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Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e., function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC50 value

Cystic fibrosis (CF) is a multiorgan disease of the lungs, sinuses, pancreas, and gastrointestinal tract that is caused by a dysfunction or deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an epithelial anion channel that regulates salt and water balance in the tissues in which it is expressed. To effectively treat the most prevalent patient population (F508del mutation), two biomolecular modulators are required: correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Despite approved potentiator and potentiator/corrector combination therapies, there remains a high need to develop more potent and efficacious correctors. Herein, we disclose the discovery of a highly potent series of CFTR correctors and the structure-activity relationship (SAR) studies that guided the discovery of ABBV/GLPG-2222 (22), which is currently in clinical trials in patients harboring the F508del CFTR mutation on at least one allele.

Defective epithelial ion transport is the hallmark of the common life-limiting genetic disease cystic fibrosis (CF). CF is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette transporter, which functions as an epithelial anion channel regulated by phosphorylation and cycles of ATP binding and hydrolysis. Here, we review selectively the structure and function of CFTR, including how the membrane-spanning domains form an anion-selective pore and the roles of the regulatory domain and nucleotide-binding domains in channel gating. We discuss the development of combination therapy for CF mutations, including CFTR correctors, which rescue misfolding to transport mutant CFTR to the plasma membrane and CFTR potentiators, which increase CFTR activity by enhancing channel gating. Finally, we highlight studies of CFTR processing and membrane trafficking, which identify proteostasis regulators that have the potential to lead to innovative new therapies for CF. Thus, understanding of CFTR has driven new insights into epithelial ion transport and transformed the treatment of CF.

The opportunistic pathogen Pseudomonas aeruginosa has two complete acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-LasI and RhlR-RhlI. LasI catalyzes the synthesis of N -3-oxododecanoyl homoserine lactone (3OC12-HSL), and LasR is a transcription factor that requires 3OC12-HSL as a ligand. RhlI catalyzes the synthesis of N -butanoyl homoserine lactone (C 4 ), and RhlR is a transcription factor that responds to C 4 . LasR and RhlR control the transcription of hundreds of P. aeruginosa genes, many of which are critical virulence determinants, and LasR is required for RhlR function. We developed an ultra-high-throughput cell-based assay to screen a library of approximately 200,000 compounds for inhibitors of LasR-dependent gene expression. Although the library contained a large variety of chemical structures, the two best inhibitors resembled the acyl-homoserine lactone molecule that normally binds to LasR. One compound, a tetrazole with a 12-carbon alkyl tail designated PD12, had a 50% inhibitory concentration (IC 50 ) of 30 nM. The second compound, V-06-018, had an IC 50 of 10 μM and is a phenyl ring with a 12-carbon alkyl tail. A microarray analysis showed that both compounds were general inhibitors of quorum sensing, i.e., the expression levels of most LasR-dependent genes were affected. Both compounds also inhibited the production of two quorum-sensing-dependent virulence factors, elastase and pyocyanin. These compounds should be useful for studies of LasR-dependent gene regulation and might serve as scaffolds for the identification of new quorum-sensing modulators.

We recently showed the inhibitory effect of a flavonoid antioxidant, silymarin, on erbB1-Shc activation in prostate cancer (PCA) DU145 cells. In the present study, we performed more detailed mechanistic and molecular modeling studies with pure silibinin to assess and define its effect on membrane signaling related to erbB1 activation in human PCA LNCaP and DU145 cells. Studies also were performed to establish the biologic responses toward extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation, cell growth, and DNA synthesis. Treatment of serum-starved cells with various doses of silibinin for 2 h followed by 125I-epidermal growth factor (EGF) showed 30–75% inhibition in ligand binding and 55–95% inhibition in its internalization in LNCaP cells and 20–64% and 12–27% inhibition in these two events in DU145 cells. Time-response studies showed similar effects. In further studies, treatment of serum-starved cultures with silibinin followed by EGF showed strong inhibitory effects on membrane and cytoplasmic signaling molecules. In the case of erbB1 activation, silibinin showed a 58–75% decrease in LNCaP and a 40–100% decrease in DU145 cells at 50, 75, and 100-μg/mL doses. Inhibitory effects of silibinin also were evident on ERK1/2 activation (20–80% inhibition) in both cell lines. Treatment of serum-starved cultures with silibinin resulted in 20–40% and 30–55% inhibition of LNCaP and DU145 cell growth, respectively, at similar doses after 1–3 d of treatment, and 10–50% cell death in both cell lines. Under 10% serum conditions, identical silibinin treatments resulted in 20–65% inhibition of cell growth in LNCaP and DU145 cells but did not cause any cell death. Similar doses of silibinin treatments for 24 h also resulted in 25–60%, 35–40%, and 36–50% inhibition of DNA synthesis when cells were cultured in 10% serum, totally serum starved, and serum starved plus stimulated with EGF, respectively. Molecular modeling of silibinin showed that it is a highly lipophilic compound, suggesting that it interacts with lipid-rich plasma membrane, including binding with erbB1, thereby competing with the EGF-erbB1 interaction. Because the ligand-erbB1 autocrine-loop is causally involved in advanced and androgen-independent PCA, the observed effects of silibinin and its strong lipophilic nature could be useful in developing this agent for the prevention and therapy of PCA. © 2001 Wiley-Liss, Inc.

