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CFTR pharmacology and its role in intestinal fluid secretion Jay R Thiagarajah and AS Verkman The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl channel expressed in epithelial cells in the airways, pancreas, intestine and other fluid-transporting tissues. Cystic fibrosis is caused by mutations in the CFTR, resulting in impaired Cl transport and plasma membrane targeting. CFTR is expressed in the lumenal membrane of enterocytes, where it functions as the principal pathway for secretion of Cl and fluid in enterotoxin-induced secretory diarrheas such as cholera. Small-molecule CFTR inhibitors reduce enterotoxin-induced intestinal fluid secretion in animal models. CFTR inhibition might also reduce intestinal fluid losses in cholera and possibly in other infectious and non-infectious diarrheas. Addresses Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521, USA Correspondence: Alan S Verkman; e-mail: [email protected]; http://www.ucsf.edu/verklab
Current Opinion in Pharmacology 2003, 3:594–599 This review comes from a themed issue on Gastrointestinal pharmacology Edited by David Grundy and Wendy Winchester 1471-4892/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2003.06.012
Abbreviations ABC ATP-binding cassette CaCC Ca2þ-activated Cl channel CFTR cystic fibrosis transmembrane conductance regulator NBD nucleotide-binding domain NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid SUR sulfonylurea receptor
Introduction The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was identified in 1989 as the genetic basis of the hereditary lethal disease cystic fibrosis. CFTR is a cAMP-activated Cl channel expressed in epithelial cells in the airways, sweat duct, testis, pancreas, intestine and other fluid-transporting tissues. In cystic fibrosis, defective epithelial cell fluid transport produces chronic lung infection and a slow deterioration in lung function, as well as pancreatic insufficiency, meconium ileus and male infertility [1]. CFTR is a large transmembrane glycoprotein containing two six-helix membrane-spanning domains, each followed by a nucleotide-binding domain (NBD), with a regulatory domain linking the first NBD and the second membrane-spanning domain (Figure 1a). CFTR activation involves ATP bindCurrent Opinion in Pharmacology 2003, 3:594–599
ing and hydrolysis at NBDs, and phosphorylation of multiple regulatory domain sites (reviewed in [2]), although many details of CFTR regulation and gating remain unknown. CFTR has homology with members of the ATP-binding cassette (ABC) family of membrane proteins (including the multi-drug resistance protein-1 and sulfonylurea receptor [SUR]) in their membrane-spanning domains and NBDs; however, unlike other ABC proteins, CFTR contains a regulatory domain. CFTR is expressed at the apical membrane of enterocytes in intestine, and is thought to be the primary pathway for Cl and hence fluid secretion into the intestinal lumen in cAMP-mediated diarrheas such as cholera [3]. Secretory diarrheas caused by Vibrio cholera and enterotoxogenic Escherichia coli, in which intestinal fluid hypersecretion leads to a massive loss of fluid and electrolytes, are a major cause of morbidity and mortality worldwide [4]. An antisecretory drug that acts directly on Cl secretion might complement oral fluid replacement, which has been the mainstay of diarrheal therapy [5]. This review is focused on small-molecule activators and inhibitors of CFTR function, and on the role that CFTR plays in intestinal fluid secretion.
CFTR activators CFTR activation can be accomplished in many ways, the most important being elevation of cytosolic cAMP (promoting CFTR phosphorylation), inhibition of phosphatase activity (blocking CFTR dephosphorylation) and direct interaction [6]. Small molecules that activate CFTR by direct interaction include flavones/isoflavones (e.g. genistein [7]), benzo[c]quinoliziniums [8], xanthines [9] and benzimidazolones [10]. Figure 1b shows the structures of IBMX, a xanthine that activates CFTR both by direct and indirect (phosphodiesterase inhibition) mechanisms; NS004, a benzimidazolone that was originally characterized as a Kþ channel opener; and UCCF029, a benzoflavone that was discovered from the screening of a flavone library [11,12]. In a recent systematic search for small-molecule CFTR activators, 60 000 compounds were screened using a cell-based fluorescence assay [13]. More than a dozen compounds with novel structures were identified with submicromolar potency for activation of human wild-type CFTR. The compounds activated CFTR without elevating cAMP or inhibiting phosphatase activity, suggesting direct CFTR binding. The structures of two such compounds (CFTRact-09 and -12), which also correct the DF508–CFTR gating defect, are shown in Figure 1b. Activation of wild-type CFTR by drug-like small molecules is relatively easy compared with activation of cystic fibrosis-causing CFTR www.current-opinion.com
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Figure 1
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CFTR activators and inhibitors. (a) CFTR structure showing membrane-spanning domains (shaded), NBD1 and NBD2, and the regulatory domain (R). (b) Chemical structures of selected CFTR activators and inhibitors.
mutants; however, the latter subject is beyond the scope of this review.
