Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism

Abstract

LRRC8 volume-regulated anion channels (VRACs) play important roles in diverse cell types and may represent therapeutic targets for diseases. To date, however, the pharmacological tools for evaluating the druggability of VRAC have been limited to inhibitors, as no activators of the channel have been reported. We performed a fluorescence-based high-throughput screen (HTS) of 1,184 FDA-approved drugs for compounds that increase VRAC activity. The most potent VRAC potentiator identified was zinc pyrithione (ZPT), which is used commercially for treating dandruff and other skin disorders. In intracellular YFP(F46L/H148Q/I152L)-quenching assays, ZPT potentiates the rate and extent of swelling-induced iodide influx dose-dependently with a half-maximal effective concentration (EC50) of 5.7 µM. Whole-cell voltage-clamp experiments revealed that co-application of hypotonic solution and 30 µM ZPT to HEK293 or HCT116 cells increases the rate of swelling-induced VRAC activation by approximately 10-fold. ZPT potentiates swelling-induced VRAC currents after currents have reached a steady state and activates currents in the absence of cell swelling. Neither ZnCl2 nor free pyrithione activated VRAC, however, treating cells with a mixture of ZnCl2 and pyrithione led to robust channel activation. Finally, the effects of ZPT on VRAC were inhibited by reactive oxygen species (ROS) scavenger NAC and NAD(P)H oxidase inhibitor DPI, suggesting the mechanism of action involves ROS generation. The discovery of ZPT as a potentiator/activator of VRAC demonstrates the utility of HTS for identifying small-molecule modulators of VRAC and adds to a growing repertoire of pharmacological tool compounds for probing the molecular physiology and regulation of this important channel.

Investigating Structural Property Relationships to Enable Repurposing of Pharmaceuticals as Zinc Ionophores

“…Pyrithione elicits a response expected of a strong ionophore in liposomal studies (Figure 8) at 0.1 mM; these effects have been described previously [33][34][35][36].…”

“…Pyrithione elicits a response expected of a strong ionophore in liposomal studies (Figure 8) at 0.1 mM; these effects have been described previously [33][34][35][36]. Table 1 provides a concise summary of the results obtained from experimental data and literature values, along with important physicochemical properties.…”

“…It is still unclear what the implications of increased intracellular zinc are for physiology or pathophysiology. Further, the lack of specificity and sensitivity of small molecule ionophores may result in the transport of other ions and this could make it more difficult to elucidate precise transport mechanisms, as evidenced with the much-explored zinc ionophore pyrithione [33][34][35][36].…”

“…Pyrithione is perhaps the oldest and most well-established medicinal zinc ionophore. While the exact mechanism is still being understood [33][34][35][36], pyrithione is known as an extremely potent agent [101], particularly when formulated as its zinc salt [36]. Pyrithione is a well characterized ionophoric agent and ultimately serves as a positive control for the experimental procedures.…”

A SWELL time to develop the molecular pharmacology of the volume-regulated anion channel (VRAC)

“…DCPIB was initially described as a VRAC inhibitor 20 years ago and is still the best-in-class inhibitor to date, even with its myriad of off-targets. Our lab has only begun to better understand how native VRAC may be modulated by small-molecule compounds utilizing the robust YFP-quenching assays previously established as a functional readout of chloride channel activity [ 72 , 82 ]. Others have used cryo-EM to identify putative binding sites of small-molecule VRAC inhibitors and using the gained structural insight to develop even more potent channel blockers [ 12 , 22 ].…”