JAK3 Inhibitors: Covalent and Noncovalent Interactions of a Cyanamide Group Investigated by Multiscale Free-Energy Simulations

Janus kinase type 3 (JAK3), an emerging target for treating autoimmune diseases, possesses a front pocket cysteine that is targeted by covalent modifiers, best represented by the marketed drug ritlecitinib (1). Recently, 2,3-dihydro-1H-inden-1-ylcyanamides have been developed as novel JAK3 inhibitors. Among them, the N-(6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)cyanamide inhibitor (2) and its methylated analogue (3), while being potent inhibitors, displayed different mechanisms of action (covalent vs noncovalent) and binding modes (Casimiro-Garcia et al., J Med Chem 2018). Prompted by this intriguing behavior, we applied a multiscale approach to characterize the reaction mechanism between the JAK3 front-pocket Cys909 and cyanamide-based inhibitors. Quantum mechanics/molecular mechanics simulations showed that 2 can readily form an isothiourea adduct with the Cys909 only when a conserved water molecule assists the reaction as a proton shuttle and that methylation of the 2,3-dihydro-1H-inden-1-ylcyanamide moiety of 2 hampers the isothiourea formation by displacing this water molecule. Metadynamics and thermodynamic integration simulations were applied to investigate the relative abundance of alternative poses accessible to 2,3-dihydro-1H-inden-1-ylcyanamides, explaining the effect of methylation on the relative binding mode preference. This multiscale approach provides new chemical insights into the mechanism of action of cyanamide inhibitors and emerges as an effective protocol to investigate the interaction between drugs and molecular targets.

贾纳斯激酶3（Janus kinase type 3, JAK3）是治疗自身免疫疾病的新兴治疗靶点，其前沿口袋含有一个可被共价修饰剂靶向的半胱氨酸残基，该类修饰剂以上市药物利特昔替尼（ritlecitinib，1）为典型代表。近期，2,3-二氢-1H-茚-1-基氰胺类化合物被开发为新型JAK3抑制剂。在该类化合物中，N-(6-(7H-吡咯并[2,3-d]嘧啶-4-基)-2,3-二氢-1H-茚-1-基)氰胺抑制剂（2）及其甲基化类似物（3）虽均为强效抑制剂，但展现出截然不同的作用机制（共价与非共价）与结合模式（Casimiro-Garcia等，《Journal of Medicinal Chemistry》, 2018）。受这一有趣现象的启发，我们采用多尺度方法，对JAK3前沿口袋Cys909与氰胺类抑制剂之间的反应机制开展表征。量子力学/分子力学（quantum mechanics/molecular mechanics, QM/MM）模拟结果表明：仅当保守水分子作为质子穿梭体辅助反应时，抑制剂2才可与Cys909快速形成异硫脲加合物；而对抑制剂2的2,3-二氢-1H-茚-1-基氰胺基团进行甲基化，会通过取代该保守水分子，阻碍异硫脲加合物的生成。此外，我们通过元动力学（metadynamics）与热力学积分（thermodynamic integration）模拟，探究了2,3-二氢-1H-茚-1-基氰胺类化合物的可及备选结合构象的相对丰度，阐明了甲基化对其相对结合模式偏好性的调控作用。本多尺度方法不仅为氰胺类抑制剂的作用机制提供了全新的化学见解，同时也为研究药物与分子靶点的相互作用提供了一套高效可行的研究范式。