Adagrasib showed no binding to HRAS(WT) or NRAS(WT) in the QRET nucleotide exchange assays (IC50 >10 µM) (Figure ). Sotorasib, on the contrary, displayed IC50 values of 1.2-1.3 µM for HRAS(WT) and NRAS(WT) (Figure ). Furthermore, TR-FRET nucleotide exchange and cell viability assays indicate that the effect of sotorasib might be stronger on NRAS(WT) compared to KRAS(WT) (Figure and S5). We could further observe a stabilization of NRAS(WT) by sotorasib but not with adagrasib (Figure and). Together these data suggest that sotorasib might have comparable efficacy against NRAS(G12C) and HRAS(G12C) mutants as against KRAS(G12C), which is in line with the very recent clinical findings. To assess the potential relevance of targeting cancers harboring NRAS(G12C) and/or HRAS(G12C) mutants we explored the Catalogue of Somatic Mutations in Cancer (COSMIC v99) (Figure ). In the case of KRAS, G12C mutations account for 11.6% (5519/47503) of all missense mutations. G12C mutations are found less frequently for NRAS and HRAS in the database, representing 2.7% (240/8835) and 1.6% (43/2614) of all NRAS and HRAS missense mutations, respectively (Figure ). While the number of patients with non-KRAS(G12C) mutations is relatively small, our analysis confirms their existence. These patients may potentially benefit from treatment with sotorasib. Further investigation is warranted to determine whether these mutations can be considered as disease-relevant drivers. 59 To further demonstrate the isoform-agnostic binding behavior of sotorasib, we performed a panel of assays to explore its binding specificity. Cellular RAS-GTP assays confirmed that sotorasib targets NRAS(G12C) and KRAS(G12C) to a similar extent, while adagrasib inhibition is KRAS-specific (Figure and). Cell viability assays further confirmed that HRAS(G12C) can be targeted by sotorasib as well (Figure ). We additionally confirmed the covalent attachment of sotorasib to NRAS(G12C) and HRAS(G12C) by a liquid chromatography-mass spectrometry (LC-MS) assay (Figure ). The highest peak (12.2 min) from the separation, was analyzed by MS (Figure ), and in case of both NRAS(G12C) (Figure ) and HRAS(G12C) (Figure ), 560.5 Da increase in molecular weight (MW) was observed in the presence of sotorasib (calculated MW 560.59 Da) (Figure and S10). Strikingly, the only residue that differs between the three RAS isoforms within the SII-P is found in position 95, which is His, Leu or Gln in case of KRAS, NRAS, and HRAS, respectively (Figure and S11).