Small-Molecule Inhibition of KRAS through Conformational Selection

Abstract

Mutations in KRAS account for about 20% of human cancers. Despite the major progress in recent years toward the development of KRAS inhibitors, including the discovery of covalent inhibitors of the G12C KRAS variant for the treatment of non-small-cell lung cancer, much work remains to be done to discover broad-acting inhibitors to treat many other KRAS-driven cancers. In a previous report, we showed that a 308.4 Da small-molecule ligand [(2R)-2-(N'-(1H-indole-3-carbonyl)hydrazino)-2-phenyl-acetamide] binds to KRAS with low micro-molar affinity [Chem. Biol. Drug Des.2019; 94(2):1441-1456]. Binding of this ligand, which we call ACA22, to the p1 pocket of KRAS and its interactions with residues at beta-strand 1 and the switch loops have been supported by data from nuclear magnetic resonance spectroscopy and microscale thermophoresis experiments. However, the inhibitory potential of the compound was not demonstrated. Here, we show that ACA22 inhibits KRAS-mediated signal transduction in cells expressing wild type (WT) and G12D mutant KRAS and reduces levels of guanosine triphosphate-loaded WT KRAS more effectively than G12D KRAS. We ruled out the direct effect on nucleotide exchange or effector binding as possible mechanisms of inhibition using a variety of biophysical assays. Combining these observations with binding data that show comparable affinities of the compound for the active and inactive forms of the mutant but not the WT, we propose conformational selection as a possible mechanism of action of ACA22.

Figure 1

(A) Structure of the ACA22 ligand. (B) NMR-derived structure of the ACA22/KRAS complex (PDB ID 6V5L). The regions involved in KRAS activation and effector binding are highlighted: phosphate (P) binding loop (residues 10–17), switch 1 (residues 30–38), effector binding region (residues 32–40), and switch 2 (residues 60–76). (C) Interaction between ACA22 (green sticks) and KRAS residues (gray sticks) surrounding pocket p1. KRAS residues poised to make hydrogen bonding and van der Waals contacts with the ligand are labeled.

Figure 2

(A) Western analysis of whole cell lysates from BHK cells expressing G12D KRAS treated for 18 h with vehicle (DMSO) or the indicated concentrations of ACA22 in a serum-starved condition. Shown are levels of p-cRaf, p-ERK, t-ERK, p-AKT, t-AKT, p-RSK, as well as vinculin as loading controls. (B) The same experiments in cells ectopically expressing WT KRAS treated with DMSO or ACA22 for 18 h, with and without stimulation by 5 ng/mL EGF for 5 min before lysis for Western analysis. The MEK inhibitor U0126 (10 μM) was used as a positive control. Note that phosphorylation levels were measured after 18 h of incubation of cells with compound in the serum-starved condition (sufficient time for potential GTP hydrolysis to deplete the GTP-RAS pool) but immediately after stimulation (i.e., without enough time for GTP hydrolysis to deplete the level of GTP-RAS). Values are normalized with respect to DMSO and averaged from three independent measurements. Statistical significance between DMSO- and compound-treated samples was obtained using unpaired Student’s t-test (GraphPad Prism); * = p ≤ 0.05.

Figure 3

(A) Effects of compound ACA22 on intrinsic and SOS-mediated nucleotide release and exchange. Top: intrinsic (left) and SOS-mediated (right) rates of nucleotide exchange reaction upon mixing 0.5 μM each of WT KRAS, BGTP (and SOS). Bottom: intrinsic (left) and SOS-mediated (right) rates of nucleotide release reaction upon mixing 0.5 μM of BGDP-loaded WT KRAS and SOS with 100 μM of GTP. Ligand concentrations: 0 (circle), 1.56 (square), 3.12 (triangle), 6.25 (inverted triangle), 12.5 (left-sided triangle), 25 (right-sided triangle), 50 (diamond), and 100 μM (star). Fluorescence intensities were normalized at 120 s, and the traces were fit with linear or single exponential functions (Igor Pro) and shown as solid lines. (B) Fluorescence polarization of BGTP-γ-S-KRAS (0.5 μM) and GST-Raf-RBD (2 μM) in the absence or presence of 50 or 100 μM compound ACA22. Data are averaged from three independent measurements.

Figure 4

Lysates from BHK cells expressing GFP-tagged G12D KRAS (A) or WT KRAS (B) were treated with vehicle (DMSO) or ACA22 for 1 h and incubated with cRaf-RBD for 1 h, then washed, denatured, and subjected to detection of G12D or WT GTP-KRAS with the anti-GFP antibody and endogenous GTP-RAS with the pan-RAS antibody. Representative blots (top) and mean ± SEM (bottom) of data from three independent experiments are shown. Values are normalized with respect to DMSO. Statistical significance between DMSO- and compound-treated samples was obtained using unpaired Student’s t-test (GraphPad Prism); * = p ≤ 0.05.