Structurally diverse small molecules for allosteric non-covalent inhibition of KRAS
Introduction
RAS proteins are small GTPases that play a crucial role in cell signaling pathways that modulate cell growth and proliferation. They act as molecular switches by cycling between active GTP-bound and inactive GDP-bound states. Mutations in RAS disrupt this cycle and result in constitutive activation, leading to uncontrolled cell growth/proliferation and cancer. RAS mutations account for ~20% of all human cancers, and of these ~85% are due to mutations in KRAS, one of the three most common human RAS isoforms. KRAS mutations account for more than 90% of pancreatic, ~45% of colon, and ~ 35% of lung cancer. Here, we describe diverse small molecule lead compounds for pan-KRAS inhibition
Methods
We used a combination of biological and biophysical assays to characterize close to a dozen ligands obtained from a virtual screening of campaign. The techniques we used included nuclear Magnetic Resonance, thermal shift assay, isothermal titration calorimetry, and fluorescence-based methods such as microscale thermophoresis in purified systems for the determination of binding affinities to wild type and oncogenic mutant KRAS including G12D, G12V, G12C, and Q61H. We employed fluorescence-based techniques to determine the ability of the compounds to inhibit KRAS activation or interaction with effector Raf. We conducted pull-down and immunoblotting assays to examine inhibition of KRAS-effector interactions and signaling through the MAPK and AKT pathways, as well as cell proliferation assays to assess effect on the growth of cancer cell lines.
Results
Directly targeting the active site of KRAS for small molecule inhibition has proven to be ineffective due primarily to its high affinity to endogenous GDP and GTP. We previously showed that RAS harbors 4 allosteric ligand binding pockets that could be explored as potential targets for allosteric inhibition by small molecules. Subsequent molecular docking yielded structurally diverse compounds that we have characterized through a combination of biophysical and cell-based assays. We found that these compounds bind to wild type and mutant KRAS, sometimes in a nucleotide dependent manner. Some of these ligands interfere with KRAS activation and/or effector binding, leading to significant inhibition of mutant KRAS signaling and cancer cell growth.
Conclusion
We show that the compounds with diverse functionalities bind to KRAS with affinities in the sub-micromolar to high micromolar range and exert different effects on its interactions with binding partners. The ligands harbor diverse functional groups and exhibit differences in binding affinity to wild type and active or inactive KRAS and therefore in inhibitory profiles.