Covalent Ligand Directed Release - more than just covalent inhibitors

Targeted covalent inhibitors are an important class of drugs and chemical probes, the majority of which specifically target cysteine residues. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here I will present α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition–elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition–elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent and chemiluminescent probes. In the reverse direction we can also use this chemistry for site-specific irreversible labeling of target cysteine, where the directing ligand leaves, allowing us to tag enzymatically active proteins with various functionalities. We used this tactic to label BTK with a variety of functional tags in vitro and in cells. Altogether this is a very versatile chemistry that unlocks many new applications in chemical biology.