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Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V . cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study. Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250 .

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50–100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application. Targeting different aspects of mutant CFTR structural defects with combination therapy leads to more potent rescue of function than that following single therapy.

Cystic Fibrosis (CF) is a hereditary condition and can cause permanent respiration problems leading to degraded life quality. The most common variation leading to CF is the F508del variation. CF can cause damage to not just the lungs but also digestive system, pancreas, and other organs. CF decreases the life expectancy of the individuals affected with the constant fear of lung complications. The current methods of treatment include using a combination of drugs to manage the symptoms. The combination of drugs has many side effects and causes damage to other organs like liver, heart or kidneys. In this study, we aim to find a drug that can relieve the symptoms of CF. We began by creating a dataset of potential drug molecules, which was subsequently refined by removing harmful compounds through an ADMET scan. All these compounds were then docked to the mutated Cystic Fibrosis Transmembrane Regulator (CFTR) protein. The compounds with the best docking affinity were Galicaftor and Bamocaftor. A currently approved drug, Ivacaftor was selected as control for the 200 ns Molecular Dynamics (MD) Simulation. The simulation revealed that the CFTR protein remained more stable and compact when complexed with Bamocaftor, when compared to Ivacaftor and Galicaftor. Moreover, the MMPBSA free energy calculations revealed that the free energy of the CFTR-bamocaftor complex is the lowest compared to the other complexes. Our findings reveal the action of bamocaftor on CFTR protein with p.Phe508del variation. However, the absence of in-vivo or in-vitro studies is a limitation, and further experimental validation is necessary to confirm its efficacy and safety.

Hyperactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) contributes to secretory diarrhea, a major cause of pediatric mortality worldwide, and autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of end-stage renal disease. Selective CFTR inhibition is a potential therapeutic strategy, with ( R )-BPO-27 emerging as a promising candidate. Here, we present a cryo-EM structure of CFTR bound to ( R )-BPO-27 at an overall resolution of 2.1 Å. Contrary to the previous hypothesis that it inhibits CFTR current by competition with ATP, we demonstrate that ( R )-BPO-27 instead directly occludes the chloride-conducting pore while permitting ATP hydrolysis, thus uncoupling the two activities. Furthermore, we find that inhibitor binding requires some degree of NBD separation, as the inhibition rate inversely correlates with the probability NBD dimerization. These findings clarify the compound’s mechanism and provide a molecular basis for optimizing its clinical potential. Hyperactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to the pathogenesis of secretory diarrheas and autosomal dominant polycystic kidney disease (ADPKD). Here, authors use cryo-EM to show that inhibitor (R)-BPO-27 inhibits CFTR via pore blockade

Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-μm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P < 0.05) and human bronchi (P < 0.05) within minutes of exposure. The mechanism involved CSE-induced reduction of CFTR gating, decreasing CFTR open-channel probability by approximately 75% immediately after exposure (P < 0.05), whereas surface CFTR expression was partially reduced with chronic exposure, but was stable acutely. CSE treatment of purified CFTR resulted in acrolein modifications on lysine and cysteine residues that likely disrupt CFTR gating. In primary HBE cells, CSE reduced airway surface liquid depth (P < 0.05) and ciliary beat frequency (P < 0.05) within 60 minutes that was restored by coadministration with ivacaftor (P < 0.005). Cigarette smoking transmits acute reductions in CFTR activity, adversely affecting the airway surface. These effects are reversible by a CFTR potentiator in vitro, representing a potential therapeutic strategy in patients with chronic obstructive pulmonary disease with chronic bronchitis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to accumulation of proteins aggregates in airways. Mutated CFTR promotes transglutaminases-mediated crosslinking of beclin 1, a positive regulator of autophagy, to induce accumulation of LC3-binding protein p62 and prevent autophagic degradation of aggregates. Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTR F508del induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS–TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro , in vivo in Scnn1b -transgenic and Cftr F508del homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTR F508del to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

