Chloride channels as drug targets

Abstract

Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABA(A) (gamma-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery.

Figure 1. Structures and mechanisms of regulation of chloride channels

a | Cystic fibrosis transmembrane conductance regulator (CFTR). Shown here are 12 membrane-spanning segments of CFTR plus two nucleotide binding domains (NBDs 1 and 2) and a regulatory R domain. CFTR activation involves cyclic AMP-dependent phosphorylation and binding of ATP molecules at the NBDs. b | The overall organization of voltage-gated chloride (ClC) channels is depicted, showing 18 segments (labelled A to R) most of which span the plasma membrane partially and in a strongly tilted configuration. Fast gating involves flipping of a pore-lining glutamate side chain into and out of the chloride pathway. Channels are arranged as dimers with a slow gate controlling the activity of both channels simultaneously. c | The calcium-activated chloride channel (CaCC) TMEM16A (anoctamin-1), with predicted topology showing eight transmembrane segments with cytosolic amino and carboxy termini. The mechanism of calcium activation is unknown. d | GABA (γ-aminobutyric acid) and glycine-gated chloride channels, showing pentameric channels formed by α, β and γ subunits. Each subunit has four transmembrane segments, with a large extracellular N terminus. The second transmembrane segment of each subunit contributes to the formation of the central pore. The N termini of the α and β subunits form the ligand binding site. Volume-sensitive chloride channels (not shown) have an unknown molecular structure. They activate upon cell swelling. CBS, cystathione β-synthase-related domain.

Figure 2. Cell-based screening assay of halide transport using a fluorescent protein mutant

Aa | Reduced yellow fluorescence protein (YFP) fluorescence following halide binding (top). Cells expressing YFP in a cytoplasmic pattern (bottom). Ab | Titration of YFP-H148Q/I152L fluorescence with chloride, iodide and nitrate at pH 7.4. Inset shows rapid indicator response following an increase in Cl⁻ concentration from 0 mM to 40 mM. Ac | pH titration at Cl⁻ concentration of 0 mM, 75 mM and 150 mM. Ba | Screening protocol for cystic fibrosis transmembrane regulator (CFTR) inhibitors. CFTR halide conductance in cells co-expressing CFTR and YFP indicator stimulated by an agonist mixture (forskolin, 3-isobutyl-1-methylxanthine (IBMX), apigenin). After addition of test compound, iodide influx is measured by YFP fluorescence. Bb | Single-well fluorescence data showing controls (no activators, no test compound) and test wells. Ca | Screen for calcium-activated chloride channel (CaCC) inhibitors. CaCC halide conduction in human colonic cells expressing native CaCC and transfected with YFP indicator is measured following stimulation by an agonist mixture (ATP, carbachol). Iodide influx quenches YFP fluorescence. Cb | Fluorescence data showing controls (no activators, no test compounds) and examples of inhibitors. CaMKII, calcium/calmodulin kinase II; PIP2, phosphatidylinositol 4,5-bisphosphate. Panel A is modified, with permission, from REF. © (2001) Elsevier Science. Panel B is modified, with permission, from REF. © (2002) American Society for Clinical Investigation. Panel C is modified, with permission, from REF. © (2008) American Society for Pharmacology and Experimental Therapeutics.

Figure 3. Cystic fibrosis transmembrane regulator (CFTR) inhibitors and their indications

Aa | Structures of thiazolidinone CFTR inhibitor CFTR_inh-172 and glycine hydrazide GlyH-101. Ab | Malonic acid hydrazide (MalH) conjugated to a macromolecular backbone (lectin or polyethylene glycol (PEG)). Ba | Intestinal fluid secretion in diarrhoea. Mechanism of enterotoxin-mediated diarrhoea showing CFTR chloride secretion following choleratoxin-induced cyclic AMP elevation. Sodium and water follow passively. Bb | CFTR_inh-172 inhibits intestinal fluid accumulation in closed mouse ileal loops. Loops were injected with saline or cholera toxin and CFTR_inh-172 was administered by intraperitoneal injection. Inset shows photograph of intestinal loops. Bc | Survival of suckling mice following oral administration of cholera toxin with or without CFTR inhibitor (MalH–lectin, 125 pmol). Vehicle control indicates no cholera toxin given. Ca | Cyst fluid secretion in polycystic kidney disease. Mechanism of fluid secretion into cysts, involving CFTR-dependent chloride secretion. Cb | CFTR inhibitors (compound a, a tetrazolo-derivatized thiazolidinone analogue; compound b, an absorbable, phenyl-derivatized glycine hydrazide analogue) cause slowing of cyst expansion in embryonic kidney organ culture. Upper panels show kidney growth and cyst formation in medium containing 8-Br-cAMP (scale bars, 1 mm). Bottom panels show day-4 kidneys in control and inhibitor-containing medium. Panel A is modified, with permission, from REF. © (2007) W. B. Saunders. Panel B is modified, with permission, from REF. © (2004) W. B. Saunders. Panel C is modified, with permission, from REF. © (2008) American Society of Nephrology.