CFTR inhibitors Two major classes of CFTR inhibitors are the arylaminobenzoates and sulphonylureas. The arylaminobenzoates, developed originally as blockers of kidney tubule Cl conductance [14], include diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)-benzoic acid
(NPPB) (Figure 1b) [15]. These compounds are nonspecific Cl channel blockers; patch-clamp and mutagenesis studies suggest voltage-dependent block of CFTR, resulting in CFTR pore occlusion. The sulfonylureas, originally used as bacteriostatic agents, were optimized for modulation of insulin release as oral hypoglycemic agents in diabetes. Glibenclamide (Figure 1b) binds to the ABC protein SUR, where it modulates Kir6.2 Kþ channel activity and insulin release from pancreatic islet
Figure 2
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Identification of CFTR inhibitors by high-throughput screening. (a) Screening approach: CFTR is stimulated by multiple agonists in stably transfected epithelial cells co-expressing human CFTR and a yellow fluorescent protein (YFP) having Cl/I-sensitive fluorescence. After addition of test compound, I influx is induced by adding an I-containing solution. (b) Representative original fluorescence data from individual wells of a 96-well plate showing controls (no activators, no test compound) and test wells. (c) Top panel: CFTRinh-172 inhibition of short-circuit current in permeabilized Fisher rat thyroid cells expressing human CFTR after stimulation by 100 mM CPT-cAMP. Bottom panel: dose-inhibition data for CFTRinh-172 and glibenclamide. www.current-opinion.com
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cells. At much higher concentrations, glibenclamide was found to inhibit CFTR [16]. Glibenclamide and the related sulfonylurea tolbutamide are likely to block CFTR function by binding to the CFTR pore in its open state [17]. Although arylaminobenzoates and sulphonylureas inhibit CFTR Cl channel function, high concentrations are generally required (> 0.1 mM) where multiple ion channels and transporters are affected. Disulfonic stilbene inhibitors of anion transport also inhibit CFTR but at very high concentrations (generally > 1 mM). A component of boiled rice also appears to inhibit CFTR Cl secretion in intestinal cells [18], although its identity remains unknown. Recently, a collection of 50 000 drug-like molecules was screened for CFTR inhibition. Fisher rat thyroid cells coexpressing human CFTR and a green fluorescent protein-
based halide sensor [19] were stimulated by a CFTRactivating cocktail and then subjected to an iodide gradient (Figure 2a). Iodide addition produced a prompt decrease in cell fluorescence after CFTR activation (Figure 2b). Inhibitors (‘active compounds’) were identified from a reduction in the fluorescence slope. The best inhibitor identified by screening and subsequent optimization was the 2-thioxo-4-thiazolidinone compound CFTRinh-172 (Figure 1b) [20]. CFTRinh-172 inhibited CFTR function at submicromolar concentrations — approximately 500 times better than glibenclamide when studied under similar conditions (Figure 2c). Further analysis showed voltage-independent inhibition of CFTR Cl conductance with prolonged mean channel-closed time but without change in unitary conductance. CFTRinh-172 did not affect other Cl channels (calcium- and volumeactivated) or ABC transporters (e.g. multi-drug resistance
Figure 3
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–
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Intestinal secretory pathways. Cl secretion involves Cl uptake at the basolateral membrane of enterocytes by the NaþKþ2Cl cotransporter (NKCC) and its exit primarily via apical membrane CFTR. Other Cl channels (such as CaCCs and ClCs) might also mediate Cl secretion. Naþ and water follow by a paracellular route. Secretion initiated by pathogens (cholera toxin, STa toxin and rotavirus) involves multiple pathways involving the release of 5-hydroxytryptamine (5-HT) from enterochromaffin cells, neuronal signaling, vasoactive intestinal peptide (VIP), acetylcholine (ACh), substance P (SP) and the release of inflammatory mediators from mast cells and neutrophils (e.g. prostaglandins and interleukins). Signals are transduced by second messengers (cAMP, cGMP, Ca2þ) to activate membrane ion channels. Secretion can be blocked by CFTR inhibitors, inhibition of neuronal signaling, 5-hydroxytryptamine receptor antagonists and anti-enkephalinases. Current Opinion in Pharmacology 2003, 3:594–599
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secretion is driven by active Cl transport from the basolateral to the apical side of enterocytes (Figure 3). Cl is taken up at the basolateral membrane via NaK2Cl cotransporter, which is driven by Naþ and Cl concentration gradients produced by the NaþKþ-ATPase and basolateral Kþ channels. Cl is electrochemically driven across the cell apical membrane primarily through the CFTR, as well as through Ca2þ-activated Cl channels (CaCCs) and other Cl channels. Both Naþ and fluid follow Cl paracellularly. As shown schematically in Figure 3, Cl and hence fluid secretion can be activated by different mechanisms. For example, secretory neuronal pathways
protein-1, SUR). Rodent pharmacology studies indicated that CFTRinh-172 has low toxicity, a large volume of distribution with slow elimination by renal glomerular filtration, little metabolism, and enterohepatic circulation with accumulation in bile and intestine [21].