Key Points There are five main classes of chloride channels: cystic fibrosis transmembrane conductance regulator (CFTR), calcium-activated, voltage-dependent, ligand-gated and volume-sensitive. Chloride channels are attractive targets for drug development for a wide range of human disorders. Fluorescence and electrophysiological high-throughput assays are now available for the discovery of chloride-channel modulators. Cell-based assays utilizing halide-sensing yellow fluorescent proteins are particularly useful for rapid, cost-effective screening. Mutations in CFTR chloride channels cause the hereditary disease cystic fibrosis, and overactivation of CFTR causes secretory diarrhoeas. Small-molecule inhibitors of normal CFTR are in development, as are potentiators and correctors of cystic fibrosis-causing mutant CFTRs. Calcium-activated chloride channels are involved in a wide range of physiological functions, including transepithelial fluid secretion, oocyte fertilization, olfactory and sensory signal transduction, smooth-muscle contraction, and neuronal and cardiac excitation. Recent advances have been made in the molecular identification of these channels and in the identification of channel activators and inhibitors. Chloride channels activated by GABA (γ-aminobutyric acid) and glycine (ionotropic receptors) modulate important physiological functions in the central and peripheral nervous system. The large diversity of ionotropic GABA and glycine receptors provide an opportunity to develop drugs to treat various neurological disorders. Volume-sensitive chloride channels remain to be identified at the molecular level. These channels may be important pharmacological targets in treating cancer and degenerative disorders. The development of drugs that target chloride channels has lagged behind those of other targets, partly because of technical challenges in screening for chloride-channel modulators. This Review examines the methods for assaying chloride-channel function and emerging drug development opportunities for each of the chloride-channel classes. Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABA A (γ-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery.

CFTR protein is an ion channel regulated by cAMP-dependent phosphorylation and expressed in many types of epithelial cells. CFTR-mediated chloride and bicarbonate secretion play an important role in the respiratory and gastrointestinal systems. Pharmacological modulators of CFTR represent promising drugs for a variety of diseases. In particular, correctors and potentiators may restore the activity of CFTR in cystic fibrosis patients. Potentiators are also potentially useful to improve mucociliary clearance in patients with chronic obstructive pulmonary disease. On the other hand, CFTR inhibitors may be useful to block fluid and electrolyte loss in secretory diarrhea and slow down the progression of polycystic kidney disease.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that promotes the exchange of chloride (Cl − ) and bicarbonate (HCO 3 − ), modulating the balance of ions and water in various tissues. CFTR is also expressed in the equatorial region of the spermatozoa’s head. However, the modulation of ionic and water transport via CFTR in spermatozoa needs further investigation. To better understand how CFTR participates in the molecular mechanisms behind spermatozoa modulation of water transport, and its impact on sperm physiology, CFTR function of human spermatozoa was inhibited with 20 µM CFTR Inh -172. The inhibition of CFTR promoted a decrease in intracellular [Cl − ] and membrane glycerol/water permeability, without any impact on sperm vitality or off-target effect in Ca 2+ channels (assessed through the evaluation of intracellular [Ca 2+ ]). To reverse the effects of CFTR inhibition, we incubated spermatozoa with CFTR-carrying seminal fluid extracellular vesicles (SF-EVs). We reported that the SF-EVs were able to restore the CFTR activity of spermatozoa previously exposed to the inhibitor, characterized by a restoration of Cl − , glycerol/water permeability, and a recovery of sperm osmoregulation capacity. Our results provide evidence for the important role of CFTR function in sperm osmoregulation and suggest that the use of CFTR-containing EVs - already employed in the treatment of cystic fibrosis - could be explored to enhance sperm quality.

The devastating inherited disease cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel. The recent approval of the CFTR potentiator drug ivacaftor (Vx-770) for the treatment of CF patients has marked the advent of causative CF therapy. Currently, thousands of patients are being treated with the drug, and its molecular mechanism of action is under intensive investigation. Here we determine the solubility profile and true stimulatory potency of Vx-770 towards wild-type (WT) and mutant human CFTR channels in cell-free patches of membrane. We find that its aqueous solubility is ~200 fold lower (~60 nanomolar), whereas the potency of its stimulatory effect is >100 fold higher, than reported, and is unexpectedly fully reversible. Strong, but greatly delayed, channel activation by picomolar Vx-770 identifies multiple sequential slow steps in the activation pathway. These findings provide solid guidelines for the design of in vitro studies using Vx-770.