Pharmacotherapy Of The Ion Transport Defect In Cystic Fibrosis

1. More than 1300 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF), a disease characterized by deficient epithelial Cl- secretion and enhanced Na+ absorption. The clinical course of the disease is determined by the progressive lung disease. Thus, novel approaches in pharmacotherapy are based primarily on correction of the ion transport defect in the airways. 2. The current therapeutic strategies try to counteract the deficiency in Cl- secretion and the enhanced Na+ absorption. A number of compounds have been identified, such as genistein and xanthine derivatives, which directly activate mutant CFTR. Other compounds may activate alternative Ca2+-activated Cl- channels or basolateral K+ channels, which supply the driving force for Cl- secretion. Apart from that, Na+ channel blockers, such as phenamil and benzamil, are being explored, which counteract the hyperabsorption of NaCl in CF airways. 3. Clinical trials are under way using purinergic compounds such as the P2Y(2) receptor agonist INS365. Activation of P2Y(2) receptors has been found to both activate Cl- secretion and inhibit Na+ absorption. 4. The ultimate goal is to recover Cl- channel activity of mutant CFTR by either enhancing synthesis and expression of the protein or by activating silent CFTR Cl- channels. Strategies combining these drugs with compounds facilitating Cl- secretion and inhibiting Na+ absorption in vivo may have the best chance to counteract the ion transport defect in cystic fibrosis.     

Targeting F508del-CFTR to develop rational new therapies for cystic fibrosis

The mutation F508del is the commonest cause of the genetic disease cystic fibrosis (CF). CF disrupts the function of many organs in the body, most notably the lungs, by perturbing salt and water transport across epithelial surfaces. F508del causes harm in two principal ways. First, the mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Second, F508del perturbs the Cl(-) channel function of CFTR by disrupting channel gating. Here, we discuss the development of rational new therapies for CF that target F508del-CFTR. We highlight how structural studies provide new insight into the role of F508 in the regulation of channel gating by cycles of ATP binding and hydrolysis. We emphasize the use of high-throughput screening to identify lead compounds for therapy development. These compounds include CFTR correctors that restore the expression of F508del-CFTR at the apical membrane of epithelial cells and CFTR potentiators that rescue the F508del-CFTR gating defect. Initial results from clinical trials of CFTR correctors and potentiators augur well for the development of small molecule therapies that target the root cause of CF: mutations in CFTR.     

Identification of synergistic combinations of F508del cystic fibrosis transmembrane conductance regulator (CFTR) modulators

Cystic fibrosis (CF) is an inherited, life-threatening disease caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), an ABC transporter-class protein and ion channel that transports ions across epithelial cell membranes. The most common mutation leads to the deletion of a single phenylalanine, and the resulting protein, F508del-CFTR, shows reduced trafficking to the membrane and defective channel gating. The ideal therapeutic approach would address both of these defects and restore channel function at the same time. We describe here the application of a combination high-throughput screening to search for synergistic modulators of F508del-CFTR. With the adapted Fischer rat thyroid-yellow fluorescent protein halide flux assay to the combination high-throughput screening platform, we identified many interesting single agents as CFTR modulators from a library of approved drugs and mechanistic probe compounds, and combinations that synergistically modulate F508del-CFTR channel function in Fischer rat thyroid cells, demonstrating the potential for combination therapeutics to address the defects that cause CF.     

Prolonged treatment of cells with genistein modulates the expression and function of the cystic fibrosis transmembrane conductance regulator

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. In the search for new CF therapies, small molecules have been identified that rescue the defective channel gating of CF mutants (termed CFTR potentiators). Here, we investigate the long-term effects of genistein, the best-studied CFTR potentiator, on the expression and function of CFTR. EXPERIMENTAL APPROACH: We pre-treated baby hamster kidney (BHK) cells expressing wild-type or F508del-CFTR (the most common CF mutant) with concentrations of genistein that potentiate (30 microM) or inhibit (100 microM) CFTR function for 2 or 24 h at 37 degrees C before examining CFTR maturation, expression and single-channel activity. KEY RESULTS: Using the iodide efflux technique, we found that genistein pre-treatment failed to restore function to F508del-CFTR, but altered that of wild-type CFTR. Pre-treatment of cells with genistein for 2 h had little effect on CFTR processing, whereas pre-treatment for 24 h either augmented (30 microM genistein) or impaired (100 microM genistein) CFTR maturation. Using immunocytochemistry, we found that all genistein pre-treatments increased the localization of CFTR protein to the cell surface. However, following the incubation of cells with genistein (100 microM) for 2 h, individual CFTR Cl(-) channels exhibited characteristics of channel block upon channel activation. CONCLUSIONS AND IMPLICATIONS: Genistein pre-treatment alters the maturation, cell surface expression and single-channel function of CFTR in ways distinct from its acute effects. Thus, CFTR potentiators have the potential to influence CFTR by mechanisms distinct from their effects on channel gating.     

