Nanomolar CFTR Inhibition by Pore-Occluding Divalent Polyethylene Glycol-Malonic Acid Hydrazides

SummaryInhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential application as antisecretory therapy in cholera. We synthesized mono- and divalent CFTR inhibitors consisting of a malonic acid hydrazide (MalH) coupled via a disulfonic stilbene linker to polyethylene glycols (PEGs; 0.2–100 kDa). IC50 values for CFTR inhibition were 10–15 μM for the monovalent MalH-PEGs, but substantially lower for divalent MalH-PEG-MalH compounds, decreasing from 1.5 to 0.3 μM with increasing PEG size and showing positive cooperativity. Whole-cell patch-clamp showed voltage-dependent CFTR block with inward rectification. Outside-out patch-clamp showed shortened single-channel openings, indicating CFTR pore block from the extracellular side. Luminally added MalH-PEG-MalH blocked by >90% cholera toxin-induced fluid secretion in mouse intestinal loops (IC50 ∼10 pmol/loop), and greatly reduced mortality in a suckling mouse cholera model. These conjugates may provide safe, inexpensive antisecretory therapy

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

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A European regulatory perspective on cystic fibrosis: current treatments, trends in drug development and translational challenges for CFTR modulators

In this article we analyse the current authorised treatments and trends in early drug development for cystic fibrosis (CF) in the European Union for the time period 2000–2016. The analysis indicates a significant improvement in the innovation and development of new potential medicines for CF, shifting from products that act on the symptoms of the disease towards new therapies targeting the cause of CF. However, within these new innovative medicines, results for CF transmembrane conductance regulator (CFTR) modulators indicate that one major challenge for turning a CF concept product into an actual medicine for the benefit of patients resides in the fact that, although pre-clinical models have shown good predictability for certain mutations, a good correlation to clinical end-points or biomarkers (e.g. forced expiratory volume in 1 s and sweat chloride) for all mutations has not yet been achieved. In this respect, the use of alternative end-points and innovative nonclinical models could be helpful for the understanding of those translational discrepancies. Collaborative endeavours to promote further research and development in these areas as well as early dialogue with the regulatory bodies available at the European competent authorities are recommended

A class of non-selective cation channels in human fibroblasts

Non-selective cation channels were detected in membrane patches of cultured human fibroblasts. The channels had a unitary conductance which ranged from 14 to 25 pS in symmetrical 130 mM NaCl and were permeable to both sodium and potassium ions. Open channel probability was dependent either on the membrane potential and the Ca2+ concentration on the intracellular side of the membrane. High Ca2+ concentrations in the millimolar range were needed to keep the channel active

Small-molecule correctors of defective ΔF508-CFTR cellular processing identified by high-throughput screening

The most common cause of cystic fibrosis (CF) is deletion of phenylalanine 508 (ΔF508) in the CF transmembrane conductance regulator (CFTR) chloride channel. The ΔF508 mutation produces defects in folding, stability, and channel gating. To identify small-molecule correctors of defective cellular processing, we assayed iodide flux in ΔF508-CFTR–transfected epithelial cells using a fluorescent halide indicator. Screening of 150,000 chemically diverse compounds and more than 1,500 analogs of active compounds yielded several classes of ΔF508-CFTR correctors (aminoarylthiazoles, quinazolinylaminopyrimidinones, and bisaminomethylbithiazoles) with micromolar potency that produced greater apical membrane chloride current than did low-temperature rescue. Correction was seen within 3–6 hours and persisted for more than 12 hours after washout. Functional correction was correlated with plasma membrane expression of complex-glycosylated ΔF508-CFTR protein. Biochemical studies suggested a mechanism of action involving improved ΔF508-CFTR folding at the ER and stability at the cell surface. The bisaminomethylbithiazoles corrected ΔF508-CFTR in ΔF508/ΔF508 human bronchial epithelia but did not correct a different temperature-sensitive CFTR mutant (P574H-CFTR) or a dopamine receptor mutant. Small-molecule correctors may be useful in the treatment of CF caused by the ΔF508 mutation

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 μg/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