Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

Niclosamide, but not ivermectin, inhibits anoctamin 1 and 6 and attenuates inflammation of the respiratory tract

“…The use of high (≥ 1 µM) concentrations of niclosamide in cell culture experiments appears inappropriate and leads to the inhibition of the SERCA pump as well as Orai Ca 2+ influx channels [ 19 , 28 , 35 ]. Artefactual inhibition of SERCA/Orai is often observed for amphipathic drugs and was also reported for the CFTR inhibitor CFTRinh-172, shown recently to compromise intracellular Ca 2+ signalling [ 50 ]. When taken orally at the established and well-tolerated standard dose of 2 g/day niclosamide leads (in humans) to a variable peak plasma concentration of ~ 250 up to 6000 ng/ml (~ 700–18000 nM) [ 3 , 72 ].…”

CAGE sequencing reveals CFTR-dependent dysregulation of type I IFN signaling in activated cystic fibrosis macrophages

“…Chemical inhibition of CFTR activity in healthy human monocytes using an allosteric CFTR inhibitor (CFTR inh -172) induced increased surface expression of CD64 and CD14 while LPS, elevated in patient serum ( 31 , 58 ) and therefore used as an in vitro proxy for the inflamed CF circulation, caused a trend toward heightened CD11b expression. It is important to exercise caution when interpreting CFTR inh -172–induced differences as entirely CFTR dependent given the multiple known off-target effects of this inhibitor, including increased induction of NF-κB signaling independently of CFTR inhibition ( 59 , 60 ). A more targeted approach, including using CRISPR-Cas9 gene editing, is far preferable.…”

Recommended Tool Compounds for Modifying the Cystic Fibrosis Transmembrane Conductance Regulator Channel Variants

“…65 4-[4-Oxo-2-thioxo-3-(3-trifluoromethyl-phenyl)-thiazolidin-5-ylidenemethyl]-benzoic acid (CFTRinh-172) and (naphthalen-2ylamino)-acetic acid (3,5-dibromo-2,4,-dihydroxy-benzylidene)-hydrazide (GlyH-101) are blockers that target but are not solely selective for CFTR protein as some of them also inhibit other transepithelial ion channels. 66,67 These compounds play crucial roles in the research of CFTR and CF model development. 63,68 CFTRinh-172.…”

“…CFTR blockers are a category of CFTR compounds that are specifically designed to inhibit the function of the CFTR channel, which can be utilized to create CFTR knock-out models to mimic CF conditions, and contribute to CF research and drug development . 4-[4-Oxo-2-thioxo-3-(3-trifluoromethyl-phenyl)-thiazolidin-5-ylidenemethyl]-benzoic acid (CFTRinh-172) and (naphthalen-2-ylamino)-acetic acid (3,5-dibromo-2,4,-dihydroxy-benzylidene)-hydrazide (GlyH-101) are blockers that target but are not solely selective for CFTR protein as some of them also inhibit other transepithelial ion channels. , These compounds play crucial roles in the research of CFTR and CF model development. , …”