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.

Figure 1

Identification of CFTR inhibitors by high-throughput screening. (a) Schematic of screening approach. CFTR was maximally stimulated by multiple agonists in stably transfected epithelial cells coexpressing human CFTR and a yellow fluorescent protein (YFP) with fluorescence sensitive to Cl^–/I^–. After addition of test compound, I^– influx was induced by adding an I^–-containing solution. (b) Representative original fluorescence data from individual wells showing controls (no activators, no test compound) and test wells. (c) Top: Chemical structure of 2-thioxo-4-thiazolidinone CFTR inhibitors. Bottom: Structures of analogs having greatest CFTR inhibitory activity. Relative potencies were 0.2 (CFTR_inh-020), 0.3 (CFTR_inh-029), 1.0 (CFTR_inh-172), 0.2 (CFTR_inh-185), 0.1 (CFTR_inh-214), and 0.1 (CFTR_inh-236).

Figure 2

Characterization of CFTR inhibition by CFTR_inh-172. (a) CFTR functional assays as in Figure 1b for indicated [CFTR_inh-172]. (b) Time course of inhibition showing CFTR-mediated I^– transport rates at different times after addition of 2 μM CFTR_inh-172. Inset: Time course of inhibition reversal showing I^– transport rates at different times after washout of 1 μM CFTR_inh-172. Mean ± SE is shown from three sets of experiments. (c) Inhibition of CFTR after stimulation by different agonists, including benzoflavone and benzimidazolone UCCF compounds (14), genistein, CPT-cAMP, 8-methoxypsoralen (8-MPO), and 8-cyclopentyl-1,3-dipropylxanthine (CPX) (all 50 μM) (SE; three sets of experiments). Black bars, agonist; white bars, agonist + 5 μM CFTR_inh-172.

Figure 3

Electrophysiological analysis of CFTR inhibition. (a) Left: CFTR_inh-172 inhibition of short-circuit current (I_sc) in permeabilized FRT cells expressing human CFTR. CFTR was stimulated by 100 μM CPT-cAMP. Right: Dose-inhibition data for CFTR_inh-172 (circles) and glibenclamide (squares) (SE; n = 3 sets of experiments). (b) CFTR_inh-172 inhibition of short-circuit current in primary culture of (nonpermeabilized) human bronchial epithelial cells. Inhibitor was added in apical bathing solution (left) or basolateral and then apical solutions (right). (c) Left: Whole-cell patch clamp of CFTR-expressing FRT cells showing membrane currents elicited at +80 mV (open circles) and –100 mV (filled circles). CFTR was stimulated by 5 μM forskolin followed by addition of 2 μM CFTR_inh-172. The alternate stimulation was interrupted (I–III) for application of graded membrane potentials (middle). Right: Current-voltage relationships under basal conditions (control, open circles), after forskolin stimulation (filled circles), and following addition of 0.2 μM CFTR_inh-172 giving approximately 50% inhibition (open triangles). I_m, membrane current; V_m, membrane potential.

Figure 4

Specificity of CFTR inhibition by CFTR_inh-172. (a) Alternative Cl^– channels. Left: UTP (100 μM) stimulated Ca²⁺-dependent Cl^– secretion measured in short-circuit current measurements on airway epithelial cells in the absence and presence of 5 μM CFTR_inh-172. Right: Volume-activated Cl^– current (hypotonic 250 mosM/kg H₂O) measured in whole-cell patch-clamp experiments on FRT cells. Currents were recorded in the absence and presence of 5 μM CFTR_inh-172. (b) MDR-1 activity. ³H-vincristine accumulation in 9HTEo-/Dx cells with upregulated MDR-1 expression. Intracellular vincristine was measured with and without verapamil (100 μM) or CFTR_inh-172 (5 μM) (SE; n = 3). (c) ATP-sensitive K⁺ channels. Left: Representative membrane potential recording from a pancreatic β cell (INS-1) perfused extracellularly with CFTR_inh-172, diazoxide (100 μM), and glibenclamide (glib; 10 μM). Right: Averaged changes in membrane potential (ΔmV) caused by indicated maneuvers (SE; n = 4). PD, potential difference.

Figure 5

Inhibition of intestinal fluid secretion. (a) Photograph of isolated mouse ileal loops at 6 hours after lumenal injection of 1 μg cholera toxin without (top) and with (middle) intraperitoneal injection of CFTR_inh-172 (250 μg/kg). Saline control (no cholera toxin) is shown for comparison (bottom). (b) Ileal loop weight at 6 hours. Mean ± SE (n = 6–8 mice) with 14–16 loops studied. For the inactive analog, the 4-carboxyphenyl substituent in CFTR_inh-172 was replaced by 3-methoxy-4-methoxyvinylphenyl (SE; six to eight mice per group; *P < 0.001, ANOVA). (c) Ratio of weight of entire small intestine at 6 hours after oral gavage before versus after luminal fluid removal (SE; four mice per group; *P < 0.001). (d) CFTR_inh-172 inhibition short-circuit current after amiloride addition and stimulation by forskolin (20 μM) in isolated rat colonic mucosa. CFTR_inh-172 was added to serosal and then mucosal surfaces as indicated. One experiment typical of four is shown.