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO−3 by a Cl −-independent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO−3 secretion and caused the secretion of Cl − by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO−3 secretion to Cl − secretion because the uptake of HCO−3 across the basolateral membrane is mediated by a 4,4 ′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive Na+:HCO−3 cotransporter. Since the stoichiometry reported for Na +:HCO−3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO−3 entry and favor the secretion of Cl −. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO−3 and Cl − secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO−3 and a Cl − channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO−3 secretion in the human airways warrants greater attention.

Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIα and RIIα. In addition, immunogold electron microscopy showed that RIIα labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIα was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Cα was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (∼100 μA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.

We previously demonstrated that the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), stimulates a sustained Cl- secretory response across T84 monolayers by opening a Ca(2+)-dependent basolateral K+ channel. In the present work, we evaluated the effects on Cl-secretion of other benzimidazolones, NS-004 and NS-1619, which have been shown to open cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast to 1-EBIO, neither NS-004 nor NS-1619 stimulated a significant Cl- secretory current (Isc). Neither NS-004 nor NS-1619 increased Isc subsequent to forskolin stimulation. However, when added after 1-EBIO, NS-004 and NS-1619 stimulated large sustained increases in Isc. In addition, NS-004 and NS-1619 potentiated the effects of carbachol. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of NS-004 and 1-EBIO on the basolateral K+ (IK) and apical Cl- (ICl) currents. Both NS-004 and 1-EBIO increased ICl, and the stimulated currents were inhibited by glibenclamide. In contrast, NS-004 failed to significantly affect IK, but subsequent addition of 1-EBIO induced a large increase in IK. The effects of 1-EBIO, NS-004, and NS-1619 on the Ca(2+)-dependent K+ channel (KCa) in T84 cells was determined in excised inside-out patches. Neither NS-004 nor NS-1619 affected K+ channel activity, whereas the subsequent addition of 1-EBIO produced a marked channel activation. Results similar to those observed in T84 monolayers were obtained from murine airway cell primary cultures: NS-004 or NS-1619 had no effect on Isc, whereas 1-EBIO stimulated a sustained Cl- secretory response. The results demonstrate that activation of CFTR alone is insufficient to evoke transepithelial Cl- secretion. Activation of the basolateral membrane K+ channel is a necessary component of the secretory response. Thus the basolateral membrane KCa may be a novel pharmacological target in cystic fibrosis therapy.

Reaction of 3,9 alpha, 17 beta-trihydroxyestra-1,3,5(10)-trien-11-one 17-acetate 3-methyl ether (3) with N3H-BF3 etherate leads mainly to lactam (4) along with the N-azido compound (5) as a minor product. Under similar conditions, 3,17 beta-dihydroxyestra-1,3,5(10)-trien-6-one 17-acetate 3-methyl ether gives lactam (12) and the tetrazole derivative (9). Similar reaction of the diacetate (8) gives only the tetrazole derivative (11). Compounds 4, 6, and 10 prevent implantation in rats at 5-, 10-, and 5-mg/kg doses, respectively. Compounds 4, 6, 9, and 10 show significant estrogenic activity at the respective contraceptive doses.Scientists at the Central Drug Research Institute in Lucknow, India, synthesized and evaluated new 6-aza-B-and 11-aza-C-homoestranes to determine the effect of structural changes around C-11 and C-6 in estradiol on its estrogenic and antifertility activities. They used sperm-positive female albino rats (180-220 g) to test the compounds anti-implantation activity and ovariectomized immature rats (25-30 g) to test their estrogenic activity. Adding N3H-BF3 etherate to 3, 9-alpha,17-beta-trihydroxyestra-1,3,5(10)-trien-11-one 17-acetate 3-methyl ether created 11-aza-3,17beta-dihydroxy-C-homoestra-1,3,5(10),8(9)-tetraen-12-one 17-acetate 3-methyl ether and the N-azido compound as a minor product. Adding N3H-BF3 etherate to 3, 17beta-dehyroxyestra-1,3,5(10)-trien-6-one 17-acetate 3-methyl ether created 6-aza-3,17beta-dihydroxy-B-homoestra-1,3,4(10)-trien-7-one 17-acetate 3-methyl ether and the tetrazolo derivative. When N3H-BF3 etherate was added to diacetate, just the tetrazole derivative emerged. 1-aza-3,17beta-dihydroxy-C-homoestra-1,3,5(10),8(9)-tetraen-12-one 17-acetate 3-methyl ether (5 mg-kg dose), 11-aza-3,17beta-dihydroxy-C-homoestra-1,3,5(10),8(9)-tetraen-12-one 3-methyl ether (10 mg/kg dose), and 6-aza-3,17beta-dihydroxy-B-homoestra-1,3,4(10)-trieno[6,7-d]tetrazole 3-methyl ether (5 mg/kg dose) all delivered subcutaneously, prevented implantation in all rats. These compounds and 6-aza-3,17beta-dihydroxy-B-homoestra-1,3,5(10)-trieno[6,7-d] tetrazole 17-acetate 3-methyl ether (10mg/kg dose) showed significant estrogenic activity.

Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP-ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously reported blocker (Singh, A. K., G. B. Afink, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP should be useful in future studies of ion channel nucleotide binding sites and possibly in preliminary steps of ion channel protein purification. In addition, we have obtained good evidence that there are at least two nucleotide binding sites located on opposite sides of the colonic Cl- channel and that occupancy of either site produces a blocked state.

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (P o ) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl − secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na + and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway.

Synthesis of 7-aryl/allyl-substituted androstene derivatives 3a through 3g has been carried out by Grignard reaction on 3 beta,17 beta-diacetoxyandrost-5-en-7-one (2) with aryl/allyl magnesium bromide. Isomeric mixture of products 3b and 3c/3e and 3f/3h was separated by column chromatography. Stereochemical assignment at C-7 has been made on the basis of 13C nuclear magnetic resonance studies and chemical considerations. Compounds 6a and 6b were synthesized by alkylation of compound 5 with beta-(N,N-diethylamino)ethyl chloride hydrochloride and 1-(2-chloroethyl)pyrrolidine hydrochloride, respectively. Compound 3g (isomeric mixture) prevented pregnancy in 60% of rats at 10 mg/kg daily dose administered orally on days 1 to 7 of pregnancy; however, its only isolable 7 beta-hydroxy isomer, 3h, was inactive at this dose.12 new C-7 aryl- and allyl-substituted androst-5-ene derivatives were synthesized, characterized, and were screened for anti-implantation activity in rats. 10 compounds were constructed by linking the aryl/allyl magnesium bromide with 3-beta, 17-beta-diacetoxyandrost-5-en-7-one by a Grignard reaction, and 2 further compounds were made by alkylation. Isomeric mixtures were separated by column chromatography, and stereochemical assignment at C-7 determined by nuclear magnetic resonance and chemical characteristics. The isomeric mixture of the N-N-dimethyl aminophenyl derivative prevented pregnancy in 6 of 10 rats tested at a 10 mg/kg dose p.o., given on Days 1-7 after coitus. The pure 7-beta-OH isomer was inactive, and the 7-alpha-OH isomer could not be obtained from column chromatography on neutral alumina.

We compared the potency and inhibitory actions of three different classes of organic acids on a Cl channel derived from colonic enterocyte plasma membrane vesicles. Chloride channels were incorporated into planar lipid bilayer membranes to examine the effects of the anthranilic acids, diphenylamine 2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), the indanyl alkanoic acids, 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden -5-yl)oxy] acetic acid (IAA-94) and its stereoenantiomer IAA-95, and the disulfonic stilbene, 4,4'-dinitro-stilbene-2,2'-disulfonic acid (DNDS). Except for DNDS, each of the blockers was equipotent from both the outer membrane and the cytoplasmic side of the channel protein. The potency order from the outmembrane side was DNDS greater than IAA-94 = IAA-95 greater than NPPB much greater than DPC. In contrast, the potency order from the cytoplasmic side was IAA-94 = IAA-95 greater than NPPB greater than DNDS much greater than DPC. DPC and NPPB caused a concentration-dependent decrease in the single-channel conductance (fast block). DNDS, IAA-94, and IAA-95 caused a flickery-type block and a concentration-dependent decrease in open-channel probability. Kinetic analysis revealed that blockade could be explained by a linear closed-opened-blocked kinetic scheme. Similarities in the electrostatic potential maps of these open-channel blockers suggest they may bind to a single shared binding site within the channel protein.