Role of CFTR in intestinal secretion Fluid secretion plays a key role in intestinal physiology. Under normal conditions, the intestine carries out the absorption of luminal fluid, electrolytes and nutrients. A small amount of basal secretion facilitates hydration of the intestinal mucosa and mixing of intestinal contents. Fluid Figure 4
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Antidiarrheal properties of a CFTR inhibitor. (a) Intestinal fluid absorption. Closed mouse ileal loops were injected with 200 ml saline and loop weight measured at indicated times. Inset: shows % absorption at 30 minutes with and without CFTRinh-172. (b) Intestinal fluid secretion. Time-course of cholera toxin-induced fluid secretion. Loops were injected with 1 mg cholera toxin. Dashed line shows control (saline-injected) loops. (c) Dose-response for inhibition of fluid accumulation. Mice were given single doses of CFTRinh-172 by intraperitoneal injection and loop weight was measured at six hours. Inset: intestinal loops six hours after cholera toxin or saline injection. Where indicated, the mouse was treated with CFTRinh-172. (d) Persistence of CFTRinh-172 inhibition. Mice were injected with 20 mg CFTRinh-172 at indicated times before or after cholera toxin administration. www.current-opinion.com
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involve the release of 5-hydroxytryptamine from enterochromaffin cells, resulting in activation of cholinergic and vasoactive intestinal peptide neurons and increased cAMP and Ca2þ levels, causing subsequent Cl channel activation. Inflammatory mediators, such as prostaglandins and interleukins, also play an important role in initiating Cl secretion, and recent studies have begun to elucidate their signaling pathways [22,23,24,25]; nucleotides and purinergic signaling also stimulate Cl secretion through Ca2þ and cAMP signaling pathways [26,27]. Evidence suggests a pivotal role for CFTR in intestinal Cl and fluid secretion. Numerous in vitro studies have shown that agonist-induced Cl secretion in intestinal sheets and cell-lines is blocked by glibenclamide and NPPB [28,29]. CFTR knockout mice develop intestinal obstruction because of defective intestinal Cl and fluid secretion and increased fluid absorption [30]. Increased expression of alternative Cl channels is found in CFTR null mice that develop mild intestinal symptoms [31]. Cholera toxin, which causes massive fluid secretion in the small intestine in normal mice, does not induce fluid secretion in CFTR knockout mice [32].
CFTR inhibitors as anti-diarrheals Secretory diarrhea is caused by intestinal infection by various bacterial and viral pathogens. Bacterial enterotoxins, such as cholera toxin from V. cholerae and STa toxin from E. coli, induce increases in the second messengers cAMP and cGMP, respectively, resulting in activation of CFTR Cl conductance (Figure 3). Other pathogens, such as entero-invasive bacteria, also stimulate Cl secretion, and recent studies on Salmonella and enteropathogenic E. coli provide evidence for the involvement of inflammatory mediators and nucleotides in secretion [25,27]. An important cause of secretory diarrhea is the rotavirus viral pathogen, which is responsible for 20% of all diarrhea-related deaths in children under the age of five [33]. Secretion in rotaviral diarrhea was thought to involve inhibition of solute and fluid absorption. However, the extensive fluid loss during symptomatic infection suggests the involvement of active secretory mechanisms. CaCCs, which provide an alternative apical exit pathway for Cl (Figure 3), have been postulated to play a role in some viral diarrheas [34]. Another diarrheal disorder, AIDS-related diarrhea, is thought to have a multi-factorial etiology, including opportunistic infection by pathogens such as enterotoxigenic bacteria (e.g. E. coli), parasites (e.g. cryptosporidium) and viruses (e.g. cytomegalovirus). CFTR inhibition has been proposed as a therapy for some forms of secretory diarrhea [35]. Indeed, previous in vitro studies showed that a diarylsulfonylurea CFTR inhibitor prevented Cl secretion in porcine mucosa [36]. The antidiarrheal efficacy of the thiazolidinone CFTRinh172 has recently been tested [20]. In a mouse closedCurrent Opinion in Pharmacology 2003, 3:594–599
ileal loop model, injected fluid was rapidly cleared and remained unaffected by CFTRinh-172 administration (Figure 4a), an important prerequisite for antidiarrheal application of a CFTR inhibitor. Injection of cholera toxin into loops produced fluid secretion over six hours after a slow onset (Figure 4b). In a dose-response study, a single intraperitoneal injection of CFTRinh-172 (just after cholera toxin infusion) reduced fluid accumulation by 90%, with an IC50 of 5 mg CFTRinh-172 (Figure 4c). Inhibition of fluid accumulation was seen when CFTRinh172 was administered three hours before or after cholera toxin (Figure 4d), which is probably a consequence of reduced fluid secretion with continued absorption. Orally administered CFTRinh-172 was also effective in blocking intestinal fluid secretion following oral cholera toxin in an open-loop model. Finally, CFTRinh-172 inhibited fluid secretion after E. coli STa toxin exposure, as well as cAMPand cGMP-stimulated Cl currents in human intestine.
Conclusions There is now good evidence for a central role of CFTRmediated Cl secretion in enterotoxin-mediated intestinal fluid secretion in infectious diarrheas, including cholera and traveler’s diarrhea. However, the relative importance of CFTR, CaCCs and other Cl channels in different forms of diarrhea, such as viral diarrhea and inflammatory bowel disease, remains unknown. CFTR is thus an attractive target for development of inhibitors with antidiarrheal efficacy in cholera and other disorders of intestinal fluid secretion. CFTR inhibitors might also be useful in creating cystic fibrosis animal models, and in pharmacologically creating the cystic fibrosis phenotype in excised human tissues.
