Graduate School of Pharmaceutical Sciences, Kyushu University.
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University.
Chem Pharm Bull (Tokyo). 2020;68(11):1074-1081. doi: 10.1248/cpb.c20-00547.
Fragment-based approach combined with electrophilic reactive compounds is a powerful strategy to discover novel covalent ligands for protein target. However, the promiscuous reactivity often interferes with identification of the fragments possessing specific binding affinity to the targeted protein. In our study, we report the fragment-based covalent drug discovery using the chemically tuned weak reactivity of chlorofluoroacetamide (CFA). We constructed a small fragment library composed of 30 CFA-appended compounds and applied it to the covalent ligand screening for cysteine protease papain as a model protein target. Using the fluorescence enzymatic assay, we identified CFA-benzothiazole 30 as a papain inhibitor, which was found to irreversibly inactivate papain upon enzyme kinetic analysis. The formation of the covalent papain-30 adduct was confirmed using electrospray ionization mass spectrometry analysis. The activity-based protein profiling (ABPP) experiment using an alkynylated analog of 30 (i.e., 30-yne) revealed that 30-yne covalently labeled papain with high selectivity. These data demonstrate potential utility of the CFA-fragment library for de novo discovery of target selective covalent inhibitors.
基于片段的方法与亲电反应性化合物相结合，是发现新型共价配体的有效策略。然而，这种广泛的反应性常常干扰了对具有特定结合亲和力的片段的鉴定。在我们的研究中，我们报告了一种基于片段的共价药物发现方法，该方法利用氯氟乙酰胺（CFA）的化学调控弱反应性。我们构建了一个由 30 个 CFA 修饰的化合物组成的小分子片段文库，并将其应用于半胱氨酸蛋白酶木瓜蛋白酶作为模型蛋白靶标的共价配体筛选。使用荧光酶测定法，我们鉴定出 CFA-苯并噻唑 30 是一种木瓜蛋白酶抑制剂，酶动力学分析表明其不可逆地使木瓜蛋白酶失活。电喷雾电离质谱分析证实了形成了共价的木瓜蛋白酶-30 加合物。使用 30 的炔基类似物（即 30-yne）的基于活性的蛋白质谱分析（ABPP）实验表明，30-yne 对木瓜蛋白酶具有高选择性的共价标记。这些数据证明了 CFA-片段文库在从头发现靶选择性共价抑制剂方面的潜在应用价值。
