Inhibition of prenylated KRAS in a lipid environment

• Published in: PloS one 2017-04, Vol.12 (4), p.e0174706-e0174706
• Main Authors: Jansen, Johanna M, Wartchow, Charles, Jahnke, Wolfgang, Fong, Susan, Tsang, Tiffany, Pfister, Keith, Zavorotinskaya, Tatiana, Bussiere, Dirksen, Cheng, Jan Marie, Crawford, Kenneth, Dai, Yumin, Dove, Jeffrey, Fang, Eric, Feng, Yun, Florent, Jean-Michel, Fuller, John, Gossert, Alvar D, Hekmat-Nejad, Mohammad, Henry, Chrystèle, Klopp, Julia, Lenahan, William P, Lingel, Andreas, Ma, Sylvia, Meyer, Arndt, Mishina, Yuji, Narberes, Jamie, Pardee, Gwynn, Ramurthy, Savithri, Rieffel, Sebastien, Stuart, Darrin, Subramanian, Sharadha, Tandeske, Laura, Widger, Stephania, Widmer, Armin, Winterhalter, Aurelie, Zaror, Isabel, Hardy, Stephen
• Format: Article
• Language: English
• Subjects: Animals; Biology and Life Sciences; Biomedical research; Cell division; Cell Line; Cell Line, Tumor; Chemistry; Gene mutation; GTPases; Humans; Interferometry; Kinases; Ligands; Lipids; Lipids - chemistry; Magnetic Resonance Spectroscopy; Mutation; NMR; Nuclear magnetic resonance; Oncogenes; Oncology; Pharmaceutical industry; Physical Sciences; Prenylation; Proteins; Proto-Oncogene Proteins p21(ras) - metabolism; Ras genes; Research and Analysis Methods; Signal transduction
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0174706

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.