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Mechanism, Structure–Activity Analysis, and In Vivo Efficacy
2 Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, 16148 Genova, Italy
Address correspondence to Alan S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143-0521. Fax: (415) 665-3847; email: verkman{at}itsa.ucsf.edu
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ABSTRACT |
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1 mM in water. Topical GlyH-101 (10 µM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 µg) reduced by 80% cholera toxin–induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTRinh-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.
Key Words: cystic fibrosis • diarrhea • high-throughput screening • patch-clamp • drug discovery
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INTRODUCTION |
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). Mutations in CFTR that reduce or inhibit its function cause the genetic disease cystic fibrosis (CF), which is characterized by chronic lung infection and pancreatic insufficiency, with progressive deterioration in lung function and death. There is considerable interest in the development and characterization of CFTR inhibitors as probes of the CFTR Cl– transport mechanism, and as reagents to reduce CFTR function in vivo for creation of animal models of CF and as clinical antidiarrheals in CFTR-dependent secretory diarrheas caused by Vibrio cholera and Escherichia coli (Thiagarajah and Verkman, 2003). CFTR inhibitors also have potential applications in polycystic kidney disease, as male contraceptives, and in reducing airway glandular secretions in infectious and allergic bronchitis/rhinitis (Gong et al., 2001; Nakanishi et al., 2001; Thiagarajah et al., 2004b).
Several CFTR inhibitors have been introduced, though most having weak potency and lacking CFTR specificity. The oral hypoglycemic agent glibenclamide inhibits CFTR Cl– conductance from the intracellular side by an open channel blocking mechanism (Sheppard and Robinson, 1997; Zhou et al., 2002) at high micromolar concentrations where it affects other Cl– and cation channels (Sturgess et al., 1988; Rabe et al., 1995; Schultz et al., 1999). Other nonselective anion transport inhibitors, including diphenylamine-2-carboxylate (DPC), 5-nitro-2(3-phenylpropyl-amino)benzoate (NPPB), and flufenamic acid, also inhibit CFTR at high concentrations by occluding the pore at an intracellular site (Dawson et al., 1999; McCarty, 2000).
Our laboratory developed a high-throughput screening assay for discovery of CFTR activators and inhibitors (Galietta et al., 2001). CFTR halide transport function is quantified from the time course of fluorescence in response to an iodide gradient in cells coexpressing a green fluorescent protein–based halide sensor (Jayaraman et al., 2000; Galietta et al., 2001) and wild-type CFTR or a CF-causing CFTR mutant. The assay was used to identify small-molecule activators of wild type and 
F508-CFTR with activating potencies down to 100 nM (Ma et al., 2002b; Yang et al., 2003). A thiazolidinone class of CFTR inhibitors was identified by screening of a collection of 50,000 small, drug-like molecules (Ma et al., 2002a). The lead compound CFTRinh-172 inhibited CFTR Cl– conductance in a voltage-independent manner, probably by binding to the NBD1 domain at the cytoplasmic surface of CFTR (Ma et al., 2002a; Taddei et al., 2004). In intact cells, CFTR Cl– channel function was 50% inhibited at CFTRinh-172 concentrations of 0.3–3 µM depending on cell type and membrane potential. CFTRinh-172 inhibited intestinal fluid secretion in response to cholera toxin and heat-stable (STa) E. coli toxin in rodents (Thiagarajah et al., 2004a), and resulted in the secretion of viscous, CF-like fluid from submucosal glands in pig and human trachea (Thiagarajah et al., 2004b).
Although thiazolidinones are potentially useful as antidiarrheals and for the creation of CF animal models, they have limited water solubility (20 µM) and inhibit CFTR by binding to its cytoplasmic-facing surface, requiring cell penetration with consequent systemic absorption when administered orally. The purpose of this work was to identify CFTR inhibitors with high water solubility that occlude the CFTR pore by binding to a site at its external surface. A low stringency, high-throughput screen of 100,000 small molecules was performed to identify novel chemical scaffolds with CFTR inhibitory activity. We identified several new classes of CFTR inhibitors, one of which was highly water soluble, blocked CFTR by occlusion of the CFTR pore near its external surface, and inhibited CFTR function in vivo in rodent models.
