As a member of small GTPase family, KRAS protein is a key physiological modulator of various cellular activities including proliferation. However, mutations of KRAS present in numerous cancer types, most frequently in pancreatic (> 60%), colorectal (> 40%), and lung cancers, drive oncogenic processes through overactivation of proliferation. The G12C mutation of KRAS protein is especially abundant in the case of these types of malignancies. Despite its key importance in human disease, KRAS was assumed to be non-druggable for a long time since the protein seemingly lacks potential drug-binding pockets except the nucleotide-binding site, which is difficult to be targeted due to the high affinity of KRAS for both GDP and GTP. Recently, a new approach broke the ice and provided evidence that upon covalent targeting of the G12C mutant KRAS, a highly dynamic pocket was revealed. This novel targeting is especially important since it serves with an inherent solution for drug selectivity. Based on these results, various structure-based drug design projects have been launched to develop selective KRAS mutant inhibitors. In addition to the covalent modification strategy mostly applicable for G12C mutation, different innovative solutions have been suggested for the other frequently occurring oncogenic G12 mutants. Here we summarize the latest advances of this field, provide perspectives for novel approaches, and highlight the special properties of KRAS, which might issue some new challenges. Electronic supplementary material The online version of this article (10.1007/s10555-020-09914-6) contains supplementary material, which is available to authorized users.

Objective and BackgroundWe aim at creating and optimizing in vitro assay methods to follow binding and potential covalent linkage of small drug‐like compounds to the G12C mutant of KRas. Oncogenic mutations of Ras proteins are found in numerous cancers. The glycine 12‐position in KRas is known to harbor several oncogenic mutations, such as cysteine, aspartate and valine. All of these mutations efficiently prevent GAP binding to mutant KRas thereby resulting in a constitutively active GTP‐bound KRas conformation that drives oncogenic processes. High‐throughput assays can facilitate identification of novel drugs that specifically target the GDP‐bound inactive KRas mutant and lock it in this inactive conformation, such as eg the recently reported ARS‐853 (1).MethodsKRas wild type and G12C was expressed and purified. Assays for thermal stability of proteins and their complexes were run on 96‐well plates using Thermofluor in a real‐time PCR instrument. Fluorescent‐based assays for thiol reactivity and (mant) GTP/GDP exchange were run on 384‐well or 96‐well plates, respectively, using a plate reader equipped with kinetics module.ResultsThermofluor assays showed that a distinct and well‐reproducible upward shift in the thermal melting point of KRas G12C upon binding to ARS‐853. The thiol‐reactivity assay was optimized with regards to protein concentration, since we found only about 25% of the expected signal was observable. The GTP/GDP exchange assay indicated that evaluation of the kinetic parameters of the exchange assay are essential to characterize functional effects of ARS‐853 that are not revealed in simple equilibrium end‐point titrations.ConclusionsCombination of assays focusing on protein stability, thiol reactivity and nucleotide‐exchange resukt in a complex test system that can distinguish drug‐like compounds that bind to KRas with or without functional effect. This test system is proposed for testing compound libraries.Support or Funding InformationSupported by the National Research, Development and Innovation Office, Hungary, project number NVKP‐16‐1‐2016‐0020This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.