Acknowledgements This work is supported by the Cystic Fibrosis Foundation, and NIH grants HL73856, HL59198, EB00415, EY13574 and DK35124. Dr Thiagarajah was supported by a Cystic Fibrosis Foundation fellowship.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Welsh MJ, Tsui LC, Boat TF, Beudet AL: Cystic Fibrosis. In The Metabolic and Molecular Basis of Inherited Disease. New York: McGraw-Hill Inc; 1995:3799-3876.
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Sheppard DN, Welsh MJ: Structure and function of the CFTR chloride channel. Physiol Rev 1999, 79:23-45.
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Barrett KE, Keely SJ: Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol 2000, 62:535-572.
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Farthing MJ: Diarrhoea: a significant worldwide problem. Int J Antimicrob Agents 2000, 14:65-69.
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Farthing MJ: History and rationale of oral rehydration and recent developments in formulating an optimal solution. Drugs 1988, 4:80-90.
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Schultz BD, Singh AK, Devor DC, Bridges RJ: Pharmacology of CFTR chloride channel activity. Physiol Rev 1999, 79:S109-S144. www.current-opinion.com
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7.
Illek B, Lizarzaburu ME, Lee V, Nantz MH, Kurth MJ, Fischer H: Structural determinants for activation and block of CFTRmediated chloride currents by apigenin. Am J Physiol 2000, 279:C1838-C1846.
8.
Dormer RL, Derand R, McNeilly CM, Mettey Y, Bulteau-Pignoux L, Metaye T, Vierfond JM, Gray MA, Galietta LJ, Morris MR et al.: Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells. J Cell Sci 2001, 114:4073-4081.
9.
Bulteau L, Derand R, Mettey Y, Metaye T, Morris MR, McNeilly CM, Folli C, Galietta LJ, Zegarra-Moran O, Pereira MM et al.: Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells. Am J Physiol 2000, 279:C1925-C1937.
10. Gribkoff VK, Champigny G, Barbry P, Dworetzky SI, Meanwell NA, Lazdunski M: The substituted benzimidazolone NS004 is an opener of the cystic fibrosis chloride channel. J Biol Chem 1994, 269:10983-10986. 11. Galietta LJ, Springsteel MR, Eda M, Niedzinski EJ, By K, Haddadin MJ, Kurth MJ, Nantz MH, Verkman AS: Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds. J Biol Chem 2001, 276:19723-19728. 12. Springsteel M, Galietta LJ, Ma T, By K, Berger TO, Yang H, Dicus CW, Choung W, Quan C, Shelat AA et al.: Benzoflavone activators of the cystic fibrosis transmembrane conductance regulator: a pharmacophore model of flavone-CFTR binding. Bioorgan Med Chem 2003, 11:4113-4120. 13. Ma T, Vetrivel L, Yang Y, Pedemonte N, Zegarra-Moran O, Galietta LJ, Verkman AS: High-affinity activators of CFTR chloride conductance identified by high-throughput screening. J Biol Chem 2002, 277:37235-37241. Describes CFTR activators with submicromolar potency and novel structures discovered by screening 60 000 random compounds. 14. Di Stefano A, Wittner M, Schlatter E, Lang HJ, Englert H, Greger R: Diphenylamine-2-carboxylate, a blocker of the ClS-conductive pathway in ClS transporting epithelia. Pflugers Arch 1985, 405:95-100. 15. Wangemann P, Wittner M, Di Stefano A, Englert HC, Lang HJ, Schlatter E, Greger R: ClS-channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship. Pflugers Arch 1986, 407:128-141. 16. Sheppard DN, Welsh MJ: Effect of ATP-sensitive KR channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents. J Gen Physiol 1992, 100:573-591. 17. Zhou Z, Hu S, Hwang TC: Probing an open CFTR pore with organic anion blockers. J Gen Physiol 2002, 120:647-662. Single-channel electrophysiological analysis of CFTR inhibition mechanisms. 18. Mathews CJ, MacLeod RJ, Zheng SX, Hanrahan JW, Bennett HP, Hamilton JR: Characterization of the inhibitory effect of boiled rice on intestinal chloride secretion in guinea-pig crypt cells. Gastroenterology 1999, 116:1342-1347. 