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Development of High‐throughput Assays for Testing of Potential Inhibitors of the Oncogenic G12C Mutant of KRas
Author(s) -
Vertessy Beata G,
Nyiri Kinga,
Steger Anett,
Koppany Gergely
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb91
Subject(s) - kras , gtp' , mutant , chemistry , point mutation , biochemistry , mutation , microbiology and biotechnology , enzyme , biology , gene
Objective and Background We 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). Methods KRas 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. Results Thermofluor 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. Conclusions Combination 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 Information Supported by the National Research, Development and Innovation Office, Hungary, project number NVKP‐16‐1‐2016‐0020 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .