In vitro activity: CFTRinh-172 dose- and time-dependently inhibits CFTR-mediated I- transportation, and effectively inhibits CFTR activation by multiple types of agonists or activators. CFTRinh-172, as a selective CFTR channel inhibitor, also completely abolishes the Cl– current in the rabbit acinar and duct cells of rabbit lacrimal gland. CFTRinh-172 also induces ROS production, mitochondrial failure, and activation of the NF-κB signaling pathway, independently of CFTR inhibition.

Kinase Assay: Assays are done using a customized screening system consisting of a 3-meter robotic arm, CO2 incubator, plate washer, liquid-handling workstation, bar code reader, delidding station, and two FLUOstar fluorescence platereaders, each equipped with two syringe pumps and HQ500/20X (500 ± 10 nm) excitation and HQ535/30M (535 ± 15 nm) emission filters. The robotic system is integrated using SAMI version 3.3 software modified for two platereaders. Custom software is written in Microsoft VBA (Visual Basic for Applications) to compute base-line–subtracted, normalized fluorescence slopes (giving halide influx rates) from stored data files. The assay is set up by loading the incubator (37°C, 90% humidity, 5% CO2) with 40–60 96-well plates containing the FRT cells, and loading a carousel with 96-well plates containing test compounds and disposable plastic pipette tips. To initiate the assay, each well of a 96-well plate is washed three times in PBS (300 μl/wash), leaving 50 μL PBS. Ten microliters of a CFTR-activating cocktail (5 μM forskolin, 100 μM IBMX, 25 μM apigenin in PBS) is added, and after 5 minutes one test compound (0.5 μL of 1 mM DMSO solution) is added to each well to give 10 μM final concentration. After 10 minutes, 96-well plates are transferred to a platereader for fluorescence assay. Each well is assayed individually for CFTR-mediated I- transport by recording fluorescence continuously (200 ms per point) for 2 seconds (base line) and then for 12 seconds after rapid (<0.5 seconds) addition of 165 μL of isosmolar PBS in which 137 mM Cl– was replaced by I-.

Cell Assay: Cell toxicity is assayed by the dihydrorhodamine method at 24 hours after cell [Fischer rat thyroid (FRT) cells] incubation with 0–1,000 μM inhibitor.

J Clin Invest. 2002 Dec;110(11):1651-8; PLoS Negl Trop Dis. 2013 Jun 27;7(6):e2293.