A non-conserved histidine residue on KRAS drives paralog selectivity of the KRASG12D inhibitor MRTX1133.

MRTX1133 is the first non-covalent inhibitor against the KRASG12D mutant that demonstrated specificity and potency in pre-clinical tumor models. Here, we used isogenic cell lines expressing a single Ras allele to evaluate the selectivity of this compound. In addition to KRASG12D, MRTX1133 showed significant activity against several other KRAS mutants as well as wildtype KRAS protein. In contrast, MRTX1133 exhibited no activity against both G12D and wildtype forms of HRAS and NRAS proteins. Functional analysis revealed that the selectivity of MRTX1133 towards KRAS is associated with its binding to H95 on KRAS, a residue that is not conserved in HRAS and NRAS. Reciprocal mutation of amino acid 95 among the three Ras paralogs resulted in reciprocal change in their sensitivity towards MRTX1133. Thus, H95 is an essential selectivity handle for MRTX1133 towards KRAS. Amino acid diversity at residue 95 could facilitate the discovery of pan-KRAS inhibitors as well as HRAS and NRAS paralog-selective inhibitors.