K-Ras^G12D Has a Potential Allosteric Small Molecule Binding Site

Abstract

KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771–785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-RasG12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-RasG12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind to four mutants in the P110 site, supporting our hypothesis that KAL-21404358 binds to the P110 site of K-RasG12D. This compound impaired the interaction of K-RasG12D with B-Raf and disrupted the RAF-MEK-ERK and PI3K-AKT signaling pathways. We synthesized additional compounds, based on the KAL-21404358 scaffold with more potent binding and greater aqueous solubility. In summary, these findings suggest that the P110 site is a potential site for binding of small molecule allosteric inhibitors of K-RasG12D.

Quinoline-Based Molecules Targeting c-Met, EGF, and VEGF Receptors and the Proteins Involved in Related Carcinogenic Pathways

“…Through a combination of computational methods and with the validation of biochemical assays, the authors proposed a mechanism of action for KAL-21404358. The quinoline compound could interfere with the protein-protein interactions, binding and stabilizing K-Ras in its inactive GDP-bound state, then, it halts the nucleotide exchange process and the subsequent activation of Ras [ 137 ].…”

Structure-based inhibitor design of mutant RAS proteins—a paradigm shift

“…Molecules that are in bold, are shown in Fig. 4 and Supplementary Figure S 2 , PDB IDs containing those molecules are shown in bold as well Binding target/site Effect Compound type PDB ID Representative example Reference SI/II-pocket (α2-helix and β1-β3 sheet) in the PPI surface Inhibition of GEF, GAP, and effector interaction Small molecule 6GJ5- 6GJ8 S1-BI-2852 Kessler-2019 [ 56 ] GEF-mediated nucleotide exchange inhibition 4DSO, 4DST, 4DSU S2-Benzimidazole DCAI Mauer-2012 [ 55 ] Inhibition of RAS-SOS binding 4EPR, 4EPY, 4EPX, 4EPW, 4EPT, 4EPV S3–‘Compound-4’ Sun-2012 [ 57 ] Inhibition of effector interaction 5OCO, 5OCT, 5OCG, 6FA1 , 6FA2, 6FA3, 6FA4 S4-ABD-4 S5-ABD-7 Qevedo-2018 [ 60 ] 6GOD, 6GOE, 6GOF, 6GOG, 6GOM, 6GQT, 6GQW, 6GQX, 6GQY Ch-3 Cruz-Migoni-2019 [ 59 ] Switch-II pocket (S-IIp) Inhibition of GTP loading to RAS Covalent small molecule 5F2E S6-ARS-853 Patricelli-2016 [ 34 ] 5V9U ARS-1620 Janes-2018 [ 42 ] 6OIM S7-AMG-510 Canon-2019 [ 27 ] 6UT0 S8-MRTX849 Fell-2020 [ 28 ] P110 pocket on the allosteric lobe of KRAS Allosteric inhibition of effector interaction Small molecule – KAL-21404358 Feng-2019 [ 67 ] Allosteric lobe of KRAS Competitive inhibition of GEF Antibody-like protein ...…”

“…NMR results showed that upon binding of KAL-21404358, an early compound hit, Switch-I (Asp-33, Ser-39), and Switch-II (Leu-56, Gly-60, Met-67, Thr-74 and Gly-75) undergo conformational changes, suggesting an allosteric effect on KRAS-G12D. This resulted in inhibition of RAS-Raf interaction, and weak depletion of phosphorylated Akt, and ERK within treated cell lines [ 67 ]. These examples show that while selectively targeting non-G12C mutants of KRAS is complicated and requires less-straightforward strategies, it is possible to achieve, by taking advantage of the small structural changes that are caused by the mutated residue.…”

Current Approaches in NSCLC Targeting K-RAS and EGFR

“…When discussing other K-RAS mutations such as mutation G12D, the aspartate side group was described as a difficult target [21]. Nevertheless, Feng et al have recently reported that K-Ras G12D has also a potential allosteric small molecule binding site [22]. Their work showed that one compound termed K-RAS allosteric ligand KAL-21404358, when bound to K-RAS G12D , impaired its interaction with B-RAF disrupting the RAF-MEK-ERK and PI3K-AKT signalling pathways [22].…”