title: Covalent Modulators of the Vacuolar ATPase.
abstract: The vacuolar H+ATPase (V-ATPase) is a complex multisubunit machine that regulates important cellular processes through controlling acidity of intracellular compartments in eukaryotes. Existing small-molecule modulators of V-ATPase either are restricted to targeting one membranous subunit of V-ATPase or have poorly understood mechanisms of action. Small molecules with novel and defined mechanisms of inhibition are thus needed to functionally characterize V-ATPase and to fully evaluate the therapeutic relevance of V-ATPase in human diseases. We have discovered electrophilic quinazolines that covalently modify a soluble catalytic subunit of V-ATPase with high potency and exquisite proteomic selectivity as revealed by fluorescence imaging and chemical proteomic activity-based profiling. The site of covalent modification was mapped to a cysteine residue located in a region of V-ATPase subunit A that is thought to regulate the dissociation of V-ATPase. We further demonstrate that a previously reported V-ATPase inhibitor, 3-bromopyruvate, also targets the same cysteine residue and that our electrophilic quinazolines modulate the function of V-ATPase in cells. With their well-defined mechanism of action and high proteomic specificity, the described quinazolines offer a powerful set of chemical probes to investigate the physiological and pathological roles of V-ATPase.
authors.forename: Ying-Chu
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute , La Jolla, California 92307, United States.
authors.forename: Keriann M
authors.initials: KM
authors.lastname: Backus
authors.affiliation: MGH Center for Systems Biology, Program in Membrane Biology & Division of Nephrology, Richard B. Simches Research Center, Massachusetts General Hospital and Department of Medicine, Harvard Medical School , Boston, Massachusetts 02114, United States.
authors.forename: Maria
authors.initials: M
authors.lastname: Merkulova
authors.forename: Christina
authors.initials: C
authors.lastname: Yang
authors.affiliation: MGH Center for Systems Biology, Program in Membrane Biology & Division of Nephrology, Richard B. Simches Research Center, Massachusetts General Hospital and Department of Medicine, Harvard Medical School , Boston, Massachusetts 02114, United States.
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute , La Jolla, California 92307, United States.
authors.forename: Benjamin F
authors.identifier: 0000-0001-5330-3492
authors.initials: BF
authors.lastname: Cravatt
authors.forename: Chao
authors.identifier: 0000-0003-0251-8156
authors.initials: C
authors.lastname: Zhang
mesh_terms: D008954:Models, Biological; D015394:Molecular Structure; D054852:Small Molecule Libraries / Q000737:chemistry*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D054852:Small Molecule Libraries; D025262:Vacuolar Proton-Translocating ATPases