Discovery of allosteric non-covalent KRAS inhibitors that bind with sub-micromolar affinity and disrupt effector binding

Abstract

Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and a notorious oncogene. Somatic mutations that render KRAS constitutively active lead to uncontrolled cell growth, survival, proliferation, and eventually cancer. KRAS is thus a critical anticancer drug target. However, despite aggressive efforts in recent years, there is no drug on the market that directly targets KRAS. In the current work, we combined molecular simulation and high-throughput virtual screening with a battery of cell-based and biophysical assays to discover a novel, pyrazolopyrimidine-based allosteric KRAS inhibitor that exhibits promising biochemical properties. The compound selectively binds to active KRAS with submicromolar affinity, slightly modulates exchange factor activity, disrupts effector Raf binding, significantly reduces signal transduction through mutant KRAS and inhibits cancer cell growth.Moreover, by studying two of its analogues, we identified key chemical features of the compound that are critical for affinity, effect on effector binding and mode of action. We propose a set of specific interactions with key residues at the switch regions of KRAS as critical for abrogating effector binding and reducing the rate of nucleotide exchange. Together, these findings not only demonstrate the viability of direct KRAS inhibition and offer guidance for future optimization efforts, but also show that pyrazolopyrimidine-based compounds may represent a first-in-class lead toward a clinically relevant targeting of KRAS by allosteric non-covalent inhibitors. Cell signalingThe inhibitory potential of compounds was tested in monoclonal Baby Hamster Kidney (BHK) cell lines stably expressing monomeric green fluorescence protein (mGFP)tagged KRAS G12D , KRAS G12V and HRAS G12V . Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Hyclone) supplemented with 10% v/v bovine calf serum and incubated with compound or vehicle (DMSO) for 3 h without serum. Cells were then harvested in lysis buffer (50 mM Tris (pH 7.5), 75 mM NaCl, 25 mM NaF, 5 mM MgCl 2 , 5 mM EGTA, 1 mM dithiothreitol, 100 µM NaVO 4 , 1% Nonidet P40 plus protease inhibitors) and subjected to Western analysis controlling protein loading by BCA (bicinchoninic acid) assay. Lysates were resolved with Bio-Rad polyacrylamide TGX 10% gel, transferred to polyvinylidene fluoride (PVDF) membrane and immunoblotted using pan-AKT (2920S), GFP (2956S), p-AKT S473 (4060L), p-cRaf S338 (9427S), p-ERK T202/Y204 (4370L), ERK1/2 (4695S) or β-actin antibodies (Cell Signaling Technology). IC 50 values were calculated with Prism 4-parameter fit. Pull-downWe pulled down GFP-RAS with GST-tagged RAS binding domain (RBD) of cRaf A85K (hereafter GST-Raf RBD ) to monitor RAS-Raf interaction. To prepare GST-Raf RBD bound to agarose beads, bacteria (BL21) transfected with a previously cloned GEX plasmid were grown in selection media to OD levels of 0.5-1.0 before protein expression was initiated with IPTG (1:1000). After 4 h, the sample was centrifuged at 6000 rpm ...