To further investigate the behavior of LY3537982 and to confirm GDC-6036 functionality, we changed the assay scheme to monitor KRAS stability in complex with these inhibitors. In these assays, no external protein (SOS cat ) is used and only KRAS and inhibitors are present. First, we used the FRET-Probe approach for KRAS(G12C) to assess the thermal stability with both GDC-6036 and LY3537982. In this assay, we observed an exceptionally high stability increase with both inhibitors (Figure ). In the 1:1 complex (25 nM) with KRAS(G12C) and these covalent inhibitors, ΔT m over 25 °C was observed, but the absolute T m value in the presence of either GDC-6036 and LY3537982 could not be determined (Figure ). Instead of artificially lowering the KRAS thermal stability, we focused our effort to study KRAS(G12C), (G12C/H95L), and (G12C/Y96D) chemical stability utilizing the FRET-Probe. In this system, time dependent KRAS (25 nM) denaturation is monitored in 1 M urea solution after KRAS preincubation with varying inhibitor concentration. In case of GDC-6036, observed IC 50 values are highly comparable to those observed with nucleotide exchange assay earlier (Figure ). GDC-6036 stabilized KRAS mutants in order G12C, G12C/H95L, and G12C/Y96D, and the observed IC 50 values were 5.9 ± 0.9, 15.0 ± 3.4, and 106 ± 6 nM, respectively. To our surprise, LY3537982 matched those values observed in the nucleotide exchange assay, even though the IC 50 values in that assay were lower than the theoretical minimum. Again, IC 50 values KRAS(G12C) and (G12C/H95L) were similarly observed in the picomolar range, 368 ± 65 and 345 ± 91 pM, and for G12C/Y96D at low nanomolar (1.6 ± 0.1 nM), respectively (Figure ). Overall, these results rule out that the SOS cat would have artificially caused the unexpectedly low IC 50 values with LY3537982, and together with the nucleotide exchange, they indicate that LY3537982 displays a comparable unchanged interaction with KRAS(G12C/H95L) as with KRAS(G12C), but some activity compromising effect is observed in case of KRAS(G12C/Y96D). On the other hand, activity of GDC-6036 is negatively affected by both H95L and Y96D co-mutations.