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MATERIALS AND METHODS |
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). For screening, cells in 96-well plates were washed three times, and then CFTR halide conductance was activated by incubation for 15 min with an activating cocktail containing 10 µM forskolin, 20 µM apigenin, and 100 µM IBMX. Test compounds (25 µM final) were added 5 min before assay of iodide influx in which cells were exposed to a 100 mM inwardly directed iodide gradient. YFP fluorescence was recorded for 2 s before and 12 s after creation of the iodide gradient. Initial rates of iodide influx were computed from the time course of decreasing fluorescence after the iodide gradient (Yang et al., 2003).
Apical Cl– Current and Short-circuit Current Measurements
FRT, T84, and human airway epithelial cells were cultured on Snapwell filters with 1 cm2 surface area (Corning-Costar) to resistances >1,000 
·cm2 as described previously (Ma et al., 2002b). Filters were mounted in an Easymount Chamber System (Physiologic Instruments). For apical Cl– current measurements on FRT cells, the basolateral hemichamber was filled with buffer containing (in mM) 130 NaCl, 2.7 KCl, 1.5 KH2PO4, 1 CaCl2, 0.5 MgCl2, 10 Na-HEPES, 10 glucose (pH 7.3). The basolateral membrane was permeabilized with amphotericin B (250 µg/ml) for 30 min before measurements. In the apical solution, 65 mM NaCl was replaced by sodium gluconate, and CaCl2 was increased to 2 mM. For short-circuit current measurements in (nonpermeabilized) T84 and human airway cells, both hemichambers contained Kreb's solution (in mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose (pH 7.3). Solutions were bubbled with 95% O2/5% CO2 and maintained at 37°C. For studies in mouse intestine, ileal segments were isolated, washed with ice-cold Kreb's buffer, opened longitudinally through the mesenteric border, and mounted in a micro-Ussing chamber (0.7 cm2 aperture area; World Precision Instruments). Hemichambers were filled with Kreb's solutions containing 10 µM indomethacin. Apical Cl–/short-circuit current was recorded using a DVC-1000 voltage-clamp (World Precision Instruments) with Ag/AgCl electrodes and 1 M KCl agar bridges.
Patch-clamp Analysis
Patch-clamp experiments were performed at room temperature on FRT cells stably expressing wild-type CFTR. Cell-attached and whole-cell configurations were used (Hamill et al., 1981). The cell membrane was clamped at specified voltages using an EPC-7 patch-clamp amplifier (List Medical). Data were filtered at 500 Hz and digitized at 2000 Hz. For whole-cell experiments, the pipette solution contained (in mM): 120 CsCl, 10 TEA-Cl, 0.5 EGTA, 1 MgCl2, 40 mannitol, 10 Cs-HEPES, and 3 mM MgATP (pH 7.3). For cell-attached experiments, EGTA was replaced with 1 mM CaCl2. The bath solution for whole-cell experiments contained (in mM): 150 NaCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 mannitol, 10 Na-TES (pH 7.4). In some experiments bath solution NaCl was reduced to 20 mM (mannitol added to maintain osmolality). In cell-attached experiments, the bath solution contained (in mM): 130 KCl, 2 NaCl, 2 CaCl2, 2 MgCl2, 10 glucose, 20 mannitol, and 10 K-Hepes (pH 7.3). Inhibitors were applied by extracellular perfusion. CFTR Cl– channel activity in cell-attached patches was analyzed as described previously (Taddei et al., 2004). The number of CFTR channels present in each patch was estimated as the maximum number of simultaneous channel openings detected in the presence of 5 µM forskolin in a continuous recording of at least three minutes.
Nasal Potential Difference Measurements in Mice
After anesthesia with intraperitoneal ketamine (90–120 mg/kg) and xylazine (5–10 mg/kg), the airway was protected by orotracheal intubation with a 21-gauge angiocatheter as previously described (Salinas et al., 2004). A PE-10 cannula pulled to a tip diameter of 0.3 mm was inserted into one nostril 5 mm distal to the anterior nares and connected though a 1 M KCl agar bridge to a Ag/AgCl electrode and high-impedance digital voltmeter (IsoMillivolt Meter; World Precision Instruments). The nasal cannula was perfused at 50 µL/min using dual microperfusion pumps serially with PBS, low chloride PBS (chloride lowered to 4.7 mM by substitution with gluconate), low chloride PBS containing forskolin (10 µM) without and then with GlyH-101 (10 µM), and then PBS. In some studies GlyH-101 (10 µM) or DIDS (100 µM) was present in all solutions. The reference electrode was a PBS-filled 21-gauge needle inserted in the subcutaneous tissue in the abdomen and connected to a second Ag/AgCl electrode by a 1 M KCl agar bridge.
