Figure 4. Fluorescent polarization assay shows the competitive displacement of GDP in KRAS* protein.
(A) Fluoro-GTP binds in GTP-binding site to yield a high fluorescence polarization (FP) value. With the displacement of small molecule inhibitors, fluoro-GTP shows a small FP value. (B) The structure and activity of selected covalent inhibitors. Covalent inhibitors selectively occupy GTP binding pocket of KRAS*, but not KRASG12V protein. Compound YZ0571-1, bearing a 2-chloroacetyl chloride moiety, has optimal activity in the fluorescence polarization assay and cell viability assays. (C) The structure and activity of selected covalent inhibitors. Covalent inhibitors selectively displace GDP from KRAS* protein, but not KRASG12V protein. (D) Compound YZ011 is covalently docked in KRAS* (The docking pose is shown in figure). WaterMap shows the thermodynamic-properties of hydration site around the protein active site. Stable hydration sites (ΔG < 3 kcal/mol) are shown in green, significantly unstable hydration sites (ΔG > 5 kcal/mol) are shown in red, and moderately unstable sites (3 kcal/mol < ΔG < 5 kcal/mol) are shown in brown. Compound YZ0711 displaces nearly all high-energy waters in the binding site.