Covalent Modulators of the Vacuolar ATPase

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.