19. Jayaraman S, Haggie P, Wachter R, Remington SJ, Verkman AS: Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem 2000, 275:6047-6050. 20. Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, Verkman AS: Thiazolidinone CFTR inhibitor identified by highthroughput screening blocks cholera-toxin induced intestinal fluid secretion. J Clin Invest 2002, 110:1651-1658. Identification and characterization of a CFTR inhibitor with submicromolar potency identified by random compound screening, and demonstration of its antidiarrheal efficacy. 21. Nagatani R, Sonawane N, Song Y, Verkman AS: Pharmacokinetics, tissue distribution and elimination
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mechanism of a thiazolidinone CFTR inhibitor in rodents. Pharm Res 2003, in press. 22. Buresi MC, Buret AG, Hollenberg MD, MacNaughton WK: Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway. FASEB J 2002, 16:1515-1525. Elucidates the mechanism by which thrombin, which is elevated in inflammatory bowel diseases, causes intestinal Cl secretion. 23. Uribe JM, McCole DF, Barrett KE: Interferon-gamma activates EGF receptor and increases TGF-alpha in T84 cells: implications for chloride secretion. Am J Physiol 2002, 283:G923-G931. 24. Oprins JC, van der Burg C, Meijer HP, Munnik T, Groot JA: Tumour necrosis factor alpha potentiates ion secretion induced by histamine in a human intestinal epithelial cell line and in mouse colon: involvement of the phospholipase D pathway. Gut 2002, 50:314-321. Shows that an important mediator of inflammatory bowel disease, tumor necrosis factor-a, potentiates histamine-stimulated Cl secretion. 25. Resta-Lenert S, Barrett KE: Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium: role of iNOS and COX-2. Gastroenterology 2002, 122:1070-1087. Shows that invasive diarrheal strains of bacteria induce Cl secretion through inflammatory mediators. 26. Kottgen M, Loffler T, Jacobi C, Nitschke R, Pavenstadt H, Schreiber R, Frische S, Nielsen S, Leipziger J: P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport. J Clin Invest 2003, 111:371-379. Elucidates the mechanism of intestinal CFTR-mediated Cl secretion by extracellular nucleotides. 27. Crane JK, Olson RA, Jones HM, Duffey ME: Release of ATP during host cell killing by enteropathogenic E. coli and its role as a secretory mediator. Am J Physiol 2002, 283:G74-G86. Provides evidence for the involvement of nucleotide release in Cl secretion induced by invasive bacteria. 28. Ko WH, Law VW, Yip WC, Yue GG, Lau CW, Chen ZY, Huang Y: Stimulation of chloride secretion by baicalein in isolated rat distal colon. Am J Physiol 2002, 282:G508-G518. 29. Duszyk M, MacVinish L, Cuthbert AW: Phenanthrolines — a new class of CFTR chloride channel openers. Br J Pharmacol 2001, 134:853-864. 30. Grubb BR, Gabriel SE: Intestinal physiology and pathology in gene-targeted mouse models of cystic fibrosis. Am J Physiol 1997, 273:G258-G266. 31. Gyomorey K, Rozmahel R, Bear CE: Amelioration of intestinal disease severity in cystic fibrosis mice is associated with improved chloride secretory capacity. Pediatr Res 2000, 48:731-734. 32. Gabriel SE, Brigman KN, Koller BH, Boucher RC, Stutts MJ: Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. Science 1994, 266:107-109. 33. de Zoysa I, Feachem R: Interventions for the control of diarrhoeal diseases among young children: rotavirus and cholera immunization. Bull World Health Organ 1985, 63:569-583. 34. Morris AP, Scott JK, Ball JM, Zeng CQ, O’Neal WK, Estes MK: NSP4 elicits age-dependent diarrhea and Ca2R-mediated IS influx into intestinal crypts of CF mice. Am J Physiol 1999, 277:G431-G444. 35. Kunzelmann K, Mall M: Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev 2002, 82:245-248. 36. O’Donnell EK, Sedlacek RL, Singh AK, Schultz BD: Inhibition of enterotoxin-induced porcine colonic secretion by diarylsulfonylureas in vitro. Am J Physiol 2000, 279:G1104-G1112.
