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 (<b>1</b>). Recently, 2,3-dihydro-1<i>H</i>-inden-1-ylcyanamides have been developed as novel JAK3 inhibitors. Among them, the <i>N</i>-(6-(7<i>H</i>-pyrrolo[2,3-<i>d</i>]pyrimidin-4-yl)-2,3-dihydro-1<i>H</i>-inden-1-yl)cyanamide inhibitor (<b>2</b>) and its methylated analogue (<b>3</b>), while being potent inhibitors, displayed different mechanisms of action (covalent vs noncovalent) and binding modes (Casimiro-Garcia et al., <i>J Med Chem</i> <b>2018</b>). 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 <b>2</b> 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-1<i>H</i>-inden-1-ylcyanamide moiety of <b>2</b> 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-1<i>H</i>-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.