Sotorasib and adagrasib are clinically validated KRAS(G12C) inhibitors occupying the same SII-P, and both rely on covalent binding to Cys12 in an inactive, GDP-bound KRAS (Figure ). To better understand the similarities and differences between these compounds, we measured the binding of sotorasib and adagrasib to KRAS(G12C) and beyond with biochemical assays. First, we used a nucleotide exchange assay to analyze the inhibitors' ability to lock RAS into its GDP-bound state and thereby block SOS cat mediated nucleotide exchange. With KRAS(G12C) used as a control, we found that IC50 values of both inhibitors were within the expected low nanomolar range (Figure , Table ). All nucleotide exchange results are listed in Table . Secondly, we used a TSA to analyze RAS thermal stability and inhibitor induced stability increase. The use of two independent external fluorescent probes, Protein-Probe (PP) and 8-anilinonaphthalene-1-sulfonic acid (ANS), provided consistent results (Figure ). Both techniques show that sotorasib induces a slightly higher increase in KRAS(G12C) thermal stability compared to adagrasib (Table ). All TSA results are listed in Table . ). A keydifference in their binding mode appears on KRAS-specific 3-helix residue H95, which is in outconformation with sotorasib and in in-conformation with adagrasib (zoom-in illustrations). Sotorasib is depicted in a green ball and stick model, with the protein highlighted in blue cartoon; adagrasib is shown in a yellow ball and stick model, with protein highlighted in purple cartoon. The illustration includes GDP in a ball and stick model with teal carbons, a Mg 2+ -ion is shown as green sphere; C-atoms of C12, M67, and H95 as orange, yellow, and blue spheres, respectively. (B) Concentration-dependent inhibition of SOS cat -mediated guanine nucleotide exchange of 100 nM RAS after 30 min preincubation with sotorasib and adagrasib at RT (mean ± SD, n = 3). (C) Adagrasib surface plasmon resonance sensorgram showing concentration-dependent binding to KRAS(WT) (mean ± SD, n = 3). (D) G-LISA assay of GTP-bound RAS (see Materials ad Methods) using starved cells incubated with adagrasib and sotorasib followed by mitogenic stimulation and monitoring of RAS-GTP levels of the KRAS(WT) and KRAS(G12C) (mean ± SD, n = 3). When analogous nucleotide exchange and TSA analyzes were performed with KRAS(WT), adagrasib showed a clearer effect compared to sotorasib (Figure , S2A, and S3). In nucleotide exchange assays with KRAS(WT), adagrasib exhibited IC50 values in the range of 200-300 nM as measured by QRET and TR-FRET (Figure , Figure , Table , and Table ). In contrast, sotorasib's effect was clearly diminished with KRAS(WT), with a IC50 falling in the micromolar range (Figure and, Table ). In TSA, both adagrasib and sotorasib stabilize KRAS(WT) only at very high concentrations (Table ). At low micromolar concentrations, sotorasib especially displayed clear luminescence quenching in these assays, we further confirmed these results using surface plasmon resonance (SPR) (Figure and). SPR revealed a concentration-dependent response of adagrasib with KRAS(WT), while no response was seen with sotorasib (Figure and S4). The observed Kd of adagrasib for KRAS(WT) was 0.78 ± 0.05 µM, in line with our observations in nucleotide exchange assays.