Current Opinion in Pharmacology 2003, 3:594–599
CFTR pharmacology and its role in intestinal fluid secretion Jay R Thiagarajah and AS Verkman The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl channel expressed in epithelial cells in the airways, pancreas, intestine and other fluid-transporting tissues. Cystic fibrosis is caused by mutations in the CFTR, resulting in impaired Cl transport and plasma membrane targeting. CFTR is expressed in the lumenal membrane of enterocytes, where it functions as the principal pathway for secretion of Cl and fluid in enterotoxin-induced secretory diarrheas such as cholera. Small-molecule CFTR inhibitors reduce enterotoxin-induced intestinal fluid secretion in animal models. CFTR inhibition might also reduce intestinal fluid losses in cholera and possibly in other infectious and non-infectious diarrheas. Addresses Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521, USA Correspondence: Alan S Verkman; e-mail: [email protected]; http://www.ucsf.edu/verklab
Current Opinion in Pharmacology 2003, 3:594–599 This review comes from a themed issue on Gastrointestinal pharmacology Edited by David Grundy and Wendy Winchester 1471-4892/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2003.06.012
Abbreviations ABC ATP-binding cassette CaCC Ca2þ-activated Cl channel CFTR cystic fibrosis transmembrane conductance regulator NBD nucleotide-binding domain NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid SUR sulfonylurea receptor
Introduction The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was identified in 1989 as the genetic basis of the hereditary lethal disease cystic fibrosis. CFTR is a cAMP-activated Cl channel expressed in epithelial cells in the airways, sweat duct, testis, pancreas, intestine and other fluid-transporting tissues. In cystic fibrosis, defective epithelial cell fluid transport produces chronic lung infection and a slow deterioration in lung function, as well as pancreatic insufficiency, meconium ileus and male infertility [1]. CFTR is a large transmembrane glycoprotein containing two six-helix membrane-spanning domains, each followed by a nucleotide-binding domain (NBD), with a regulatory domain linking the first NBD and the second membrane-spanning domain (Figure 1a). CFTR activation involves ATP bindCurrent Opinion in Pharmacology 2003, 3:594–599
ing and hydrolysis at NBDs, and phosphorylation of multiple regulatory domain sites (reviewed in [2]), although many details of CFTR regulation and gating remain unknown. CFTR has homology with members of the ATP-binding cassette (ABC) family of membrane proteins (including the multi-drug resistance protein-1 and sulfonylurea receptor [SUR]) in their membrane-spanning domains and NBDs; however, unlike other ABC proteins, CFTR contains a regulatory domain. CFTR is expressed at the apical membrane of enterocytes in intestine, and is thought to be the primary pathway for Cl and hence fluid secretion into the intestinal lumen in cAMP-mediated diarrheas such as cholera [3]. Secretory diarrheas caused by Vibrio cholera and enterotoxogenic Escherichia coli, in which intestinal fluid hypersecretion leads to a massive loss of fluid and electrolytes, are a major cause of morbidity and mortality worldwide [4]. An antisecretory drug that acts directly on Cl secretion might complement oral fluid replacement, which has been the mainstay of diarrheal therapy [5]. This review is focused on small-molecule activators and inhibitors of CFTR function, and on the role that CFTR plays in intestinal fluid secretion.
CFTR activators CFTR activation can be accomplished in many ways, the most important being elevation of cytosolic cAMP (promoting CFTR phosphorylation), inhibition of phosphatase activity (blocking CFTR dephosphorylation) and direct interaction [6]. Small molecules that activate CFTR by direct interaction include flavones/isoflavones (e.g. genistein [7]), benzo[c]quinoliziniums [8], xanthines [9] and benzimidazolones [10]. Figure 1b shows the structures of IBMX, a xanthine that activates CFTR both by direct and indirect (phosphodiesterase inhibition) mechanisms; NS004, a benzimidazolone that was originally characterized as a Kþ channel opener; and UCCF029, a benzoflavone that was discovered from the screening of a flavone library [11,12]. In a recent systematic search for small-molecule CFTR activators, 60 000 compounds were screened using a cell-based fluorescence assay [13]. More than a dozen compounds with novel structures were identified with submicromolar potency for activation of human wild-type CFTR. The compounds activated CFTR without elevating cAMP or inhibiting phosphatase activity, suggesting direct CFTR binding. The structures of two such compounds (CFTRact-09 and -12), which also correct the DF508–CFTR gating defect, are shown in Figure 1b. Activation of wild-type CFTR by drug-like small molecules is relatively easy compared with activation of cystic fibrosis-causing CFTR www.current-opinion.com
CFTR pharmacology and its role in intestinal fluid secretion Thiagarajah and Verkman 595
Figure 1
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(b) CFTR activators
Cell surface IBMX
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R
Cytoplasm
NS004
UCCF-029
CFTRact-09
CFTRact-12
CFTR inhibitors
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Current Opinion in Pharmacology
CFTR activators and inhibitors. (a) CFTR structure showing membrane-spanning domains (shaded), NBD1 and NBD2, and the regulatory domain (R). (b) Chemical structures of selected CFTR activators and inhibitors.
mutants; however, the latter subject is beyond the scope of this review.
