Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin–induced intestinal fluid secretion

Abstract

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR)

“…NiNTA-purified CFTR retained significant (P<0.003) sensitivity to azide, an inhibitor of mitochondrial F-type ATPase, whereas the antiFlag step removed this azide-sensitive contaminant (P<0.0005). CFTR Inhibitor 172 (Inh172) [38–40] was reported to partially inhibit CFTR ATPase function [41]. Preincubation with Inh172 significantly inhibited CFTR ATPase activity in the direct assay (P<0.002).…”

Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

“…Because transepithelial Cl - secretion required the stimulation of apical Cl - channels and/or basolateral K + channels, the involvement of different ion channels in nobiletin-stimulated Cl - secretion was examined with various selective ion channel inhibitors. First, DIDS, a CaCC inhibitor [34] and CFTR inh -172, a potent selective inhibitor of CFTR [35], were used to study the involvement of the two major types of Cl - channels expressed in airway epithelia [36]. The apical and basolateral nobiletin-stimulated I SC was sensitive to CFTR inh -172, but insensitive to DIDS.…”

A Cell-Based High-Throughput Screen for Novel Chemical Inducers of Fetal Hemoglobin for Treatment of Hemoglobinopathies

“…It is an enabling technology and the number of researchers that have applied this powerful tool in biomedical research has increased. Successful examples include discoveries of novel inhibitors of protein arginine methyltransferase [17], metalloform-selective methionine aminopeptidase [18], and cystic fibrosis transmembrane conductance regulator [19].…”