Identification of natural coumarin compounds that rescue defective DeltaF508-CFTR chloride channel gating

1. Deletion of phenylalanine at position 508 (DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the DeltaF508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects. 2. We identified a class of natural coumarin compounds that can correct the defective DeltaF508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing DeltaF508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L). 3. Dose-dependent potentiation of defective DeltaF508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTR(inh)-172. Two potent compounds, namely imperatorin and osthole, have activation K(d) values of approximately 10 micromol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of DeltaF508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of DeltaF508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout. 4. In conclusion, the natural coumarin DeltaF508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the DeltaF508 mutation.     

Cross talk of cAMP and flavone in regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and Na+/K+/2Cl- cotransporter in renal epithelial A6 cells

We have reported that in renal epithelial A6 cells flavones stimulate the transepithelial Cl- secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and/or the Na+/K+/2Cl- cotransporter. On the other hand, it has been established that cAMP activates the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter. However, no information is available on the interaction between cAMP and flavones on stimulation of the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter. To clarify the interaction between cAMP and flavones, we studied the regulatory mechanism of the CFTR Cl- channel and the Na+/K+/2Cl- cotransporter by flavones (apigenin, luteolin, kaempherol, and quercetin) under the basal and cAMP-stimulated conditions in renal epithelial A6 cells. Under the basal (cAMP-unstimulated) condition, these flavones stimulated the Cl- secretion by activating the Na+/K+/2Cl- cotransporter without any significant effects on the CFTR Cl- channel activity. On the other hand, these flavones diminished the activity of the cAMP-stimulated Na+/K+/2Cl- cotransporter without any significant effects on the CFTR Cl- channel activity. Interestingly, the level of the flavone-induced Cl- secretion under the basal condition was identical to that under the cAMP-stimulated condition. Based on these results, it is suggested that although both cAMP and flavones activate the Na+/K+/2Cl- cotransporter, these flavones have more powerful effects than cAMP on the Na+/K+/2Cl- cotransporter.     

Antihypertensive 1,4-dihydropyridines as correctors of the cystic fibrosis transmembrane conductance regulator channel gating defect caused by cystic fibrosis mutations

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel gene. CF mutations like deltaF508 cause both a mistrafficking of the protein and a gating defect. Other mutations, like G551D, cause only a gating defect. Our aim was to find chemical compounds able to stimulate the activity of CFTR mutant proteins by screening a library containing approved drugs. Two thousand compounds were tested on Fischer rat thyroid cells coexpressing deltaF508-CFTR and a halide-sensitive yellow fluorescent protein (YFP) after correction of the trafficking defect by low-temperature incubation. The YFP-based screening allowed the identification of the antihypertensive 1,4-dihydropyridines (DHPs) nifedipine, nicardipine, nimodipine, isradipine, nitrendipine, felodipine, and niguldipine as compounds able to activate deltaF508-CFTR. This effect was not derived from the inhibition of voltage-dependent Ca2+ channels, the pharmacological target of antihypertensive DHPs. Indeed, methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-2(trifluoromethylphenyl)pyridine-5-carboxylate (BayK-8644), a DHP that is effective as an activator of such channels, also stimulated CFTR activity. DHPs were also effective on the G551D-CFTR mutant by inducing a 16- to 45-fold increase of the CFTR Cl- currents. DHP activity was confirmed in airway epithelial cells from patients with CF. DHPs may represent a novel class of therapeutic agents able to correct the defect caused by a set of CF mutations.     

Targeting CFTR: how to treat cystic fibrosis by CFTR-repairing therapies

Several novel compounds recently appeared as promising leads to develop effective drugs against the basic defect in Cystic fibrosis (CF) and the first rationale therapies for CF relying on the understanding of the basic defect started to hit the clinical setting. Most of these efforts are focused on correcting the F508del mutation (occurring in ≈90% of CF patients) which causes misfolding of the CF transmembrane conductance regulator (CFTR) protein, the intracellular retention of such abnormal conformation by the endoplasmic reticulum quality control and premature degradation, thus precluding CFTR from reaching the cell membrane where it normally functions as a cAMP-stimulated Cl- channel. Here, several rationale therapeutic strategies are briefly reviewed, namely, mutation-specific (or "CFTR-repairing") approaches (with a particular focus on the cellular defect associated with F508del-CFTR), manipulation of other ionic (non-CFTR) conductances and gene therapy. Still more innovative strategies, such as manipulation of the proteostasis network, displacement of molecular chaperones, targeting mutant CFTR by in silico small-molecule screens and systems biology approaches are also discussed.     

