Furthermore, we conducted additional simulations of both GDP-bound KRAS(G12C) and G12C/Y96D SII-P apo structures, to observe if the mutation itself has any impact on the binding pocket (Figures and). The resistance-mediating double mutation G12C/Y96D appears to constrict the SII-P compared to G12C (Figure ). This is highlighted by the reduced availability of the SII-P (expressed as the % of the frames where it is found) and its decreased volume (expressed as median volumes). The pocket with a median volume of 64.1Å 3 is only available in 27.1% of the analyzed frames according to SiteMap, compared to 96.0Å 3 and 59.6% in the context of KRAS(G12C). Remarkably, with sotorasib bound in the SII-P of KRAS(G12C), the pocket volume median is increased by a factor of two to a median volume of 211.6Å 3 , confirming the dynamic and inducible nature of this pocket. A detailed view on the simulation results allows us to identify a putative molecular mechanism of the inaccessibility of the SII-P. The mutated Y96D appears to promote a novel intra-protein interaction, a salt-bridge between D96 and R68; thereby influencing the size and accessibility of the pocket (Figure and). In addition, this suggests a competitive mechanism where the D96-R68 interaction may preclude the inhibitor's own interaction with R68. With sotorasib, this interaction was indeed compromised in simulations with G12C/Y96D (Figure ). The negative effect of the Y96D mutation on nucleotide exchange might also be connected to the interaction of SOS E1002 in its "active" conformation (Figure ). Consequently, the de novo salt-bridge between D96 and R68 may reduce the KRAS-SOS interaction. In a next step we evaluated the effect of mutations of the residues R68 and Q99 in a cell viability assay (Figure ). Q99 is located close to the SII-P in the α3-helix (Figure ), and has been shown to confer resistance to adagrasib, while not affecting the clinical activity of sotorasib. We could indeed confirm this with a ~20-fold shift in the potency of adagrasib in the context of KRAS(G12C/Q99L)-expressing cells (Figure and Table ). In addition, our MD simulations with KRAS(WT) show mainly water-mediated interactions of both inhibitors with Q99 (Figure ). As the Q99L mutation only affects adagrasib binding, this mutation may distort the neighboring H95 (in) conformation, which is critical for adagrasib, thus having an indirect effect on adagrasib binding (Figure ). Clinically reported mutations at the R68 residue in KRAS(G12C) include R68S and R68M. The R68S mutation blocks drug binding of both adagrasib and sotorasib, leading to resistance to these inhibitors. In contrast, R68M has been reported to confer resistance to sotorasib but remain sensitive to adagrasib. Again, we could confirm the clinical observation by demonstrating that sotorasib's effect on cell viability was drastically reduced in cells expressing KRAS(G12C/R68M), while adagrasib's effect remained unchanged (Figure ). These results are in line with our simulation observations, where only sotorasib displays direct interactions to R68 with high frequency (Figure ). We speculate that while the integrity of the SII-P is retained by the similar-sized methionine mutation (R68M), the shorter serine (R68S) might be detrimental to the pockets integrity. The R68S mutation likely leads to a compromised shielding of the hydrophobic SII-P from solvent exposure, affecting the pocket interactions with both inhibitors (Figure ). Further research is needed to disclose if these R68 mutations could have an impact on nucleotide exchange via affecting an important interaction with SOS (Figure ).