Figure 4. Lung pathophysiology in cystic fibrosis (CF) and activators of ΔF508-CFTR, the most common CF-causing mutation

Aa | CF transmembrane regulator (CFTR) normally functions as a cyclic AMP-activated chloride channel at the apical plasma membrane of selected epithelial cells (top). ΔF508-CFTR is misfolded, retained at the endoplasmic reticulum (ER) and rapidly degraded (bottom). Ab | Lung pathophysiology in CF showing reduced chloride and bicarbonate secretion by submucosal glands, producing a viscous, acidic airway surface liquid (ASL) that promotes bacterial colonization. Ba | Structures of nanomolar-potency ΔF508-CFTR potentiators (correctors of defective channel gating). Bb | Short-circuit current analysis of ΔF508-CFTR-expressing epithelial cells (following low-temperature rescue to permit targeting of ΔF508-CFTR to the plasma membrane), showing small response to forskolin (fsk, 20 µM), followed by activation by PG-01 and inhibition by 10 µM CFTR_inh-172. The small response to a high concentration of forskolin represents the ΔF508-CFTR gating defect, as forskolin alone fully activates wild-type CFTR. PG-01 strongly increases ΔF508-CFTR chloride conductance, with the increase inhibited by CFTR_inh-172. Patch-clamp analysis indicated that PG-01 increases ΔF508-CFTR chloride current with open probability similar to that of activated wild-type CFTR. Ca | Structures of ΔF508-CFTR correctors (correctors of defective folding/cellular processing). Cb | Short-circuit analysis of cells as in (Bb), but cultured for 24 hours in the absence (top trace) or presence (bottom trace) of corr-4a. ΔF508-CFTR activated by 20 µM forskolin and 50 µM genistein. I_sc, short-circuit current; PKA, protein kinase A. Panel Bb is modified, with permission, from REF. © (2005) American Society for Pharmacology and Experimental Therapeutics. Panel Cb is modified, with permission, from REF. © (2005) The American Society for Clinical Investigation.

Figure 5. Cellular physiology of calcium-activated chloride channels (CaCCs) and small-molecule inhibitors

a | Cellular roles of CaCCs. Cytoplasmic calcium elevation following various stimuli activates CaCCs directly or through calcium/calmodulin kinase II (CaMKII)-mediated phosphorylation. CaCC activation facilitates epithelial cell chloride secretion, and by depolarizing the plasma membrane it modulates neuroexcitation and smooth-muscle contraction. b | Inhibitors of human intestinal CaCC identified by high-throughput screening. c | Whole-cell currents in HT-29 cells following CaCC stimulation by ATP and carbachol, in the absence (control) and presence of indicated compounds. ‘Basal’ refers to absence of activators. ER, endoplasmic reticulum; IP₃, inositol 1,4,5-trisphosphate. Panel b is modified, with permission, from REF. © (2008) American Society for Pharmacology and Experimental Therapeutics.

Figure 6. Physiology of selected voltage-gated (ClC)-type chloride channels

a | ClC-1 in skeletal muscle stabilizes membrane potential. ClC-1 loss-of-function mutations causes myotonia. b | ClC-Kb in the basolateral membrane of kidney distal tubular cells facilitates transepithelial sodium chloride absorption, through coordinated activity with the apical bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, apical potassium channel (to recycle potassium in the tubule lumen) and basolateral Na⁺/K⁺-ATPase. c | ClC-5 in kidney proximal tubule epithelial cells facilitates endocytosis and endosomal acidification. ClC-5 loss-of function mutations cause proteinuria and kidney stones (Dent’s disease). Organellar ClCs such as ClC-5 and ClC-7 function as electrogenic Cl⁻/H⁺ exchangers. d | ClC-7 chloride transport in bone osteoclasts facilitates net secretion of HCl into the lacuna for bone demineralization. ClC-7 loss-of-function mutations cause osteopetrosis. ACh, acetylcholine.

Figure 7. Ligand-gated chloride channels

Schematic of GABA (γ-aminobutyric acid) inhibitory synapse. Release of GABA from presynaptic membrane triggers the transient activation of ionotropic GABA receptors. The low intracellular chloride concentration in the postsynaptic neuron, generated by the action of the K⁺/Cl⁻ cotransporter (KCC2), drives chloride influx through GABA-activated chloride channels causing membrane hyperpolarization. Benzodiazepines, anaesthetics, ethanol and other compounds act on GABA receptors to potentiate neurotransmitter effect. V_m, transmembrane potential.