An electrophilic warhead library for mapping the reactivity and accessibility of tractable cysteines in protein kinases

Highlights

• Covalent fragment library with diverse warheads for profiling cysteine reactivity was developed.

• Affinity profiles of various kinases from each major phylogenetic branch were characterised.

• Reactivity and accessibility of JAK3 C909 and MELK C70 were evaluated by the covalent probe library.

• Suitable warheads were equipped to a known hinge binder core fragment for retrospective and prospective validation.

• Potent JAK3 and MELK inhibitors were identified with low-nanomolar activity.

Abstract

Targeted covalent inhibitors represent a viable strategy to block protein kinases involved in different disease pathologies. Although a number of computational protocols have been published for identifying druggable cysteines, experimental approaches are limited for mapping the reactivity and accessibility of these residues. Here, we present a ligand based approach using a toolbox of fragment-sized molecules with identical scaffold but equipped with diverse covalent warheads. Our library represents a unique opportunity for the efficient integration of warhead-optimization and target-validation into the covalent drug development process. Screening this probe kit against multiple kinases could experimentally characterize the accessibility and reactivity of the targeted cysteines and helped to identify suitable warheads for designed covalent inhibitors. The usefulness of this approach has been confirmed retrospectively on Janus kinase 3 (JAK3). Furthermore, representing a prospective validation, we identified Maternal embryonic leucine zipper kinase (MELK), as a tractable covalent target. Covalently labelling and biochemical inhibition of MELK would suggest an alternative covalent strategy for MELK inhibitor programs.