CFTR inhibitors Two major classes of CFTR inhibitors are the arylaminobenzoates and sulphonylureas. The arylaminobenzoates, developed originally as blockers of kidney tubule Cl conductance [14], include diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)-benzoic acid
(NPPB) (Figure 1b) [15]. These compounds are nonspecific Cl channel blockers; patch-clamp and mutagenesis studies suggest voltage-dependent block of CFTR, resulting in CFTR pore occlusion. The sulfonylureas, originally used as bacteriostatic agents, were optimized for modulation of insulin release as oral hypoglycemic agents in diabetes. Glibenclamide (Figure 1b) binds to the ABC protein SUR, where it modulates Kir6.2 Kþ channel activity and insulin release from pancreatic islet
Figure 2
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(b)
Cl– YFP 10 min YFP
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Identification of CFTR inhibitors by high-throughput screening. (a) Screening approach: CFTR is stimulated by multiple agonists in stably transfected epithelial cells co-expressing human CFTR and a yellow fluorescent protein (YFP) having Cl/I-sensitive fluorescence. After addition of test compound, I influx is induced by adding an I-containing solution. (b) Representative original fluorescence data from individual wells of a 96-well plate showing controls (no activators, no test compound) and test wells. (c) Top panel: CFTRinh-172 inhibition of short-circuit current in permeabilized Fisher rat thyroid cells expressing human CFTR after stimulation by 100 mM CPT-cAMP. Bottom panel: dose-inhibition data for CFTRinh-172 and glibenclamide. www.current-opinion.com
Current Opinion in Pharmacology 2003, 3:594–599
596 Gastrointestinal
cells. At much higher concentrations, glibenclamide was found to inhibit CFTR [16]. Glibenclamide and the related sulfonylurea tolbutamide are likely to block CFTR function by binding to the CFTR pore in its open state [17]. Although arylaminobenzoates and sulphonylureas inhibit CFTR Cl channel function, high concentrations are generally required (> 0.1 mM) where multiple ion channels and transporters are affected. Disulfonic stilbene inhibitors of anion transport also inhibit CFTR but at very high concentrations (generally > 1 mM). A component of boiled rice also appears to inhibit CFTR Cl secretion in intestinal cells [18], although its identity remains unknown. Recently, a collection of 50 000 drug-like molecules was screened for CFTR inhibition. Fisher rat thyroid cells coexpressing human CFTR and a green fluorescent protein-
based halide sensor [19] were stimulated by a CFTRactivating cocktail and then subjected to an iodide gradient (Figure 2a). Iodide addition produced a prompt decrease in cell fluorescence after CFTR activation (Figure 2b). Inhibitors (‘active compounds’) were identified from a reduction in the fluorescence slope. The best inhibitor identified by screening and subsequent optimization was the 2-thioxo-4-thiazolidinone compound CFTRinh-172 (Figure 1b) [20]. CFTRinh-172 inhibited CFTR function at submicromolar concentrations — approximately 500 times better than glibenclamide when studied under similar conditions (Figure 2c). Further analysis showed voltage-independent inhibition of CFTR Cl conductance with prolonged mean channel-closed time but without change in unitary conductance. CFTRinh-172 did not affect other Cl channels (calcium- and volumeactivated) or ABC transporters (e.g. multi-drug resistance
Figure 3
Intestinal lumen
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–
Guanylin receptor
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? ClC Cl– other Cl– channels
cGMP
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–
5-HT 5-HT
VIP
Enkephalin
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Inflammatory mediators
K+ channels ACh
SP
Mu receptor Blood
Enteric neurons –
Entero-invasive bacteria Inflammatory bowel disease
Loperamide Diphenoxylate Current Opinion in Pharmacology
Intestinal secretory pathways. Cl secretion involves Cl uptake at the basolateral membrane of enterocytes by the NaþKþ2Cl cotransporter (NKCC) and its exit primarily via apical membrane CFTR. Other Cl channels (such as CaCCs and ClCs) might also mediate Cl secretion. Naþ and water follow by a paracellular route. Secretion initiated by pathogens (cholera toxin, STa toxin and rotavirus) involves multiple pathways involving the release of 5-hydroxytryptamine (5-HT) from enterochromaffin cells, neuronal signaling, vasoactive intestinal peptide (VIP), acetylcholine (ACh), substance P (SP) and the release of inflammatory mediators from mast cells and neutrophils (e.g. prostaglandins and interleukins). Signals are transduced by second messengers (cAMP, cGMP, Ca2þ) to activate membrane ion channels. Secretion can be blocked by CFTR inhibitors, inhibition of neuronal signaling, 5-hydroxytryptamine receptor antagonists and anti-enkephalinases. Current Opinion in Pharmacology 2003, 3:594–599
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CFTR pharmacology and its role in intestinal fluid secretion Thiagarajah and Verkman 597
secretion is driven by active Cl transport from the basolateral to the apical side of enterocytes (Figure 3). Cl is taken up at the basolateral membrane via NaK2Cl cotransporter, which is driven by Naþ and Cl concentration gradients produced by the NaþKþ-ATPase and basolateral Kþ channels. Cl is electrochemically driven across the cell apical membrane primarily through the CFTR, as well as through Ca2þ-activated Cl channels (CaCCs) and other Cl channels. Both Naþ and fluid follow Cl paracellularly. As shown schematically in Figure 3, Cl and hence fluid secretion can be activated by different mechanisms. For example, secretory neuronal pathways
protein-1, SUR). Rodent pharmacology studies indicated that CFTRinh-172 has low toxicity, a large volume of distribution with slow elimination by renal glomerular filtration, little metabolism, and enterohepatic circulation with accumulation in bile and intestine [21].
