Small-Molecule Inhibition of KRAS through Conformational Selection

Mutations in KRASaccount for about 20% of human cancers. Despitethe major progress in recent years toward the development of KRASinhibitors, including the discovery of covalent inhibitors of theG12C KRAS variant for the treatment of non-small-cell lung cancer,much work remains to be done to discover broad-acting inhibitors totreat many other KRAS-driven cancers. In a previous report, we showedthat a 308.4 Da small-molecule ligand [(2R)-2-(N'-(1H-indole-3-carbonyl)hydrazino)-2-phenyl-acetamide] bindsto KRAS with low micro-molar affinity [Chem. Biol. Drug Des. 2019; 94(2):1441-1456]. Bindingof this ligand, which we call ACA22, to the p1 pocketof KRAS and its interactions with residues at beta-strand 1 and theswitch loops have been supported by data from nuclear magnetic resonancespectroscopy and microscale thermophoresis experiments. However, theinhibitory potential of the compound was not demonstrated. Here, weshow that ACA22 inhibits KRAS-mediated signal transductionin cells expressing wild type (WT) and G12D mutant KRAS and reduceslevels of guanosine triphosphate-loaded WT KRAS more effectively thanG12D KRAS. We ruled out the direct effect on nucleotide exchange oreffector binding as possible mechanisms of inhibition using a varietyof biophysical assays. Combining these observations with binding datathat show comparable affinities of the compound for the active andinactive forms of the mutant but not the WT, we propose conformationalselection as a possible mechanism of action of ACA22.