Intestinal Fluid Secretion Measurements
Mice (CD1 strain, 25–35 g) were deprived of food for 24 h and anaesthetized with intraperitoneal ketamine (40 mg/kg) and xylazine (8 mg/kg). Body temperature was maintained at 36–38°C using a heating pad. Following a small abdominal incision three closed ileal loops (length 20–30 mm) proximal to the cecum were isolated by sutures. Loops were injected with 100 µl of PBS or PBS containing cholera toxin (1 µg) without or with GlyH-101 (2.5 µg). The abdominal incision was closed with suture and mice were allowed to recover from anesthesia. At 4 h the mice were anaesthetized, intestinal loops were removed, and loop length and weight were measured to quantify net fluid secretion. Mice were killed by an overdose of ketamine and xylazine. All protocols were approved by the UCSF Committee on Animal Research.
Synthesis Procedures
Procedures were developed for synthesis of glycine hydrazide analogues (see Table I and Fig. 3 B). All synthesized compounds had >98% purity (TLC/HPLC) and were confirmed by mass and 1H nmr spectrometry (typical data for GlyH-101 given below).
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). A solution of above product (2.29 g, 10 mmol) in ethanol (10 ml) was refluxed with hydrazine hydrate (0.6 g, 12 mmol) for 10 h. Solvent and excess reagent were distilled under vacuum. The product was recrystallized from ethanol to yield 1.8 g of N-(2-naphthalenyl)glycine hydrazide (compound III) (82%, mp 147–148°C). A mixture of compound III (2.15 g, 10 mmol) and 3,5-dibromo-2,4-dihydroxybenzaldehyde (3 g, 10 mmol) in ethanol (5 ml) was refluxed for 3 h. The hydrazone that crystallized upon cooling was filtered, washed with ethanol, and recrystallized from ethanol to give 3.8 g (78%) of GlyH-101. Melting point (mp) >300°C, ms (ES–): M/Z 492 (M-H)–1; 1H nmr (DMSO-d6): 
4.1(s, 2H, CH2), 6.5–7.5 (m, 9H, aromatic, NH), 8.5 (s, 1H, CH=N), 10.4 (s, 1H, NH-CO), 11.9 (s, 1H, OH), 12.7(s, 1H, OH). Compounds GlyH-102–109, GlyH-114–127, and AceH-401–404 were synthesized similarly by condensing appropriate hydrazides with substituted benzaldehydes or acetophenones.
N-(6-quinolinyl)-[(3,5-dibromo-2,4-dihydroxyphenyl) methylene] Glycine Hydrazide (GlyH-126) and Related Quinolinyl-Glycine Hydrazides
To a stirred solution of 6-aminoquinoline (compound IV) (0.72 g, 5 mmol) in acetonitrile (20 ml) was added 33% aqueous glyoxylic acid (1.85 g, 20 mmol) solution. A solution of NaBH3CN (0.64 g, 10.2 mmol) in acetonitrile (20 ml) was then added at 3°C over 20 min and the reaction mixture was warmed to room temperature and stirred for 48 h. Acetonitrile was evaporated under vacuum, water (20 ml) was added to the residue, the solution was alkalinized to pH 9.5, and unreacted amine was extracted with ether. Concentrated HCl (25 ml) was added to the aqueous solution and the mixture was stirred at 25°C for 1 h. Solvent was evaporated under vacuum. The resultant residue of N-(6-quinolinyl)glycine was dissolved in dry ethanol (50 ml) saturated with dry HCl, stirred overnight and then refluxed for 3 h. Ethanol was evaporated, the ester hydrochloride was suspended in dry ether, and ammonia gas was bubbled. The ammonium chloride was filtered and ether was removed by evaporation to give ethyl N-(6-quinolinyl)glycinate (0.5 g, 87%, mp 122–123°C, Ramamurthy and Bhatt, 1989). N-(6-quinolinyl)glycine hydrazide (compound VI), synthesized by hydrazinolysis of the above ester, was reacted with 3,5-dibromo-2,4-dihydroxybenzaldehyde to give GlyH-126. Similar procedures were used for synthesis of GlyH-127.
