Covalent Modulators of the Vacuolar ATPase

Abstract

The vacuolar H+ ATPase (V-ATPase) is a complex multi-subunit 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.

Covalent inhibitors: an opportunity for rational target selectivity

“…Zhang, Cravatt and coworkers reported the discovery of a highly selective covalent inhibitor that targets a cysteine in the nucleotide-binding site of the vacuolar H + ATPase (V-ATPase, Figure 3B). [24] The discovery of this V-ATPase was achieved by profiling a library prepared to target the kinase EphB3. Impressively, this molecule had remarkable proteomic selectivity for V-ATPase with essentially one band out of crude lysates being labeled by the inhibitor as revealed by fluorescence imaging and chemical proteomic activity-based profiling.…”

Senescent stromal cells promote cancer resistance through SIRT1 loss-potentiated overproduction of small extracellular vesicles

“…Lysosomal pH was detected as described previously 76 , with minor modifications. Cells (5 × 10 3 ) plated within 24 well plates were treated with either DMSO, or 100 nM Bafilomycin-A, for 1 h, at which point the cells were rinsed extensively with media and allowed to recover for 1 h. The cells were then treated with LysoTracker Green DND-26 (Thermo Fisher) for 1 h, washed twice with PBS, and lysed in 200 μl of RIPA buffer (10 mM Tris, 140 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate and 1 μg/ml each aprotinin and leupeptin).…”

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

“…We recently described an efficient strategy to globally assess the ligandability of cysteines in native biological systems that leverages broadly reactive, electrophilic small-molecule fragments referred to as "scouts" Bar-Peled et al, 2017). Two scout fragments bearing an a-chloroacetamide (KB02) or acrylamide (KB05) (Figure 2A, 2B) -reactive groups frequently found in covalent chemical probes and drugs (Baillie, 2016;Chen et al, 2017;Honigberg et al, 2010;Ostrem et al, 2013;Xu et al, 2019;Yang et al, 2014;Zeng et al, 2017) -were used to construct in-depth cysteine ligandability maps across primary human T cells in both control and activated states. We analyzed scout fragment-cysteine interactions using two complementary chemical proteomic methods that provided a balance of confidence in quantitative accuracy (isoTOP-ABPP) with greater multiplexing capacity (TMT-ABPP) (Figures 2A and S2A, B).…”