Combining Fundamental Kinetics and Standard Alkylation Assays to Prioritize Lead-Like KRAS G12C Inhibitors.

We measure the fundamental rate constants of internally discovered KRAS G12C inhibitors to demonstrate how kinetic analyses can be integrated with standard biochemical and cell-based assays for more optimal biophysical compound prioritization. In this proof-of-principle study, we characterize three irreversible covalent inhibitors targeting the mutant cysteine at the switch II binding pocket. We estimate the three fundamental kinetic rate constants ( , , ) that define the contributions of affinity and inactivation to the overall alkylation rate for a more complete biophysical characterization. These parameters are typically unavailable and are generally approximated by a single overall alkylation rate constant ( ), where the relative contributions of affinity and inactivation remain unknown. We demonstrate that the alkylation rate constant sacrifices valuable mechanistic information leading to higher risk of suboptimal compound prioritization. Estimation of the three fundamental kinetic rate constants was made possible by developing label-free surface plasmon resonance (SPR) methodologies adapted to measure transient binding using standard SPR equipment. Binding enthalpy was measured by Eyring transition state analysis, which can also benefit compound prioritization. We illustrate how these methodologies can enable more reliable prioritization of lead-like compounds when combined with standard orthogonal assays in a typical lead optimization setting.