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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.

SUBMITTER: Chen YC

PROVIDER: S-EPMC5274637 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Covalent Modulators of the Vacuolar ATPase.

Chen Ying-Chu YC Backus Keriann M KM Merkulova Maria M Yang Christina C Brown Dennis D Cravatt Benjamin F BF Zhang Chao C

Journal of the American Chemical Society 20161230 2

The vacuolar H<sup>+</sup> 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 ...[more]

PMID: 28010062

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Project description:Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F1-γ-subunit of the two-sector F1Fo-ATP synthase allow for Fo-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal Fo-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.

Dataset Information

Covalent Modulators of the Vacuolar ATPase.

Publications

Covalent Modulators of the Vacuolar ATPase.

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