In addition to the irreplaceable role of KRAS H95 for adagrasib, our MD simulations showed close contacts and high interaction frequency of both inhibitors with Tyr-96 (Figure ), an additional secondary mutation site that has been reported to confer resistance in the clinic. To validate the individual importance of KRAS H95 and Y96, we created two single mutant constructs; KRAS(H95L) reflecting the native SII-P present in NRAS(WT), and KRAS(Y96D) linked to the acquired resistance with G12C targeting inhibitors. During the characterization of these mutants without inhibitor, we found KRAS(Y96D) to have a negative impact on SOS cat induced nucleotide exchange (Figure ). Even more importantly KRAS(Y96D), but not KRAS(H95L), showed significantly reduced thermal stability in comparison to KRAS(WT) (Figure and Table ). When the nucleotide exchange assay was performed with inhibitors, we observed a reduced inhibition with sotorasib using KRAS(H95L) compared to KRAS(WT) (Figure and Table ). In the case of adagrasib, however, both mutants were resistant to nucleotide exchange inhibition, indicating non-existing binding at the tested concentrations.