Contribution of Noncovalent Recognition and Reactivity to the Optimization of Covalent Inhibitors: A Case Study on KRasG12C

Covalent drugs can carry electrophilic groups that enable them to chemically modify their targets, offering the potential to target previously undruggable proteins with high potency. The covalent binding of drug-sized molecules involves both noncovalent recognition through secondary interactions and a chemical reaction that forms a covalent complex. Optimizing their covalent mechanism of action requires consideration of both types of interactions. These binding steps can be characterized by an equilibrium dissociation constant (KI) for noncovalent interactions and a reaction rate constant (kinact) for covalent binding, with both being influenced by the ligand’s warhead and scaffold. To investigate the contribution of these two steps, we focused on the KRASG12C mutant, a key oncogenic variant of KRAS. Using a synthetically accessible nonchiral core derived from ARS-1620, we incorporated four different warheads and a previously described KRAS-specific basic side chain. This enabled the synthesis of novel covalent KRASG12C inhibitors, which we tested for their binding affinity and biological effects through various biophysical and biochemical assays. Our findings helped us understand how scaffold and warhead influence both the noncovalent and covalent binding processes. We found that the atropisomeric core of ARS-1620 is not essential for inhibiting KRASG12C, the basic side chain has minimal impact on binding, and the warheads primarily influence covalent reactivity without affecting noncovalent binding. This analysis reveals key structural determinants for effective covalent inhibition and may guide the design of future covalent therapeutic agents.