Covalent drugs might bear electrophiles to chemically modify their targets and have
the potential to target previously undruggable proteins with high potency. Covalent
binding of drug-size molecules includes a noncovalent recognition provided by secondary
interactions and a chemical reaction leading to covalent complex formation. Optimization
of their covalent mechanism of action should involve both types of interactions. Noncovalent
and covalent binding steps can be characterized by an equilibrium dissociation constant
(KI) and a reaction rate constant (kinact), respectively, and they are affected by
both the warhead and the scaffold of the ligand. The relative contribution of these
two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant
of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620
that was equipped with four different warheads and a previously described KRAS-specific
basic side chain. Combining these structural changes, we have synthesized novel covalent
KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by
various biophysical and biochemical assays. These data allowed us to dissect the effect
of scaffold and warhead on the noncovalent and covalent binding event. Our results
revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C
inhibition, the basic side chain has little effect on either binding step, and warheads
affect the covalent reactivity but not the noncovalent binding. This type of analysis
helps identify structural determinants of efficient covalent inhibition and may find
use in the design of covalent agents.
