Structure Kinetics Relationships and Molecular Dynamics Show Crucial Role for Heterocycle Leaving Group in Irreversible Diacylglycerol Lipase Inhibitors

Drug discovery programs of covalent
irreversible, mechanism-based
enzyme inhibitors often focus on optimization of potency as determined
by IC50-values in biochemical assays. These assays do not
allow the characterization of the binding activity (Ki) and reactivity (kinact)
as individual kinetic parameters of the covalent inhibitors. Here,
we report the development of a kinetic substrate assay to study the
influence of the acidity (pKa) of heterocyclic
leaving group of triazole urea derivatives as diacylglycerol lipase
(DAGL)-α inhibitors. Surprisingly, we found that the reactivity
of the inhibitors did not correlate with the pKa of the leaving group, whereas the position of the nitrogen
atoms in the heterocyclic core determined to a large extent the binding
activity of the inhibitor. This finding was confirmed and clarified
by molecular dynamics simulations on the covalently bound Michaelis–Menten
complex. A deeper understanding of the binding properties of covalent
serine hydrolase inhibitors is expected to aid in the discovery and
development of more selective covalent inhibitors.

共价不可逆、基于机制的酶抑制剂的药物发现项目，通常以生化检测中测得的半数抑制浓度（IC₅₀）为基准优化抑制剂药效。此类检测无法分别表征共价抑制剂的结合活性（抑制常数Ki）与反应活性（动力学常数kinact）这两项独立动力学参数。本研究报道了一种动力学底物分析法，用于探究作为二酰甘油脂肪酶（DAGL）-α抑制剂的三唑脲类衍生物，其杂环离去基团的酸度（pKa）对抑制剂活性的影响。令人意外的是，本研究发现抑制剂的反应活性与离去基团的pKa并无关联，而杂环核心内氮原子的位置在很大程度上决定了抑制剂的结合活性。通过对共价结合的米氏（Michaelis–Menten）复合物开展分子动力学模拟，该结论得到了验证与进一步阐明。对共价丝氨酸水解酶抑制剂结合特性的深入理解，将助力开发选择性更强的共价抑制剂。