A collagenase-based dissociation technique has been developed to routinely establish monolayer cultures of freshly isolated porcine vas deferens epithelium. Cells isolated from each tissue are transferred to 25-cm2tissue culture flasks and grown in a standard cell culture medium. Flasks reach confluency in 3–4 days, and cells are subsequently seeded onto permeable supports. Cultured cells display a monolayer cobblestone appearance and are immunoreactive to anti-ZO-1 and anti-cytokeratin antibodies. Electron microscopy is employed to demonstrate the presence of junctional complexes and microvilli. When evaluated in modified Ussing chambers, cultured monolayers exhibit a basal lumen negative potential difference, high electrical resistance (>1,000 Ω · cm2), and respond to norepinephrine, vasopressin, ATP, adenosine, and histamine, with changes in short-circuit current indicative of anion secretion. Responses are significantly attenuated in Cl−- and/or HCO[Formula: see text]-free solutions. Attempts to further optimize culture conditions have shown that chronic exposure to insulin increases proliferation rates. Thus the culture method described will reliably produce viable neurotransmitter-responsive cell monolayers that will allow for the characterization of vas deferens epithelial function and associated control mechanisms.

We previously characterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as the clinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine (ZOX), as pharmacological activators of the intermediate-conductance Ca2+-activated K+channel, hIK1. The mechanism of activation of hIK1, as well as the highly homologous small-conductance, Ca2+-dependent K+channel, rSK2, was determined following heterologous expression in Xenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced a concentration-dependent activation of hIK1, with half-maximal ( K1/2) values of 84 μM and 98 μM, respectively. Similarly, CZ activated rSK2 with a K1/2of 87 μM. In the absence of CZ, the Ca2+-dependent activation of hIK1 was best fit with a K1/2of 700 nM and a Hill coefficient ( n) of 2.0. rSK2 was activated by Ca2+with a K1/2of 700 nM and an n of 2.5. Addition of CZ had no effect on either the K1/2or n for Ca2+-dependent activation of either hIK1 or rSK2. Rather, CZ increased channel activity at all Ca2+concentrations ( Vmax). Event-duration analysis revealed hIK1 was minimally described by two open and three closed times. Activation by 1-EBIO had no effect on τo1, τo2, or τc1, whereas τc2and τc3were reduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. In conclusion, we define 1-EBIO, CZ, and ZOX as the first known activators of hIK1 and rSK2. Openers of IK and SK channels may be therapeutically beneficial in cystic fibrosis and vascular diseases.

We evaluated the effects of clotrimazole and clofibrate on Ca(2+)- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the colonic cell line, T84. We used 1-ethyl-2-benzimidazolinone (1-EBIO) to activate the Ca(2+)-dependent K+ channel (KCa) in these cells to induce a sustained Cl- secretory current (Isc). Clotrimazole potently inhibited the KCa-dependent Isc, with an inhibition constant (Ki) of 0.27 +/- 0.02 microM. Clofibrate also inhibited the 1-EBIO-induced Isc albeit with lower affinity (Ki = 6.5 +/- 1.2 microM). Clotrimazole (10 microM) inhibited the Isc response to the Ca(2+)-mediated agonist, carbachol, by 82%. Similarly, both clotrimazole and clofibrate inhibited cAMP-mediated Cl- secretion, with Ki values of 5.2 +/- 1.0 and 6.7 +/- 1.1 microM, respectively. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of clotrimazole and clofibrate on the basolateral K+ (IK) and apical Cl- (ICl) currents following stimulation by either 1-EBIO or forskolin. Both clotrimazole and clofibrate inhibited the 1-EBIO- and forskolin-induced IK without affecting ICl. We determined the effects of clotrimazole and clofibrate on KCa using 86Rb+ uptake studies into membrane vesicles. Both clotrimazole and clofibrate inhibited the 1-EBIO-induced 86Rb+ uptake, with Ki values of 0.31 +/- 0.08 and 10.8 +/- 5.5 microM, respectively. Similarly, clotrimazole inhibited the Ca(2+)-induced 86Rb+ uptake with a Ki of 0.51 +/- 0.15 microM. Charybdotoxin inhibited both the 1-EBIO- and Ca(2+)-induced 86Rb+ uptakes with similar affinities (Ki values of 0.57 +/- 0.07 and 0.47 +/- 0.08 nM, respectively), suggesting 1-EBIO and Ca2+ activate the same channel (KCa) in this assay. In excised, single-channel recordings both clotrimazole and clofibrate inhibited KCa, demonstrating a direct inhibition of the channel by these compounds. We demonstrate that clotrimazole blocks the intestinal KCa, thereby inhibiting Cl- secretion. These results suggest that clotrimazole may be useful as an antidiarrheal.

