Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl- channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 microM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl- conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 microM at +60 mV to 5.6 microM at -60 mV. Apparent potency was reduced by lowering extracellular Cl- concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (-60 mV holding potential, 5 microM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5-8.0, where it exists predominantly as a monovalent anion with solubility approximately 1 mM in water. Topical GlyH-101 (10 microM) 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 microg) reduced by approximately 80% cholera toxin-induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTR(inh)-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.

Figures

Figure 1.

Identification of novel classes of CFTR inhibitors by high-throughput screening. (A) Screening procedure. FRT cells coexpressing a halide-sensing yellow fluorescent protein (YFP-H148Q) and human wild-type CFTR were incubated with activating cocktail for 15 min before addition of test compounds at 25 μM. Iodide influx was induced by rapid addition of an iodide-containing solution. (B) Structures of novel classes of CFTR inhibitors identified by screening of a collection of 100,000 drug-like small molecules. (C) Dose inhibition of the glycine hydrazide GlyH-101 determined by fluorescence assay (left) and apical Cl− current analysis (right). Apical Cl− current was induced by 100 μM CPT-cAMP. CPT-cAMP and GlyH-101 were added to both apical and basolateral solutions.

Figure 2.

CFTR inhibition by the glycine hydrazide GlyH-101. Studies were done in FRT cells coexpressing human wild-type CFTR and a halide-sensing yellow fluorescent protein (YFP). (A) Kinetics of CFTR inhibition. After CFTR stimulation by a mixture of activators (10 μM forskolin, 100 μM IBMX, 20 μM apigenin), iodide influx (mean ± SEM, n = 3) was measured by the fluorescence assay at indicated times after addition of GlyH-101 (10 μM). Inset shows reduction in apical Cl− current (activated by 100 μM CPT-cAMP) after rapid addition of GlyH-101 (5 μM). (B) Kinetics of reversal of inhibition after GlyH-101 washout. FRT cells were incubated with the mixture of activators containing 5 μM GlyH-101 for 5 min. GlyH-101 was removed and cells were washed three times with PBS. Iodide influx was measured at specified times after GlyH-101 washout in the presence of the activator mixture. (C) Inhibition of iodide influx (mean ± SEM, filled bars) by GlyH-101 (50 μM, open bars) after CFTR stimulation by indicated agonists (all 50 μM, except for forskolin 10 μM, CPT-cAMP 500 μM).

Figure 3.

Glycine hydrazide (GlyH) CFTR inhibitors. (A) Classes of GlyH-101 analogues prepared for analysis of structure–activity relationships showing sites of modification (brackets). Substitutions to benzaldehyde phenyl rings not shown. (B) Reaction schemes for the synthesis of GlyH-101, 126, 201, and 301. Reagents and conditions (see materials and methods for details): (a) ICH2COOEt, NaOAc, 95°C; (b) N2H4.H2O, EtOH/reflux; (c) 3,5-di-Br-2,4-di-OH-Ph-CHO, EtOH/reflux; (d) 3,5-di-Br-2,4-di-OH-Ph-COCl, pyridine, 22°C; (e) N2H4.H2O, Pd/C (10%), DMF/reflux; (f) glyoxalic acid, 10°C; (g) Na2BH3CN/CH3CN, 48 h; dry HCl, EtOH. (C) Structure (left) and apical Cl− current analysis (right, done as in Fig. 1 C) of OxaH-110 in FRT cells.

Figure 4.

General structures of the synthesized compounds. See Table I for list of substituents and CFTR inhibition activities.

Figure 5.

