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

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.


1 H
/ 13 C NMR Spectra and HPLC Traces of Compounds

Figure S1 Overlaid 1 HFigure S2
Figure S1 Overlaid 1 H, 15 N-SOFAST-HMQC (which is a fast version of HSQC) NMR spectra of KRAS G12C interacting with the reference molecules (ARS-853 and ARS-1620), molecules 1-4a,b are shown.The free KRAS G12C protein spectra are colored blue, while the ligand-bound spectra are red.Residues showing the most significant chemical shift perturbation are shown.

Figure S3 Figure
Figure S3Determination of KI and kinact for the interaction of 1a-4b covalent probes with KRAS G12C .Calculated kobs values were plotted against the concentration of the probes and and KI and kinact were calculated directly from non-linear regression according to the kobs -c function as follows:k obs = k inact •c K I +c.

Figure S5
Figure S5 Transformation steps in the thermodynamic integration.The softcore atoms are shown with dashed lines with colours matching the corresponding steps.

Table S2
Calculated kobs values for the 1a-4b compounds according to the covalent engagement of KRAS G12C at different measured concentrations of the covalent probes (from 20 M to 160 M).Data are shown as results of duplicated experiments.