S513591. eCollection 2025.
The ubiquitin-specific protease 7 (USP7), also known as herpes-associated ubiquitin-specific protease (HAUSP) is an interesting target due to its role in the tumor suppressor p53 pathway. In recent years targeted covalent inhibitors have gained significant importance in pharmaceutical research. Thus, we have investigated a small library of 129 ligands bearing different types of covalent reactive groups ("warheads") from various kinase drug discovery projects for their reactivity towards the catalytic cysteine of USP7, as well as their influence on its melting temperature. These compounds mainly encompassed α,β-unsaturated amides specifically acrylamides, SAr reacting compounds, aryl fluorosulfates and sulfonyl fluorides.
We analyzed an array of 129 electrophilic compounds which had been designed as covalent kinase inhibitors in a DSF-based (differential scanning fluorimetry) screen against USP7. The hits were evaluated for their ability to cause similar thermal shifts for a CYS-deficient USP7 control mutant (USP7asoc), where only the catalytic Cys223 was retained. Additionally, covalent binding was evaluated by intact protein mass spectrometry (MS).
The DSF screen revealed that, predominantly 18 of the 129 tested compounds decreased the melting temperature of USP7 and its mutant USP7asoc. For 8 of these, the hypothesized covalent binding mode was corroborated with native and mutant USP7 by intact protein MS. Nearly all identified hits have a covalent warhead that reacts via nucleophilic aromatic substitution (SAr).
The screening and evaluation of the kinase library revealed several initial hits of interest. Seven SAr warheads and one acrylamide warhead compound covalently modified the target protein (USP7) and showed clear shifts in the melting temperatures ranging from -6.0 °C to +1.7 °C.
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），因其在肿瘤抑制因子p53通路中的作用而成为一个有趣的靶点。近年来，靶向共价抑制剂在药物研究中变得尤为重要。因此，我们研究了一个包含129种配体的小型文库，这些配体来自各种激酶药物发现项目，带有不同类型的共价反应基团（“弹头”），考察它们对USP7催化半胱氨酸的反应活性以及对其解链温度的影响。这些化合物主要包括α,β-不饱和酰胺，特别是丙烯酰胺、亲核芳香取代（SAr）反应性化合物、芳基氟硫酸盐和磺酰氟。
我们分析了129种亲电化合物，这些化合物被设计为共价激酶抑制剂，通过基于差示扫描荧光法（DSF）的筛选来检测其对USP7的作用。对筛选出的命中化合物，评估其对仅保留催化性半胱氨酸（Cys223）的CYS缺陷型USP7对照突变体（USP7asoc）引起类似热位移的能力。此外，通过完整蛋白质质谱（MS）评估共价结合情况。
DSF筛选显示，在129种测试化合物中，主要有18种降低了USP7及其突变体USP7asoc的解链温度。其中8种化合物，通过完整蛋白质MS证实了其与天然型和突变型USP7的假定共价结合模式。几乎所有鉴定出的命中化合物都有一个通过亲核芳香取代（SAr）反应的共价弹头。
对激酶文库的筛选和评估揭示了几个感兴趣的初步命中化合物。7种SAr弹头化合物和1种丙烯酰胺弹头化合物共价修饰了靶蛋白（USP7），并使解链温度出现了-6.0℃至+1.7℃的明显变化。
