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.