Oxamic Hydrazides (OxaH-110–113)
The oxamic hydrazides were synthesized by heating a mixture of 2-naphthaleneamine with diethyl oxalate in toluene. The resultant N-substituted oxamic acid ethyl ester was treated with hydrazine hydrate followed by condensation with substituted benzaldehydes or acetophenones to yield compounds OxaH-110–113.
3,5-Dibromo-4-hydroxy-[2-(2-naphthalenamine)aceto] Benzoic Acid Hydrazide (GlyH-202) and Related GlyH-201 and Oxa-203–204
N-(2-naphthalenyl)glycine hydrazide (compound III, 2.15 g, 10 mmol) was reacted with 3,5-dibromo-4-hydroxybenzoyl chloride (3.14 g, 10 mmol) (Gilbert et al., 1982) in pyridine (10 ml) for 5 h. Pyridine was removed and the residue was diluted with water. The product was recrystallized from ethanol to yield a gray powder 3.8 g (77%), mp > 300°C. Compounds GlyH-201 and Oxa-203–204 were synthesized by similar procedure.
N-2-naphthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methyl] Glycine Hydrazide (GlyH-301) and Related Glycine Hydrazides (GlyH-302, OxaH-303–304)
A mixture of GlyH-101 (1.5 g, 3 mmol), hydrazine hydrate (0.15 ml, 3 mmol), and Pd/C catalyst (0.1 g, 10% Pd) in 5 ml of dimethylformamide was refluxed for 6–8 h (Verma et al., 1984). The reaction mixture was filtered, diluted with cold water, and extracted with diethyl ether. GlyH-301 was crystallized from ether to yield 0.9 g (60%), mp 258–260°C. Compounds GlyH-302 and OxaH-303–304 were prepared similarly.
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7, 5, 5, and 5 µM for compounds a–d, respectively. Fig. 1 C shows representative fluorescence (left) and apical Cl– current (right) data for compound d. We next screened 100–250 commercially available analogues of each compound class to determine whether active structural analogues exist, an important prerequisite for follow-up compound optimization by synthesis of targeted analogues. Whereas few or no active analogues of compounds a, b and c were found, initial screening of 285 analogues of compound d (substituted glycine hydrazides, GlyH) revealed 34 analogues that inhibited CFTR-mediated iodide influx by >25% at 25 µM.
Prior to extensive structure–activity analysis of synthesized GlyH analogues and characterization of inhibition mechanism, we determined the time course of GlyH-101 action and reversibility, and whether inhibition was effective for different CFTR-activating mechanisms. Fig. 2 A shows prompt inhibition of iodide influx in the fluorescence and apical Cl– current (inset) assays upon GlyH-101 addition. Interestingly 50% of the inhibition occurred within the 1-s addition/mixing time, with further inhibition over 1 min. Fig. 2 B indicates complete reversal of inhibition after GlyH-101 washout with >75% reversal over 5 min. Fig. 2 C shows effective CFTR inhibition by GlyH-101 after activation by different types of agonists, including potent direct activators of CFTR that do not elevate cytosolic cAMP or inhibit phosphatase activity (CFTRact-02, 08, and 10; Ma et al., 2002b).
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Chemistry and Structure–Activity Relationships of Glycine Hydrazides
The GlyH-101 structure was modified systematically to establish structure–activity relationships and to identify analogues with improved CFTR inhibitory activity. Fig. 3 A shows the various classes of structural analogues that were synthesized and tested for CFTR inhibition. Structural modifications were done on both ends of the glycine hydrazide backbone (Fig. 3 A, left, top, and middle). Replacing the glycine methylene group by a carbonyl group and replacing nitrogen by oxygen generated oxamic acid hydrazides (OxaH, right, top) and acetic acid hydrazides (AceH, right, middle), respectively. The hydrazone group modification produced two important series of compounds (middle, bottom and right, bottom). Also shown are compounds containing an additional methyl group at the hydrazone bond (top, middle), and containing a 6-quinolinyl group replacing the naphthalenyl group (left, bottom).