Role of CFTR in intestinal secretion Fluid secretion plays a key role in intestinal physiology. Under normal conditions, the intestine carries out the absorption of luminal fluid, electrolytes and nutrients. A small amount of basal secretion facilitates hydration of the intestinal mucosa and mixing of intestinal contents. Fluid Figure 4
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Antidiarrheal properties of a CFTR inhibitor. (a) Intestinal fluid absorption. Closed mouse ileal loops were injected with 200 ml saline and loop weight measured at indicated times. Inset: shows % absorption at 30 minutes with and without CFTRinh-172. (b) Intestinal fluid secretion. Time-course of cholera toxin-induced fluid secretion. Loops were injected with 1 mg cholera toxin. Dashed line shows control (saline-injected) loops. (c) Dose-response for inhibition of fluid accumulation. Mice were given single doses of CFTRinh-172 by intraperitoneal injection and loop weight was measured at six hours. Inset: intestinal loops six hours after cholera toxin or saline injection. Where indicated, the mouse was treated with CFTRinh-172. (d) Persistence of CFTRinh-172 inhibition. Mice were injected with 20 mg CFTRinh-172 at indicated times before or after cholera toxin administration. www.current-opinion.com
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involve the release of 5-hydroxytryptamine from enterochromaffin cells, resulting in activation of cholinergic and vasoactive intestinal peptide neurons and increased cAMP and Ca2þ levels, causing subsequent Cl channel activation. Inflammatory mediators, such as prostaglandins and interleukins, also play an important role in initiating Cl secretion, and recent studies have begun to elucidate their signaling pathways [22,23,24,25]; nucleotides and purinergic signaling also stimulate Cl secretion through Ca2þ and cAMP signaling pathways [26,27]. Evidence suggests a pivotal role for CFTR in intestinal Cl and fluid secretion. Numerous in vitro studies have shown that agonist-induced Cl secretion in intestinal sheets and cell-lines is blocked by glibenclamide and NPPB [28,29]. CFTR knockout mice develop intestinal obstruction because of defective intestinal Cl and fluid secretion and increased fluid absorption [30]. Increased expression of alternative Cl channels is found in CFTR null mice that develop mild intestinal symptoms [31]. Cholera toxin, which causes massive fluid secretion in the small intestine in normal mice, does not induce fluid secretion in CFTR knockout mice [32].
CFTR inhibitors as anti-diarrheals Secretory diarrhea is caused by intestinal infection by various bacterial and viral pathogens. Bacterial enterotoxins, such as cholera toxin from V. cholerae and STa toxin from E. coli, induce increases in the second messengers cAMP and cGMP, respectively, resulting in activation of CFTR Cl conductance (Figure 3). Other pathogens, such as entero-invasive bacteria, also stimulate Cl secretion, and recent studies on Salmonella and enteropathogenic E. coli provide evidence for the involvement of inflammatory mediators and nucleotides in secretion [25,27]. An important cause of secretory diarrhea is the rotavirus viral pathogen, which is responsible for 20% of all diarrhea-related deaths in children under the age of five [33]. Secretion in rotaviral diarrhea was thought to involve inhibition of solute and fluid absorption. However, the extensive fluid loss during symptomatic infection suggests the involvement of active secretory mechanisms. CaCCs, which provide an alternative apical exit pathway for Cl (Figure 3), have been postulated to play a role in some viral diarrheas [34]. Another diarrheal disorder, AIDS-related diarrhea, is thought to have a multi-factorial etiology, including opportunistic infection by pathogens such as enterotoxigenic bacteria (e.g. E. coli), parasites (e.g. cryptosporidium) and viruses (e.g. cytomegalovirus). CFTR inhibition has been proposed as a therapy for some forms of secretory diarrhea [35]. Indeed, previous in vitro studies showed that a diarylsulfonylurea CFTR inhibitor prevented Cl secretion in porcine mucosa [36]. The antidiarrheal efficacy of the thiazolidinone CFTRinh172 has recently been tested [20]. In a mouse closedCurrent Opinion in Pharmacology 2003, 3:594–599
ileal loop model, injected fluid was rapidly cleared and remained unaffected by CFTRinh-172 administration (Figure 4a), an important prerequisite for antidiarrheal application of a CFTR inhibitor. Injection of cholera toxin into loops produced fluid secretion over six hours after a slow onset (Figure 4b). In a dose-response study, a single intraperitoneal injection of CFTRinh-172 (just after cholera toxin infusion) reduced fluid accumulation by 90%, with an IC50 of 5 mg CFTRinh-172 (Figure 4c). Inhibition of fluid accumulation was seen when CFTRinh172 was administered three hours before or after cholera toxin (Figure 4d), which is probably a consequence of reduced fluid secretion with continued absorption. Orally administered CFTRinh-172 was also effective in blocking intestinal fluid secretion following oral cholera toxin in an open-loop model. Finally, CFTRinh-172 inhibited fluid secretion after E. coli STa toxin exposure, as well as cAMPand cGMP-stimulated Cl currents in human intestine.
Conclusions There is now good evidence for a central role of CFTRmediated Cl secretion in enterotoxin-mediated intestinal fluid secretion in infectious diarrheas, including cholera and traveler’s diarrhea. However, the relative importance of CFTR, CaCCs and other Cl channels in different forms of diarrhea, such as viral diarrhea and inflammatory bowel disease, remains unknown. CFTR is thus an attractive target for development of inhibitors with antidiarrheal efficacy in cholera and other disorders of intestinal fluid secretion. CFTR inhibitors might also be useful in creating cystic fibrosis animal models, and in pharmacologically creating the cystic fibrosis phenotype in excised human tissues.
Acknowledgements This work is supported by the Cystic Fibrosis Foundation, and NIH grants HL73856, HL59198, EB00415, EY13574 and DK35124. Dr Thiagarajah was supported by a Cystic Fibrosis Foundation fellowship.
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