Patch-clamp analysis of GlyH-101 inhibition mechanism. (A) Superimposed whole-cell membrane currents evoked by voltages from −100 to +100 mV (20-mV steps) in CFTR-expressing FRT cells after maximal CFTR stimulation by 5 μM forskolin. Holding potential was −100 mV and interpulse duration was 4 s. Data shown before (left) and after (right) 10 μM GlyH-101. (B) Current–voltage relationships in the absence of inhibitors (control, open circles), after addition of 10 μM (filled squares) and 30 μM (open squares) GlyH-101, after washout of 10 μM GlyH-101 (recovery, shaded circles), and after addition of 5 μM CFTRinh-172 (filled circles). (C) Dose–response for inhibition of CFTR Cl− current by GlyH-101 at indicated membrane potentials. Each point is the mean ± SEM of four to five experiments. Data were fitted to the Hill equation. Fitted Ki: 1.4 ± 0.4, 3.8 ± 0.2, 5.0 ± 0.3, and 5.6 ± 0.4 μM for voltages of +60, +20, −20, and −60 mV, respectively. Ki at −20 and −60 mV significantly greater than at +20 (P < 0.05) and +60 mV (P < 0.02). (D) Effect of reducing extracellular Cl− concentration on apparent GlyH-101 potency. GlyH-101 dose–response in high (150 mM) vs. low (20 mM) extracellular Cl− (mean ± SEM, four sets of experiments). Membrane potential was +20 mV.

Figure 6.

Single channel analysis of CFTR inhibition by GlyH-101. (A) Representative traces (left) and corresponding amplitude histograms (right) obtained from a cell-attached patch. Pipette potential (Vp) was −60 mV. CFTR was stimulated with forskolin (5 μM) in the absence and presence of GlyH-101 at indicated concentrations. Dashed lines show zero current level (channels closed) with downward deflections indicating channel openings (Cl− ions moving from pipette into the cell). Apparent open channel probability decreased from 0.48 in the absence of inhibitor to 0.14 at 5 μM GlyH-101. (B) Mean channel open times (mean ± SEM, five sets of experiments) as a function of GlyH-101 concentration from cell-attached patch-clamp experiments (*, P < 0.05; **, P < 0.01 vs. control).

Figure 7.

Determination of pH-dependent ionic equilibria of GlyH-101 by spectrophotometric titration of GlyH-101 analogues. (A) Chemical structures (left) and corresponding pH-dependent absorbance changes (right) of compounds (10 μM) in NaCl (100 mM) containing MES, HEPES, boric acid, and citric acid (each 10 mM) titrated to indicated pH using HCl/NaOH. Absorbance changes measured at wavelengths of 346, 348, 346, and 236 nm (top to bottom). (B) Deduced ionic equilibria of GlyH-101 showing pKa values.

Figure 8.

GlyH-101 inhibits forskolin-induced hyperpolarization in nasal potential difference in mice. (A, left) Nasal PD recording showing responses to amiloride (100 μM) and low Cl− (4.7 mM) solutions. Where indicated, the low Cl− solutions contained forskolin (10 μM) without or with GlyH-101 (10 μM). (A, right) Averaged PD values (mean ± SEM, n = 5). (B) Paired analysis of experiments as in A showing PD changes (ΔPD) after forskolin (10 μM), CFTRinh-172 (20 μM), and GlyH-101 (10 μM). (C) A series of experiments was done in which all solutions contained DIDS (100 μM) or GlyH-101 (10 μM). ΔPD (mean ± SEM) for low Cl− and forskolin-induced hyperpolarizations. *, P < 0.005 for reduced ΔPD compared with control. CFTRinh-172 data taken from Salinas et al. (2004).

Figure 9.

GlyH-101 inhibits cholera toxin/cAMP-dependent intestinal fluid secretion. (A–C) Inhibition of short-circuit current by GlyH-101 after CFTR stimulation in T84 cells (A), human airway cells (B), and mouse ileum (C). Following constant baseline current, amiloride (10 μM, apical solution) and CPT-cAMP (0.1 mM, both solutions) were added, followed by indicated concentrations of GlyH-101 (both solutions). Indomethacin (10 μM) was present in all solutions in ileum studies. Experiments representative of three to five measurements. (D) Closed intestinal loop model of cholera toxin–induced fluid secretion. Intestinal luminal fluid, shown as loop weight/length (g/cm, SEM, six mice), measured at 4 h after injection of saline (control), cholera toxin (1 μg), or cholera toxin given together with GlyH-101 (2.5 μg). *, P