Development of novel inhibitors of swelling‐activated LRRC8 anion channels

Abstract

Cells critically regulate their volume in response to hypoosmotic swelling by transporting chloride and small organic osmolytes out of the cell through so‐called volume‐regulated anion channels (VRACs). The 5 genes encoding VRACs were recently identified in two independent genome‐wide siRNA screens and are named LRRC8 (for Leucine Rich Repeat Containing) A‐E. LRRC8‐containing VRACs have recently been implicated in drug uptake, adipocyte size, and insulin signaling. Unfortunately, the pharmacological tools available to study the physiology and therapeutic potential of LRRC8 channels is limited to low‐affinity small‐molecule inhibitors that exhibit promiscuous activity toward other anion channels, cation channels, and myriad other off‐targets. To circumvent this barrier, we are employing high‐throughput screening (HTS) of small‐molecule libraries and medicinal chemistry to develop more potent and specific inhibitors of LRRC8 channels. HTS will be performed using a fluorescence assay based on halide quenching of a YFP mutant (i.e. F46L, H148Q, I152L) termed Ozzy. Briefly, HEK‐293 cells stably transfected with Ozzy will be plated in black‐walled, clear‐bottomed 384‐well plates, treated with hypotonic solution containing compounds from the Vanderbilt Institute of Chemical Biology library, and then treated with sodium iodide (NaI). Cell swelling opens endogenously expressed VRAC channels, through which iodide enters the cell and quenches Ozzy fluorescence. Small‐molecule inhibitors should block the iodide‐induced quenching of Ozzy. Checkerboard analysis using the best‐in‐class VRAC inhibitor, DCPIB, yielded a Z′ value of 0.74, indicating the assay is reproducible and robust enough to ensure high‐confidence hit‐picking in a large‐scale screen. The assay also reports dose‐dependent inhibition of iodide influx by DCPIB with an IC50 of 11 micromolar, which is close to values derived from electrophysiological experiments. We are currently using this assay to interrogate an analog library based on the DCPIB scaffold, with the goal of identifying more potent and specific inhibitors of VRAC.Support or Funding InformationDepartment of Anesthesiology Faculty Development FundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.