Fig. 2: biochemical characterizations of KM12 and KM12-AM.
From: Identification and characterization of binders to a cryptic and functional pocket in KRAS
A Overlay and overlap of KM12-AM (PDB code 9GLZ) and cRAF1RBD-CRD (PDB code 6QUU) binding modes on RAS (left panel) and cRAF constructs used in this study (right panel). B cRAF proteins mentioned in (A) were tested in the GMPPnP loaded KRASG12D/KM12-AM TR-FRET assay and potential competition determined by quantification of IC50 (concentration needed for 50% maximal inhibition achieved with the respective proteins tested) and Ainf (maximum amplitude of the effect) values, as described in Methods. While CRD-containing constructs appear to result in (close to) complete competition with binding of KM12-AM, RBD alone results in only partial displacement (~50%). The assay is validated by titration of untagged KM12-AM or GMPPnP-loaded KRASG12D that both fully displace with IC50 (Ki) in range with their affinity as measured by SPR (see Tables 1 and 2). TR-FRET curves, Ki and Ainf values shown (n = 2 biological independent replicates) are derived from one representative experiment. This experiment was repeated at least two times with similar results. Source Data is provided as a source Data file. C A-B-A SPR experiment studies using MRTX1133 (right panels) or not (DMSO, left panels) as a switch II pocket binder during A-step, for KD determination of either cRafRBD (by steady-state affinity measurement, top panels) or KM12 (by kinetic affinity measurement, bottom panels) added in B-Step, on immobilized GMPPnP-loaded KRASG12D, as described in Methods. For steady-state affinity measurements, insets representing the corresponding Req versus concentration curves have been incorporated. For kinetic affinity measurements, sensorgrams are reported as overlay of fitted (in black for each concentration) and experimental (one color per concentration) curves. KD, Rmax and Chi2 values derived from one representative experiment are reported on the right side of each panel.
