Covalent ligands are of great interest as therapeutic drugs or biochemical tools. Here, we reported the discovery of highly selective and irreversible inhibitors of lipoprotein-associated phospholipase A2 (Lp-PLA2) using a covalent fragment-based approach. The crystal structure of Lp-PLA2 in complex with a covalent fragment not only reveals the covalent reaction mechanism but also provides a good starting point to design compound 8, which has a more than 130,000-fold and 3900-fold increase in potency and selectivity, respectively, compared to those of the covalent fragment. Furthermore, fluorescent probes with high selectivity and sensitivity are developed to characterize Lp-PLA2 and its enzymatic activity in vitro or even in living cells in a way more convenient than immunoblotting tests or immunofluorescence imaging. Overall, we provide a paradigm for application of the covalent fragment-based strategy in covalent ligand discovery and the advantage of enol–cyclocarbamate as a new warhead in designing covalent inhibitors of serine hydrolases.

中文翻译：

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通过基于共价片段的方法鉴定高选择性脂蛋白相关的磷脂酶A2（Lp-PLA2）抑制剂。

共价配体作为治疗药物或生化工具备受关注。在这里，我们报道了使用基于共价片段的方法发现脂蛋白相关磷脂酶A2（Lp-PLA2）的高度选择性和不可逆抑制剂的发现。Lp-PLA2与共价片段复合的晶体结构不仅揭示了共价反应机理，而且为设计化合物8提供了良好的起点与共价片段相比，其效价和选择性分别提高了130,000倍和3900倍以上。此外，开发了具有高选择性和敏感性的荧光探针，以比免疫印迹测试或免疫荧光成像更方便的方式表征Lp-PLA2及其在体外甚至在活细胞中的酶活性。总体而言，我们为基于共价片段的策略在共价配体发现中的应用提供了范例，并且为设计丝氨酸水解酶的共价抑制剂提供了新的战斗部-烯醇-环氨基甲酸酯。