Discovery of Glycine Hydrazide Pore-occluding CFTR Inhibitors

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 μM, 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 μM at +60 mV to 5.6 μM 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 μM 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 ∼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.

Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl− Secretion by Human Airway Epithelia

“…The cAMP-elevating agonist forskolin (Calbiochem) was prepared as a 20 mM stock solution in dimethyl sulfoxide (DMSO), and an aliquot was added at final concentrations of 2-50 M. CFTR blocker glibenclamide (17) was prepared as a 300 mM stock solution in DMSO and added to solutions at 1 mM. GLYH101 (18) and CFTRinh172 (19) were provided by Dr. Alan Verkman (University of California, San Francisco), prepared as a 20 mM stock solution in DMSO, and added to solutions at concentrations noted in the text. The Ca 2ϩ -ATPase blocker thapsigargin (20) was prepared as a 1 mM stock in DMSO and used at 5 M. TPEN was added to Ca 2ϩ -free Ringer's containing 100 M EGTA or Cl Ϫ -free ϩ Ca 2ϩ -free Ringer's at 1 mM and dissolved by continuous stirring for 1 h. TPEN was then used at either 1 or 0.5 mM as mentioned specifically in the text.…”

The Therapeutic Potential of Small-molecule Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…Like CFTR inh -172, 106 GlyH-101 strongly inhibited cholera toxin-induced fluid secretion in the small intestine of mice. 111 However, in marked contrast to CFTR inh -172, which must be administered intraperitoneally, 106 GlyH-101 was active when added directly to the lumen of the small intestine. 111 These data argue that GlyH-101 and related compounds might be developed into a non-absorbable drug therapy for secretory diarrhoea.…”

“…111 However, in marked contrast to CFTR inh -172, which must be administered intraperitoneally, 106 GlyH-101 was active when added directly to the lumen of the small intestine. 111 These data argue that GlyH-101 and related compounds might be developed into a non-absorbable drug therapy for secretory diarrhoea. Towards this goal, Sonawane et al 117 The data of Sonawane et al 119 suggest that non-absorbable divalent polyethylene glycolmalonic acid hydrazides are likely to be especially valuable in the development of rational new therapies for enterotoxin-induced secretory diarrhoea.…”

“…106 Third, efficacy: CFTR inh -172 inhibited cholera toxin-induced fluid secretion in the small intestine of mice, highlighting its value as a lead compound for drug discovery. 106 Although CFTR inh -172 has the drawbacks of limited water solubility (~20 μM) and reduced potency in intact cells and tissues (K i ~5 μM), 111 it has become a very widely used research tool. To date, it remains the most selective CFTR inhibitor available.…”

Identification of New Small Molecule Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Protein: In Vitro and in Vivo Studies

“…1,2) At present, two classes of potent CFTR inhibitors (thiazolidinone and glycine hydrazide) have been identified by highthroughput screening. 6,7) Both are effective in reducing cholera toxin-induced intestinal fluid secretion in mice. 6,7) The lead thiazolidinone compound, CFTR inh -172, reversibly inhibited CFTR with K i ϳ0.3 mM, presumably by binding to the CFTR's nucleotide binding domain 1.…”