Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

Targeting non-catalytic cysteine residues with irreversible electrophiles improved the selectivity and potency of various inhibitors. This strategy has been applied to scaffolds targeting diverse proteins and in all cases results in an irreversible bond between the cysteine thiol and the electrophile. Recently, several acrylamide-based irreversible kinase inhibitors have been advanced to clinical trials, and the potency and selectivity of these compounds is greatly dependent on the covalent modification of a cysteine residue found in the enzyme active site. However, concerns about off-target modification motivated us to seek out strategies to develop reversible cysteine-targeting small molecule inhibitors.;Here, we describe the development of cysteine-targeting inhibitors that form a covalent bond with their kinase target, but do so in a reversible manner. The covalent bond to a cysteine thiol is long-lived in the context of the folded active-site, but readily broken with off target proteins. We show that electron-deficient olefins, including acrylamides, can be chemically tuned to react with cysteine thiols in a rapidly reversible manner. By installing a nitrile group, the intrinsic reactivity of the olefins towards thiols increases while at the same time the formation of irreversible adducts is eliminated. These properties result in an inhibitor with sustained occupancy of the active site of the kinase due to covalent bond formation as well as high selectivity for its target and minimal off-target effects. We specifically combined 2-cyanoacrylates and 2-cyanoacrylamides with a scaffold that binds promiscuously to protein kinases and to obtain highly selective and reversible, yet covalent inhibitors of the C-terminal domain of p90 ribosomal S6 kinases (RSK-CTD).;Our results establish a chemical framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides. Future studies will seek to define the druggable cysteinome and apply this technology to generate novel small molecules inhibitors for several cysteine containing therapeutic targets. We believe that the application of reversible covalent technology to drug discovery and development will yield compounds with both dramatically increased potency and minimal toxicity.