A study published in Nature (2013) describes the development of small molecules that irreversibly bind to the oncogenic mutant K-Ras(G12C). These compounds target a newly identified allosteric site, the switch-II pocket (S-IIP), which is not present in the wild-type protein. The S-IIP is located between the central β-sheet of Ras and the α2 (switch-II) and α3 helices. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, shifting the nucleotide preference from GTP to GDP and impairing binding to Raf. This allosteric regulation is achieved by exploiting the unique nucleophilicity of the cysteine residue in the mutant, allowing selective binding to the mutant over the wild-type protein. The study used a disulphide-fragment-based screening approach called tethering to identify a chemical starting point. Fragments 6H05 and 2E07 showed the greatest modification of K-Ras(G12C). Further optimization led to the development of acrylamides and vinyl sulphonamides, which showed improved potency. These compounds were tested for off-target specificity using intact protein mass spectrometry, showing selective modification of K-Ras(G12C) over bovine serum albumin (BSA). Structural analysis revealed that the S-IIP is a previously unrecognized pocket in Ras, and compounds binding to this site disrupt the active conformation of Ras. The study also showed that these compounds reduce the affinity of K-Ras(G12C) for GTP, increasing its preference for GDP. This effect was confirmed through EDTA-catalyzed off-exchange reactions, where the presence of the compounds significantly decreased GTP affinity relative to GDP. The compounds were tested in lung cancer cell lines, showing decreased viability and increased apoptosis in cell lines expressing the G12C mutation. The half-maximum effective concentration (EC50) for compound 12 in H1792 cells was found to be tenfold lower than that of compound 10, indicating its higher potency. The study also demonstrated that these compounds block SOS-catalyzed nucleotide exchange, further impairing Ras signaling. Overall, the study provides a proof-of-concept for the use of genotype-specific inhibitors targeting the S-IIP pocket in K-Ras(G12C)-driven cancer. The findings suggest that these compounds could serve as a starting point for drug discovery efforts targeting K-Ras(G12C) and potentially other Ras alleles.A study published in Nature (2013) describes the development of small molecules that irreversibly bind to the oncogenic mutant K-Ras(G12C). These compounds target a newly identified allosteric site, the switch-II pocket (S-IIP), which is not present in the wild-type protein. The S-IIP is located between the central β-sheet of Ras and the α2 (switch-II) and α3 helices. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, shifting the nucleotide preference from GTP to GDP and impairing binding to Raf. This allosteric regulation is achieved by exploiting the unique nucleophilicity of the cysteine residue in the mutant, allowing selective binding to the mutant over the wild-type protein. The study used a disulphide-fragment-based screening approach called tethering to identify a chemical starting point. Fragments 6H05 and 2E07 showed the greatest modification of K-Ras(G12C). Further optimization led to the development of acrylamides and vinyl sulphonamides, which showed improved potency. These compounds were tested for off-target specificity using intact protein mass spectrometry, showing selective modification of K-Ras(G12C) over bovine serum albumin (BSA). Structural analysis revealed that the S-IIP is a previously unrecognized pocket in Ras, and compounds binding to this site disrupt the active conformation of Ras. The study also showed that these compounds reduce the affinity of K-Ras(G12C) for GTP, increasing its preference for GDP. This effect was confirmed through EDTA-catalyzed off-exchange reactions, where the presence of the compounds significantly decreased GTP affinity relative to GDP. The compounds were tested in lung cancer cell lines, showing decreased viability and increased apoptosis in cell lines expressing the G12C mutation. The half-maximum effective concentration (EC50) for compound 12 in H1792 cells was found to be tenfold lower than that of compound 10, indicating its higher potency. The study also demonstrated that these compounds block SOS-catalyzed nucleotide exchange, further impairing Ras signaling. Overall, the study provides a proof-of-concept for the use of genotype-specific inhibitors targeting the S-IIP pocket in K-Ras(G12C)-driven cancer. The findings suggest that these compounds could serve as a starting point for drug discovery efforts targeting K-Ras(G12C) and potentially other Ras alleles.