title: Inhibition of prenylated KRAS in a lipid environment.
abstract: 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.
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Johanna M
authors.identifier: http://orcid.org/0000-0003-3937-6243
authors.initials: JM
authors.lastname: Jansen
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Charles
authors.initials: C
authors.lastname: Wartchow
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Jahnke
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Susan
authors.initials: S
authors.lastname: Fong
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Tsang
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Keith
authors.initials: K
authors.lastname: Pfister
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Zavorotinskaya
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Dirksen
authors.initials: D
authors.lastname: Bussiere
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Jan Marie
authors.initials: JM
authors.lastname: Cheng
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Crawford
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Dai
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Dove
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Eric
authors.initials: E
authors.lastname: Fang
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America.
authors.forename: Yun
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Florent
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Fuller
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Alvar D
authors.initials: AD
authors.lastname: Gossert
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Hekmat-Nejad
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Chrystèle
authors.initials: C
authors.lastname: Henry
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Julia
authors.initials: J
authors.lastname: Klopp
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: William P
authors.initials: WP
authors.lastname: Lenahan
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Andreas
authors.initials: A
authors.lastname: Lingel
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Ma
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Arndt
authors.initials: A
authors.lastname: Meyer
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Mishina
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Jamie
authors.initials: J
authors.lastname: Narberes
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Gwynn
authors.initials: G
authors.lastname: Pardee
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Savithri
authors.initials: S
authors.lastname: Ramurthy
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Rieffel
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America.
authors.forename: Darrin
authors.initials: D
authors.lastname: Stuart
authors.affiliation: Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Sharadha
authors.initials: S
authors.lastname: Subramanian
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: Tandeske
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Stephania
authors.initials: S
authors.lastname: Widger
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Armin
authors.initials: A
authors.lastname: Widmer
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Winterhalter
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Isabel
authors.initials: I
authors.lastname: Zaror
authors.affiliation: Department of Oncology, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
authors.forename: Stephen
authors.initials: S
authors.lastname: Hardy
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D054876:Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*
chemical_list: D008055:Lipids; D016283:Proto-Oncogene Proteins p21(ras)