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels

1. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl(-) channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl(-) channel activity of wild-type CFTR and delF508-CFTR mutant. 2. The effects of n-alkanols like octanol on CFTR activity were measured by iodide ((125)I) efflux and patch-clamp techniques on three distinct cellular models: (1). CFTR-expressing Chinese hamster ovary cells, (2). human airway Calu-3 epithelial cells and (3). human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. 3. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated (125)I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. (125)I efflux and Cl(-) currents induced by octanol were blocked by glibenclamide but insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, as expected for a CFTR Cl(-) current. 4. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. 5. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanol<1-octanol<2-octanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF.     

Pharmacological treatment of the ion transport defect in cystic fibrosis

Cystic fibrosis (CF) is a lethal monogenetic disease characterised by impaired water and ion transport over epithelia. The lung pathology is fatal and causes death in 95% of CF patients. The genetic basis of the disease is a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most common mutation, DeltaF508, results in a protein that cannot properly be folded in the endoplasmic reticulum, is destroyed and hence does not reach the apical cell membrane. This paper will discuss those pharmacological approaches that are directed at correcting the defect in ion transport. At present, no clinically effective drug is available, although research has defined areas in which progress might be made. These are the following: (1) the drug 4-phenylbutyrate (4PBA) increases the expression of DeltaF508-CFTR in the cell membrane, probably by breaking the association between DeltaF508-CFTR and a chaperone; (2) a number of xanthines, in particular 8-cyclopentyl-1, 3-dipropylxanthine (CPX), are effective in activating CFTR, presumably by direct binding and also possibly by correcting the trafficking defect; (3) the isoflavone genistein can activate both wild-type and mutant CFTR, probably through direct binding to the channel; (4) purinergic agonists (ATP and UTP) can stimulate chloride secretion via a Ca(2+)-dependent chloride channel and in this way compensate for the defect in CFTR, but stable analogues will be required before this type of treatment has clinical significance; (5) treatment with inhaled amiloride may correct the excessive absorption of Na(+) ions and water by airway epithelial cells that appears connected to the defect in CFTR; although clinical tests have not been very successful so far, amiloride analogues with a longer half-life may give better results. The role of CFTR in bicarbonate secretion has not yet been established with certainty, but correction of the defect in bicarbonate secretion may be important in clinical treatment of the disease. Currently, major efforts are directed at developing a pharmacological treatment of the ion transport defect in CF, but much basic research remains to be done, in particular, with regard to the mechanism by which defective CFTR is removed in the endoplasmic reticulum by the ubiquitin-proteasome pathway, which is a central pathway in protein production and of significance for several other diseases apart from CF.     

CFTR potentiators partially restore channel function to A561E-CFTR,a cystic fibrosis mutant with a similar mechanism of dysfunction as F508del-CFTR

Background and Purpose

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease.

Experimental Approach

Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques.

Key Results

Low temperature incubation delivered a small proportion of A561E-CFTR protein to the cell surface. Like F508del-CFTR, low temperature-rescued A561E-CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E-CFTR also exhibited thermoinstability, losing function more quickly than F508del-CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561E-CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561E- and F508del-CFTR Cl⁻ channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches.

Conclusions and Implications

Like F508del-CFTR, A561E-CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561E-CFTR. Transformational drug therapy for A561E-CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability.     

