Fig. 5
From: Targeting KRASG13C with cyclic linker based inhibitors to explore warhead orientation
The nucleotide-analogues lock RAS in an inactive state. (A): GEF-catalyzed nucleotide exchange of KRASWT:GDP, KRASG13C:GDP and KRASG13C-7a and 7b. In the assay, 5 µM of the preparatively modified or unmodified KRAS (residues 1–169) was incubated with 10 µM mant-dGDP, followed by the addition of SOS at varying concentrations (0.25 µM and 0.5 µM; black and red curves, respectively). (B): Pull-down-Assay. Whereas KRASG13C:GDP can be pulled down by GST-tagged Raf RBD in the presence of GppNHp and GppNHp/SOS, KRASG13C-7a/7b:GDP cannot become activated and consequently cannot bind to the Raf RBD, neither in the presence of GppNHp only nor GppNHp and SOS. The unprocessed SDS gel is shown in Figure S2.