We evaluated the effects of the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), on Cl- secretion across T84 monolayers. 1-EBIO stimulated a sustained Cl- secretory response at a half-maximal effective concentration of 490 microM. Charybdotoxin (CTX) inhibited the 1-EBIO-induced short-circuit current (Isc) with an inhibitory constant (Ki) of 3.6 nM, whereas 293B, an inhibitor of adenosine 3',5'-cyclic monophosphate-activated K+ channels, had no effect on the current induced by 1-EBIO. In contrast, CTX failed to inhibit the 293B-sensitive forskolin-induced Isc. The above results suggested that 1-EBIO may be activating the basolateral membrane Ca(2+)-dependent K+ channel (KCa) in these cells. This was further confirmed using nystatin to permeabilize the apical membrane in the presence of a mucosa-to-serosa K+ gradient and determining the effects of 1-EBIO on the basolateral K+ current (IK). Under these conditions, 1-EBIO induced a large increase in IK that was blocked by CTX. In membrane vesicles prepared from T84 cells, 1-EBIO stimulated 86Rb+ uptake in a CTX-sensitive manner; the Ki for inhibition by CTX was 3.5 nM. Similar to our intact monolayer studies, this 86Rb+ uptake was not blocked by 293B. The effects of 1-EBIO on the KCa in T84 cells was determined in excised inside-out patches. 1-EBIO (100 microM) increased the product of the number of channels and the open channel probability from 0.09 +/- 0.03 to 1.17 +/- 0.27 (n = 8); this effect on KCa activity required a minimal level of free Ca2+. Similar to its effect on T84 cells, 1-EBIO stimulated a sustained Cl- secretory current in rat colonic epithelium, which was partially blocked by CTX. Finally, 1-EBIO stimulated a sustained Cl- secretory response in primary cultures of murine tracheal epithelium. We conclude that the benzimidazolone, 1-EBIO, stimulates Cl- secretion in secretory epithelia via the direct activation of a Kca. 1-EBIO is the first pharmacological opener of this important class of epithelial K+ channels to be identified.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel that is regulated by protein kinase A and cytosolic nucleotides. Previously, Sheppard and Welsh reported that the sulfonylureas glibenclamide and tolbutamide reduced CFTR whole cell currents. The aim of this study was to quantify the effects of tolbutamide on CFTR gating in excised membrane patches containing multiple channels. We chose tolbutamide because weak (i.e., fast-type) open channel blockers introduce brief events into multichannel recordings that can be readily quantified by current fluctuation analysis. Inspection of current records revealed that the addition of tolbutamide reduced the apparent single-channel current amplitude and increased the open-channel noise, as expected for a fast-type open channel blocker. The apparent decrease in unitary current amplitude provides a measure of open probability within a burst (P0 Burst), and the resulting concentration-response relationship was described by a simple Michaelis-Menten inhibition function. The concentration of tolbutamide causing a 50% reduction of Po Burst (540 +/- 20 microM) was similar to the concentration producing a 50% inhibition of short-circuit current across T84 colonic epithelial cell monolayers (400 +/- 20 microM). Changes in CFTR gating were then quantified by analyzing current fluctuations. Tolbutamide caused a high-frequency Lorentzian (corner frequency, fc > 300 Hz) to appear in the power density spectrum. The fc of this Lorentzian component increased as a linear function of tolbutamide concentration, as expected for a pseudo-first-order open-blocked mechanism and yielded estimates of the on rate (koff = 2.8 +/- 0.3 microM-1 s-1), the off rate (kon = 1210 +/- 225 s-1), and the dissociation constant (KD = 430 +/- 80 microM). Based on these observations, we propose that there is a bimolecular interaction between tolbutamide and CFTR, causing open channel blockade.

Development of chloride channel modulators. Chloride channels are ubiquitously distributed, biophysically varied and functionally diverse. Despite the known contribution of chloride channels to the physiology of various cell types and the pathology of several diseases, high affinity ligands are not available to study these channels. Here we report the iterative and integrated use of ion channel kinetic analysis and computational chemical methods in the development of high affinity blockers of the outwardly rectifying chloride channel (ORCC). Kinetic analysis, with emphasis on estimation of the block time constant as determined from critical closed time plots, was used to guide the synthesis of new disulfonic stilbene derivatives. Computational chemical methods were used to deduce the important features of the disulfonic stilbene molecule necessary for potent blockade of ORCC and ultimately led to the discovery of the calixarenes. Para-sulfonated calixarenes were found to be potent blockers of ORCC with subnanomolar inhibition constants and exceptionally long block times.

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