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), which was further esterified and reacted with hydrazine hydrate and benzaldehyde. The oxamic acid hydrazides were synthesized starting from aromatic amines and diethyl oxalate. Modifications were made initially on the N-aryl (R1) and benzaldehyde (R3) positions (see Table I and Fig. 4 for definition of Ri and CFTR inhibition). Good CFTR inhibition was found when R3 contained 3,5-dibromo and at least one hydroxyl substituent at the 4-position (GlyH-102, 105, 114); addition of a second hydroxyl group increased inhibition (GlyH-101, 104, 115–116). Inhibition was greatly reduced when R3 contained 4-bromophenyl or 4-carboxyphenyl substituents (GlyH-120–121). In addition, the 4-hydroxyl group in GlyH-101 was important for inhibition since its 4-methoxy analogue, GlyH-103, had little activity. Similar structure–activity results were found for GlyH-115 and GlyH-122.
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R2 was next modified (replacing methylene), keeping 2-naphthalenyl as R1 and dibromo-dihydroxyphenyl as R3. Introduction of a carbonyl group in GlyH-101 and GlyH-102 at R2, giving OxaH-110 and OxaH-111, gave two to threefold greater inhibitory potency. Replacement of CH2 by CHCH3 (GlyH-106–107) had minimal effect on CFTR inhibition. In another structural variation, addition of a methyl group at R4 to GlyH-102, yielding GlyH-109, gave improved CFTR inhibition. Modification of the N=C group in GlyH-101 and GlyH-102 to NH-CH2 in GlyH-301 and GlyH-302, or to NH-CO in GlyH-201 and GlyH-202, reduced CFTR inhibitory potency.
Fig. 3 C shows apical Cl– current analysis of CFTR inhibition in FRT cells for the most active analogue OxaH-110. Dose–response data for some of the most potent CFTR inhibitors gave Ki values (in µM, ±SEM, n = 3) of 2.7 ± 0.3 (OxaH-110), 4.3 ± 0.9 (GlyH-101), 4.7 ± 0.9 (GlyH-102), 4.7 ± 0.3 (GlyH-109), and 6.7 ± 0.9 (GlyH-115).
Patch-clamp Analysis of CFTR Inhibition Mechanism
The mechanism of CFTR block by GlyH-101 was studied using the whole-cell configuration of the patch-clamp technique. After maximal activation of CFTR in stably transfected FRT cells by 5 µM forskolin, current–voltage relationships were measured at GlyH-101 concentrations from 0 to 50 µM. Representative original current recordings are shown in Fig. 5 A. In the absence of inhibitor (left), membrane current increased linearly with voltage and did not show relaxation phenomena, as expected for pure CFTR Cl– currents. Extracellular perfusion with 10 µM GlyH-101 produced a reduction in current that was strongly dependent on membrane potential (Fig. 5 A, right). At more positive membrane potentials, outward positive currents (Cl– movement into the cell) were reduced compared with inward currents. Fig. 5 B shows current–voltage relationships for GlyH-101 concentrations of 0 (control), 10, and 30 µM, and after washout of 10 µM GlyH-101 (recovery). Data for the thiazolidinone CFTRinh-172 (5 µM) is shown for comparison. The current–voltage relationship was linear in the absence of inhibitor, after GlyH-101 washout, and after inhibition by CFTRinh-172, whereas GlyH-101 inhibition at submaximal concentrations produced inward rectification. Fig. 5 C summarizes percentage CFTR current block as a function of GlyH-101 concentration at different membrane voltages. GlyH-101 inhibitory potency was reduced at more negative voltages, with apparent Ki of 1.4, 3.8, 5.0, and 5.6 µM for voltages of +60, +20, –20, and –60 mV, respectively (Hill coefficients, nH = 0.5, 0.7, 1.3, 1.8). In additional studies to investigate whether the GlyH-101 binds to a site in the CFTR pore, Cl– flux in the CFTR pore was altered by reducing extracellular Cl– concentration to 20 mM. The potency of GlyH-101 inhibition of CFTR Cl– current was significant reduced at the low extracellular Cl– (Fig. 5 D).
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5.5 for titration of the first phenolic hydroxyl. Removal of one ortho hydroxyl (GlyH-102) eliminated the descending portion of the curve, confirming the pKa of 5.5 for the first para hydroxyl and 8.5 for the second ortho hydroxyl. Removal of the aromatic ring containing the resorcinolic hydroxyls (ethyl N-[2-naphthalenyl] glycinate; Fig. 7 A, bottom) indicated a pKa 4.7 for the residual secondary amine. From these data, the deduced equilibria among the ionic forms of GlyH-101 is shown in Fig. 7 B. GlyH-101 exists primarily as a singly charged anion at pH between 6 and 8.
