Extended Data Fig. 2: Biochemical and cellular effects of the KRAS inhibitor.
From: Pan-KRAS inhibitor disables oncogenic signalling and tumour growth
a,b, KRAS variants were loaded with GDP or GCP and subjected to isothermal titration calorimetry (ITC) with the KRASi. c, The association (on) and dissociation (off) rate constants of drug binding to GDP-loaded KRAS variants (mean ± s.e.m., n = 3). d, Effect of KRASi treatment on nucleotide exchange under intrinsic conditions. e, KRAS variants were loaded with the non-hydrolyzable GTP analogue GMPPNP (GNP) and reacted with a GST-tagged RBD domain of CRAF. The mixtures were analyzed by GST pull-down and immunoblotting to determine the ability of the drug to displace effectors from the active state of KRAS. Pull-down reactions were analyzed by immunoblotting and quantified by densitometry. f, Extracts from HEK293 cells expressing the indicated variants treated with the KRASi for 2 h were subjected to RBD pull down and immunoblotting. The levels of total and GTP-bound KRAS were quantified by densitometry. g, The indicated KRAS variants were loaded with GDP and subjected to ITC in the presence of increasing concentrations of the KRASi. h, Effect of KRASi treatment on GTP hydrolysis by KRAS under intrinsic conditions or in the presence of the GAP related domain (GRD) of NF1 (mean ± s.e.m., n = 3). i, The indicated KRAS G12C mutant cell lines were treated for 2 h and their extracts were subjected to RBD pull-down and immunoblotting to determine the level of active KRAS. j, H2122 cells were treated as shown and analyzed to determine the effect on KRAS activation. A representative of two independent repeats is shown in a, b, d-f, i and j.
