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.

Figure 1

Potent, specific and rapid labeling of a cellular protein by the electrophilic probe 1 in 293 cells. (A) Structures of an electrophilic quinazoline 2 and its clickable analog (1). (B) Dose response of live cell labeling revealed potent engagement of probe 1 to an unknown cellular target of ~70 kDa (p70). (C) Time-course experiment showed rapid engagement of 1 to p70. (D) Dose-dependent blockade of probe labeling by pre-treatment of 2.

Figure 2

Identification of p70 as the vacuolar ATPase subunit A. (A) Streptavidin pull-down enriched p70. (B) LC-MS/MS analysis identified the target protein as ATP6V1A. (C) Immunoblot analysis using a house-made antibody confirmed the protein pulled down with probe 1 was ATP6V1A.

Figure 3

Identification of the probe-labeling site in ATP6V1A. (A) Sequence alignment of human and mouse ATP6V1A highlighting the mutated sites. (B) Mutation of four conserved Cys residues failed to significantly affect probe labeling of 3xFLAG-tagged ectopic ATP6V1A (<). (C) Mutation of C138 abolished probe labeling of human ATP6V1A while the converse mutation of S138C enabled labeling of the mouse protein. (D) Five mutations in distinct regions of NHR all diminished probe binding substantially. The top panels in B–D are in-gel fluorescence images (FL) and the bottom are western blots showing levels of FLAG-tagged ATP6V1A. NT: non-transfected.

Figure 4

Chloroacetamide quinazolines exhibit exquisite proteomic selectivity, expose the mechanism of action of 3-bromopyruvate, and inhibit vesicle re-acidification. (A) Quantitative isoTOP-ABPP experiments revealed that pre-treatment with 2 in vitro fully protected C138 from iodoacetamide alkyne labeling while iodoacetamide alkyne labeling of a second Cys (C277) in ATP6V1A was insensitive to 2. Samples were treated with either 2 (Light-Red) or DMSO (Heavy-Blue) and the ratio of MS1 chromatographic peaks compared (R=20; >95% decrease in MS1 peak intensity). (B) C138 exhibits a maximal competition R value of 20 while all other Cys have R values less than 4. (C) 3-bromopyruvate (3-BP) caused dose-dependent blockade of ATP6V1A labeling by probe 1. (D) The two quinazolines at 10 μM had no significant effect on steady-state vesicle pH. (E) Probe 1 caused dose-dependent inhibition of vesicle re-acidification.