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PD; Fig. 8 B) indicated 4 mV hyperpolarization after forskolin with depolarization of similar magnitude after GlyH-101; for comparison, data are shown for CFTRinh-172 from a previous study (Salinas et al., 2004). In a separate series of experiments, nasal PDs were measured as in A except that all solutions contained DIDS or GlyH-101. Fig. 8 C shows partial inhibition by DIDS of the (CFTR-independent) hyperpolarization produced by low Cl– (left), and substantial inhibition by GlyH-101 of the forskolin-induced hyperpolarization (right). Together these results indicate rapid inhibition of upper airway CFTR Cl– conductance by topical GlyH-101.
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2–5 µM for inhibition of cAMP-stimulated short-circuit current by GlyH-101 in T84 cells (A), primary human bronchial cell cultures (B), and intact mouse ileum (C). Inhibition was 100% at higher GlyH-101 concentrations. Cholera toxin–induced intestinal fluid secretion was measured in an in vivo closed-loop model in which loops for each mouse were injected with saline (control), cholera toxin (1 µg), or cholera toxin (1 µg) + GlyH-101 (2.5 µg). GlyH-101 was added to the lumen (rather than systemically) based on initial studies showing poor intestinal absorption and little effect of systemically administered compound. Referenced to the saline control, the cholera toxin–induced increase in fluid secretion over 4 h, quantified from loop weight-to-length ratio, was 80% reduced by GlyH-101 (Fig. 9 D). To rule out the possibility that GlyH-101 inhibited intestinal fluid secretion by blocking the binding of cholera toxin to its cell receptor, T84 cell fluorescence was measured after 1 h incubation with FITC-labeled cholera toxin B subunit (50 µg/ml) at 37°C. GlyH-101 at 50 µM did not inhibit cholera toxin binding/uptake (not depicted).
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DISCUSSION |
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2 µM. Although the most potent thiazolidinone CFTRinh-172 has Ki of 0.2–0.3 µM in permeabilized cell preparations, its Ki is 2–5 µM in many intact epithelial cells because of the interior negative membrane potential that reduces its concentration in cytoplasm (Thiagarajah et al., 2004a). Thus, the glycine hydrazides are as or more potent than the thiazolidinones, and like the thiazolidinones, they block CFTR in nasal and intestinal epithelia in vivo.
Patch-clamp studies indicated that CFTR inhibition by GlyH-101 is sensitive to membrane potential. At sub-maximal concentrations of GlyH-101, there was marked inward rectification in the CFTR current–voltage relationship, indicating that Cl– flux from the extracellular to the intracellular side of the membrane is more strongly blocked than that in the opposite direction. The apparent Ki increased approximately fourfold as applied potential was varied from +60 to –60 mV. Since GlyH-101 is negatively charged at pH 6–8, the simplest interpretation of these data is that GlyH-101 inhibition involves direct interaction with the channel pore at the extracellular side of the membrane. Accordingly, negative membrane potentials reduce the inhibitory efficacy of the negatively charged GlyH-101 by electrostatic repulsion, which drives the compound away from the pore. In contrast, the open channel blocker glibenclamide, which is thought to act from the intracellular side of the CFTR pore (Sheppard and Robinson, 1997), produces outward rectification of CFTR current–voltage relationship (Zhou et al., 2002). The reduced GlyH-101 potency at low extracellular Cl– concentration provided further evidence that GlyH-101 binds to a site at the external CFTR pore.
Analysis of GlyH-101 dose–response data also revealed an increase in apparent Hill coefficient at more negative membrane potentials, suggesting the possibility of more than one inhibitor binding site within the pore and/or cooperative interaction between inhibitor molecules, as reported previously for other ion channels (Pottosin et al., 1999; Brock et al., 2001). In support of the hypothesis that GlyH-101 is an open channel blocker, cell-attached patch-clamp experiments revealed fast closures within bursts of channel openings. The frequency of fast closures increased with GlyH-101 concentration, producing a reduction in mean channel open time as found for glibenclamide (Sheppard and Robinson, 1997). The appearance of closure events on the millisecond time scale classifies GlyH-101 as an "intermediate"-type channel blocker, similar to glibenclamide; in contrast, "fast" blockers reduce apparent single channel conductance, and "slow" blockers cause closures of many seconds duration (Hille, 1992). In whole-cell patch-clamp and apical Cl– current experiments, CFTR Cl– conductance was nearly completely inhibited at high concentrations (
30 µM) of GlyH-101. Together these results suggest that the GlyH-101 inhibition mechanism involves direct CFTR pore occlusion at a site at or near the extracellular-facing pore surface.
