Fig. 1: Monobody 12VC1 is selective to the G12C and G12V RAS mutants.
From: Selective and noncovalent targeting of RAS mutants for inhibition and degradation
a Yeast display binding measurements of 12VC1 to the wild-type RAS isoforms and KRAS mutants in GTPγS (50 nM) or GDP (100 nM) bound forms. Each data point represents one technical replicate. Binding of biotinylated RAS was detected with neutravidin-Dylight 650. Binding signals in arbitrary units (arb. units.) are the median fluorescence intensity of the fluorescent population in the 75–95th percentile of the Dylight 650 detection channel of a flow cytometer. The mean and SD of three technical replicates are plotted. b BLI sensorgrams of 12VC1 binding against different KRAS mutants and WT in GTPγS or GDP-loaded state. The experimental BLI traces (black) for G12C and G12V in the GTPγS-bound state were globally fitted (red) using the 1:1 binding kinetic model. Steady-state global analysis was performed for KRAS(WT). The KD values shown are the mean ± SD from n = 3, technical replicates. N.D., not determined due to too weak binding. c Colocalization of mCherry fused 12VC1 (pseudo-color magenta) with overexpressed EGFP fused KRAS(WT) and mutants (pseudo-color green). Scale bar denotes 10 μm. The graphs on the right show the fluorescence intensity profiles under the yellow lines across the microscopy images. d Pull-down assay with biotinylated 12VC1 and GST-RBD of lysates of cell lines containing KRAS(G12C) and (G12V) and (WT) with and without ARS1620 and EGF treatments. Captured proteins (12VC1 pull down and RBD pull down) were probed using immunoblotting with a pan-RAS antibody. The amounts of RAS, total ERK, and pERK in the input lysate (WCL) were assessed using immunoblotting. Representative data shown (technical replicate, n = 2). e Affinity purified-mass spectrometry (AP-MS) analysis using 12VC1 as a capture reagent. The SAINT score50 and enrichment score of each protein that was uniquely present in the affinity purified sample of KRAS(G12V) harboring cell line (PATU8902) over non-KRAS mutant cell line (A375) are plotted. The data showed that 12VC1 captured overwhelmingly more KRAS(G12V) from PATU8902 than from A375. The red dot represents SAINT score and enrichment score of KRAS(G12V). The dashed line signifies the cutoff for 5% false detection rate (FDR).