New horizons in the treatment of cystic fibrosis

Cystic fibrosis (CF) is a lethal, recessive, genetic disease affecting approximately 1 in 2500 live births among Caucasians. The CF gene codes for a cAMP/PKA-dependent, ATP-requiring, membrane chloride ion channel, generally found in the apical membranes of many secreting epithelia and known as CFTR (cystic fibrosis transmembrane conductance regulator). There are currently over 1700 known mutations affecting CFTR, many of which give rise to a disease phenotype. Around 75% of CF alleles contain the ΔF508 mutation in which a triplet codon has been lost, leading to a missing phenylalanine at position 508 in the protein. This altered protein fails to be trafficked to the correct location in the cell and is generally destroyed by the proteasome. The small amount that does reach the correct location functions poorly. Clearly the cohort of patients with at least one ΔF508 allele are a major target for therapeutic intervention. It is now over two decades since the CF gene was discovered and during this time the properties of CFTR have been intensely investigated. At long last there appears to be progress with the pharmaco-therapeutic approach. Ongoing clinical trials have produced fascinating results in which clinical benefit appears to have been achieved. To arrive at this point ingenious ways have been devised to screen very large chemical libraries for one of two properties: (i) agents promoting trafficking of mutant CFTR to, and insertion into the membrane, and known as correctors or (ii) agents which activate appropriately located mutant CFTR, known as potentiators. The best compounds emerging from these programmes are then used as chemical scaffolds to synthesize other compounds with appropriate pharmaceutical properties, hopefully with their pharmacological activity maintained or even enhanced. In summary, this approach attempts to make the mutant CFTR function in place of the real CFTR. A major function of CFTR in healthy airways is to maintain an adequate airway surface liquid (ASL) layer. In CF the position is further confounded since epithelial sodium channels (ENaC) are no longer regulated and transport salt and water out of the airways to exacerbate the lack of ASL. Thus an additional possibility for treatment of CF is to use agents that inhibit ENaC either alone or as adjuncts to CFTR correctors and/or potentiators. Yet a further way in which a pharmacological approach to CF can be considered is to recruit alternative chloride channels, such as calcium-activated chloride channel (CaCC), to act as surrogates for CFTR. A number of P2Y(2) receptor agonists have been investigated that operate by increasing Ca(2+)(i) which in turn activates CaCC. Some of these compounds are currently in clinical trials. The knowledge base surrounding the structure and function of CFTR that has accumulated in the last 20 years is impressive. Translational research feeding from this is now yielding compounds that provide real prospects for a pharmacotherapy for this disease.     

Cystic fibrosis: a new target for 4-Imidazo[2,1-b]thiazole-1,4-dihydropyridines

The pharmacology of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel has attracted significant interest in recent years with the aim to search for rational new therapies for diseases caused by CFTR malfunction. Mutations that abolish the function of CFTR cause the life-threatening genetic disease cystic fibrosis (CF). The most common cause of CF is the deletion of phenylalanine 508 (ΔF508) in the CFTR chloride channel. Felodipine, nifedipine, and other antihypertensive 1,4-dihydropyridines (1,4-DHPs) that block L-type Ca(2+) channels are also effective potentiators of CFTR gating, able to correct the defective activity of ΔF508 and other CFTR mutants ( Mol. Pharmacol. 2005 , 68 , 1736 ). For this purpose, we evaluated the ability of the previously and newly synthesized 4-imidazo[2,1-b]thiazoles-1,4-dihydropyridines without vascular activity and inotropic and/or chronotropic cardiac effects ( J. Med. Chem. 2008 , 51 , 1592 ) to enhance the activity of ΔF508-CFTR. Our studies indicate compounds 17, 18, 20, 21, 38, and 39 as 1,4-DHPs with an interesting profile of activity.     

4-Chlorobenzo[F]isoquinoline (CBIQ), a novel activator of CFTR and DeltaF508 CFTR

4-Chlorobenzo[F]isoquinoline (CBIQ) is a novel compound, here shown to activate both CFTR (cystic fibrosis transmembrane conductance regulator) Cl- ion channels and KCNN4, intermediate conductance, calcium-sensitive K+-channels, present in transporting epithelia by the use of heterologous expression systems. Earlier studies with other benzoquinolines, namely 7,8- and 5,6 benzoquinoline, showed they too could activate CFTR and KCNN4, but the evidence was only indirect. However this study also shows that CBIQ can also activate DeltaF508 CFTR, the most common mutant form of CFTR present in approximately 75% of patients with cystic fibrosis. This property is not shared with the other benzoquinolines. As activation of CFTR and KCNN4 work in unison to promote epithelial chloride secretion, CBIQ is a new chemical scaffold for developing agents that may be useful in cystic fibrosis.     

The Cdc42 inhibitor secramine B prevents cAMP-induced K+ conductance in intestinal epithelial cells

Cyclic AMP- (cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl- transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMP-stimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba2+-sensitive K+ channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca2+-mediated chloride secretion pathway, which requires a separate K+ channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMP-dependent but not Ca2+-dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K+ conductance in intestinal epithelial cells.     