Synthesis and characterization of a series of glycine hydrazide analogues indicated the important structural determinants for CFTR inhibition. Synthesis work was directed to alter polarity, planarity, and hydrophilic/hydrophobic properties of the GlyH-101, systematically modifying different portions of the molecule as diagrammed in Fig. 3 A. Slight changes in planarity by replacing the glycinyl methylene group (R2) with carbonyl group improved inhibition activity, whereas reduction of Schiff base group (N=C) reduced activity. An extensive set of modifications of the benzaldehyde moiety (R3) indicated the requirement of two bromines separated by one para-hydroxyl group. Replacement of the 2-naphthalenyl group by (hetero)aromatic groups reduced inhibition activity. Many GlyH-101 analogues having substitutions that reduced overall polarity were inactive, suggesting that the presence of a hydrophobic group at one end at R1 and an anion group at R3 are key requirements for CFTR inhibition activity. Molecular docking computations should be informative when structural information about the CFTR pore becomes available.
In summary, the glycine hydrazides represent a new class of potent CFTR inhibitors with a novel external pore-occluding mechanism producing inward rectification and a reduction in mean channel open time. The large series of structural analogues with varying activities should permit the synthesis of engineered analogues with specified ADME (administration, distribution, metabolism, excretion) and other properties, such as membrane impermeability. The antidiarrheal efficacy of GlyH-101 when added in the intestinal lumen rather than systemically is particularly interesting, and suggests the possibility of developing a nonabsorbable drug for reducing intestinal fluid loss in secretory diarrheas produced by Vibrio cholera and Escherichia coli.
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ACKNOWLEDGMENTS |
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Olaf S. Andersen served as editor.
Submitted: 11 March 2004
Accepted: 4 June 2004
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F508-CFTR chloride channel gating. J. Biol. Chem. 278:35079–35085.
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B. Illek, Z. Fu, C. Schwarzer, T. Banzon, S. Jalickee, S. S. Miller, and T. E. Machen Flagellin-stimulated Cl- secretion and innate immune responses in airway epithelia: role for p38 Am J Physiol Lung Cell Mol Physiol, October 1, 2008; 295(4): L531 - L542. [Abstract] [Full Text] [PDF]
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 L. Chen, C. A. Bosworth, T. Pico, J. F. Collawn, K. Varga, Z. Gao, J. P. Clancy, J. A. Fortenberry, J. R. Lancaster Jr., and S. Matalon DETANO and Nitrated Lipids Increase Chloride Secretion across Lung Airway Cells Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 150 - 162. [Abstract] [Full Text] [PDF]
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 R. G. Painter, R. W. Bonvillain, V. G. Valentine, G. A. Lombard, S. G. LaPlace, W. M. Nauseef, and G. Wang The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils J. Leukoc. Biol., June 1, 2008; 83(6): 1345 - 1353. [Abstract] [Full Text] [PDF]
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N. D. Sonawane, O. Zegarra-Moran, W. Namkung, L. J. V. Galietta, and A. S. Verkman {alpha}-Aminoazaheterocyclic-Methylglyoxal Adducts Do Not Inhibit Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Activity J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 529 - 535. [Abstract] [Full Text] [PDF]
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S. G. Brown, M. Gallacher, R. E. Olver, and S. M. Wilson The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L942 - L954. [Abstract] [Full Text] [PDF]
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R. De La Fuente, W. Namkung, A. Mills, and A. S. Verkman Small-Molecule Screen Identifies Inhibitors of a Human Intestinal Calcium-Activated Chloride Channel Mol. Pharmacol., March 1, 2008; 73(3): 758 - 768. [Abstract] [Full Text] [PDF]
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P. Pariwat, S. Homvisasevongsa, C. Muanprasat, and V. Chatsudthipong A Natural Plant-Derived Dihydroisosteviol Prevents Cholera Toxin-Induced Intestinal Fluid Secretion J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 798 - 805. [Abstract] [Full Text] [PDF]
