Chiara Folli

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

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.

IL-4 Is a Potent Modulator of Ion Transport in the Human Bronchial Epithelium In Vitro

Recent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6–48 h causes a marked inhibition of the amiloride-sensitive Na+ channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca2+-dependent Cl− channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Rα. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the γ amiloride-sensitive Na+ channel mRNA, one of the subunits of the amiloride-sensitive Na+ channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na+ absorption and increasing Cl− secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid.

Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker.

The thiazolidinone CFTRinh‐172 was identified recently as a potent and selective blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Here, we characterized the CFTRinh‐172 inhibition mechanism by patch‐clamp and short‐circuit analysis using cells stably expressing wild‐type and mutant CFTRs. CFTRinh‐172 did not alter CFTR unitary conductance (8 pS), but reduced open probability by >90% with K i≈0.6 μM. This effect was due to increased mean channel closed time without changing mean channel open time. Short‐circuit current experiments indicated similar CFTRinh‐172 inhibitory potency (K i≈0.5 μM) for inhibition of Cl− current in wild‐type, G551D, and G1349D CFTR; however, K i was significantly reduced to 0.2 μM for ΔF508 CFTR. Our studies provide evidence for CFTR inhibition by CFTRinh‐172 by a mechanism involving altered CFTR gating.

Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07

This study compares the effect of three chemically unrelated cystic fibrosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D‐CFTR mutant. We measured Cl− transport as the amplitude of short‐circuit current in response to the membrane permeable cAMP analogue 8‐(4‐chlorophenylthio)adenosine‐3′‐5′‐cyclic monophosphate (CPT‐cAMP) alone or in combination with a CFTR opener. The correction of G551D‐CFTR defect was quantified by comparison with maximal activity elicited in cells expressing wild type CFTR. To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. In both types of epithelia, cAMP caused activation of Cl− transport that was inhibited by glibenclamide and not by 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonic acid. After normalising for CFTR expression, the response of FRT‐G551D epithelia was 1% that of wild type monolayers. Addition of genistein (10–200 μM), but not of 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX, 1–100 μM) or of the benzo[c]quinolizinium MPB‐07 (10–200 μM) to FRT‐G551D epithelia pre‐treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. The genistein dose‐response curve was bell‐shaped due to inhibitory activity at the highest concentrations. The dose‐dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. On human nasal epithelia the correction of G551D‐CFTR defective conductance obtained with genistein was 20% that of wild type. The impressive effect of genistein suggests that it might correct the Cl− transport defect on G551D patients.

Double Mechanism for Apical Tryptophan Depletion in Polarized Human Bronchial Epithelium

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-γ in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-γ. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-γ caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-γ treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na+-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na+-dependent amino acid transporter and an IFN-γ-inducible degradation by IDO.

2-(dialkylamino)-4H-1-benzopyran-4-one derivatives modify chloride conductance in CFTR expressing cells.

Some 2-(diethylamino)-7-hydroxy-4H-1-benzopyran-4-one derivatives, potentially useful as activators of the cystic fibrosis transmembrane conductance regulator (CFTR), were prepared. The synthesized compounds were tested, together with others 2-(dialkylamino)-7-hydroxybenzopyran-4-one derivatives, by measuring their capacity to modify the kinetics of iodide influx in Fisher rat thyroid cells expressing wild type CFTR and the halide-sensitive yellow fluorescent protein. Among the tested compounds the dinitrile derivatives 8 and 9 are endowed with an activity comparable to the reference compound apigenin.