Pharmacological interventions for the correction of ion transport defect in cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated and ATP-gated Cl^- channel expressed in the apical plasma membrane of epithelial cells in the airways, digestive and reproductive tracts. Cystic fibrosis (CF) caused by mutations in the CFTR gene is characterised by chronic airway obstructions and infections, pancreatic failure, male infertility and elevated levels of salt in sweat. A pharmacological therapy would help to restore the defective transepithelial Cl^- transport observed in CF cells. Therefore, searching for potent and specific small molecules or peptides able to stimulate transepithelial Cl^- transport through direct interaction with CFTR or via CFTR-independent mechanisms has become a crucial end point in the field. With the growing understanding of the pharmacology of CFTR activity and processing, a number of academic investigators and biopharmaceutical companies have developed high-throughput screening assays, and reported active seeking of CFTR activators or modulators of airway functions in order to treat CF. This article provides an updated overview of the new emerging molecules and discusses the corresponding patent literature.     

The kappa-opioid receptor agonist asimadoline inhibits epithelial transport in mouse trachea and colon

The potent kappa-opioid receptor agonist n-methyl-N-[(1S)-1-phenyl-2-((3S)-3-hydroxypyrrolidin-1-yl)-ethyl]-2,2-diphenyl-acetamide hydrochloride (asimadoline, EMD 61753) was initially developed for the treatment of chronic pain. Because opioids are well known to reduce secretion and to cause constipation, we investigated the effects on epithelial transport in murine trachea and colon. In Ussing chamber experiments, asimadoline (100 microM) decreased short-circuit currents in airways and colon epithelium. The inhibition of I(SC) was not blocked by naloxone (10 microM) or nor-binaltorphimine (10 microM), suggesting that the response was not mediated by kappa-opioid receptors. The effect of asimadoline on I(SC) was concentration-dependent with half-maximal inhibition of I SC at 23.7 (9.5-49.3) microM and was sensitive to the K+ channel blocker charybdotoxin (10 nM). The amiloride-sensitive Na+ current was reduced by asimadoline, but not in cAMP stimulated tissues. Asimadoline strongly inhibited transient Ca2+-dependent Cl- secretion, activated by the muscarinic receptor agonist carbachol (100 microM) or the purinergic agonist ATP (100 microM). Thus, asimadoline inhibits epithelial transport independent of kappa-opioid receptors, by inhibition of basolateral Ca2+-activated and charybdotoxin-sensitive K+ channels.     

Rescue of DeltaF508 and other misprocessed CFTR mutants by a novel quinazoline compound

Cystic fibrosis (CF) is most commonly caused by deletion of Phe508 in the cystic fibrosis transmembrane conductance regulator protein (DeltaF508 CFTR). The misfolded DeltaF508 CFTR protein is retained in the endoplasmic reticulum (misprocessed mutant) and is rapidly degraded. Studies on misprocessed mutants of P-glycoprotein (P-gp), a sister protein of CFTR, however, have shown that specific substrates and modulators can act as specific chemical/pharmacological chaperones to rescue the protein. A major goal in CF research is the identification of compounds that can be used at low concentrations to rescue misprocessed CFTR mutants. Here, we show that a novel quinazoline derivative, 4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzenesulfonyl)piperazin-1-yl]ethyl}quinazoline (CF(cor)-325), rescued DeltaF508 CFTR. Incubation of BHK cells stably expressing human DeltaF508 CFTR with 1-10 microM CF(cor)-325 resulted in maturation and delivery of a functional molecule to the cell surface as determined by the iodide efflux assay. The misprocessed CFTR mutants R258G, S945L, and H949Y were also rescued by CF(cor)-325 in either BHK or HEK 293 cells. CF(cor)-325 appeared to be specific for DeltaF508 CFTR because another quinazoline derivative, prazosin, did not rescue the misprocessed CFTR mutants. CF(cor)-325 could also rescue misprocessed mutants of P-gp. The compound was a P-gp inhibitor as it inhibited vinblastine-stimulated ATPase activity. P-gp-mediated vinblastine resistance was also reduced about 10-fold with 300 nM CF(cor)-325. These results show that CF(cor)-325 is a particularly important lead compound for treatment of CF because low concentrations can be used to rescue many misprocessed CFTR mutants.     

Therapeutic approaches to repair defects in deltaF508 CFTR folding and cellular targeting

The deltaF508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene is the most common mutation in CF. The mutant CFTR protein is defective with respect to multiple functions including cAMP-regulated chloride conductance, nucleotide transport, and regulatory actions on other ion channels. Since the deltaF508 protein is also temperature-sensitive and unstable during translation and folding in the endoplasmic reticulum (ER), most of the nascent chains are targeted for premature proteolysis from the ER. This paper focuses on the events that occur in the ER during folding and reviews potential targets for therapeutic intervention.