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M. D. Fuller, C. H. Thompson, Z.-R. Zhang, C. S. Freeman, E. Schay, G. Szakacs, E. Bakos, B. Sarkadi, D. McMaster, R. J. French, et al. State-dependent Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by a Novel Peptide Toxin J. Biol. Chem., December 28, 2007; 282(52): 37545 - 37555. [Abstract] [Full Text] [PDF]
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C. Routaboul, C. Norez, P. Melin, M.-C. Molina, B. Boucherle, F. Bossard, S. Noel, R. Robert, C. Gauthier, F. Becq, et al. Discovery of {alpha}-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1023 - 1035. [Abstract] [Full Text] [PDF]
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B. Yangthara, A. Mills, V. Chatsudthipong, L. Tradtrantip, and A. S. Verkman Small-Molecule Vasopressin-2 Receptor Antagonist Identified by a G-Protein Coupled Receptor "Pathway" Screen Mol. Pharmacol., July 1, 2007; 72(1): 86 - 94. [Abstract] [Full Text] [PDF]
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W. M. Xu, Q. X. Shi, W. Y. Chen, C. X. Zhou, Y. Ni, D. K. Rowlands, G. Yi Liu, H. Zhu, Z. G. Ma, X. F. Wang, et al. Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility PNAS, June 5, 2007; 104(23): 9816 - 9821. [Abstract] [Full Text] [PDF]
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X. Liu, M. Luo, L. Zhang, W. Ding, Z. Yan, and J. F. Engelhardt Bioelectric Properties of Chloride Channels in Human, Pig, Ferret, and Mouse Airway Epithelia Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 313 - 323. [Abstract] [Full Text] [PDF]
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A. Perez, A. C. Issler, C. U. Cotton, T. J. Kelley, A. S. Verkman, and P. B. Davis CFTR inhibition mimics the cystic fibrosis inflammatory profile Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L383 - L395. [Abstract] [Full Text] [PDF]
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L. Chen, R. P. Patel, X. Teng, C. A. Bosworth, J. R. Lancaster Jr., and S. Matalon Mechanisms of Cystic Fibrosis Transmembrane Conductance Regulator Activation by S-Nitrosoglutathione J. Biol. Chem., April 7, 2006; 281(14): 9190 - 9199. [Abstract] [Full Text] [PDF]
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J. J. Wine Acid in the airways. Focus on "Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis" Am J Physiol Cell Physiol, March 1, 2006; 290(3): C669 - C671. [Full Text] [PDF]
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Y. Song, D. Salinas, D. W. Nielson, and A. S. Verkman Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis Am J Physiol Cell Physiol, March 1, 2006; 290(3): C741 - C749. [Abstract] [Full Text] [PDF]
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 M. Schuier, H. Sies, B. Illek, and H. Fischer Cocoa-Related Flavonoids Inhibit CFTR-Mediated Chloride Transport across T84 Human Colon Epithelia J. Nutr., October 1, 2005; 135(10): 2320 - 2325. [Abstract] [Full Text] [PDF]
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 M. H. Levin and A. S. Verkman CFTR-Regulated Chloride Transport at the Ocular Surface in Living Mice Measured by Potential Differences Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1428 - 1434. [Abstract] [Full Text] [PDF]
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S.-Y. Lee and C. O. Lee Inhibition of Na+-K+ Pump and L-Type Ca2+ Channel by Glibenclamide in Guinea Pig Ventricular Myocytes J. Pharmacol. Exp. Ther., January 1, 2005; 312(1): 61 - 68. [Abstract] [Full Text] [PDF]
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Y. Song, N. D. Sonawane, D. Salinas, L. Qian, N. Pedemonte, L. J. V. Galietta, and A. S. Verkman Evidence against the Rescue of Defective {Delta}F508-CFTR Cellular Processing by Curcumin in Cell Culture and Mouse Models J. Biol. Chem., September 24, 2004; 279(39): 40629 - 40633. [Abstract] [Full Text] [PDF]
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D. N. Sheppard CFTR Channel Pharmacology: Novel Pore Blockers Identified by High-throughput Screening J. Gen. Physiol., July 26, 2004; 124(2): 109 - 113. [Full Text] [PDF]
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