Chloride transport modulators as drug candidates

Abstract

Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride-transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers, and cotransporters, some having broad anion specificity. Loss-of-function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt-wasting nephropathy, myotonia, osteopetrosis, hearing loss, and goiter. Although impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for the development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension, and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.

Keywords: chloride channel; chloride exchanger; drug discovery; epithelium; solute carrier proteins.

Figure 1.

Functional, cell-based screening assay for anion transporters using halide-sensing cytoplasmic fluorescent sensor. A: schematic showing adherent cell expressing a generic plasma membrane anion channel, exchanger, and cotransporter. The cell cytoplasm is stained with a halide-sensing fluorescent molecule. The assay is initiated by addition of halide X⁻ (generally I⁻ or Cl⁻) to the extracellular medium. Transport of X⁻ from the extracellular solution into the cytoplasm reduces sensor fluorescence. B: time course of halide sensor fluorescence following addition of X⁻, showing effect of a preadded transport inhibitor, inactive compound, and activator. X⁻, halide; F_halide, halide sensor fluorescence.

Figure 2.

Discovery of potentiators of defective F508del-CFTR chloride channel gating by high-throughput screening. A: screening procedure. Cells coexpressing F508del-CFTR and the halide-sensitive fluorescent protein YFP-H148Q/I152L were grown for 24 h at reduced temperature of 27°C (to increase plasma membrane F508del-CFTR expression). After washing, test compounds (2.5 µM) and forskolin (20 µM) were added, and iodide influx was assayed at 37°C from the time course of yellow fluorescent protein (YFP) fluorescence after adding iodide to the extracellular solution. B: examples time courses of YFP fluorescence in control wells (saline, negative control; 50 µM genistein, positive control) with examples of inactive and active test compounds. Chemical structure of an active compound emerging from the screening is shown. Adapted from Ref. (CC BY 4.0).

Figure 3.

Small molecule inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel expressed in intestinal epithelial cells. A: short-circuit current (Isc) in CFTR-expressing epithelial cells showing CFTR activation by the cell-permeable cAMP analog CPT-cAMP and inhibition by (R)-BPO-27 (structure shown on left). B: (R)-BPO-27 reduced fluid accumulation in closed, mid-jejunal intestinal loops in mice in which fluid secretion was induced by luminal cholera toxin (means ± SE, 4–8 intestinal loops per group, **P < 0.01 compared with cholera toxin with no inhibitor). Photographs of intestinal loops shown at the right. Adapted with permission from Ref. .

Figure 4.

Small molecule inhibitor of calcium-activated chloride channel TMEM16A expressed in vascular smooth muscle. A: short-circuit current (Isc) in TMEM16A-expressing epithelial cells showing TMEM16A activation by ionomycin and inhibition by TM_inh-23 (structure shown on left). B: daily mean systolic blood pressure (SBP; measured by telemetry) in spontaneously hypertensive rats with twice daily TM_inh-23 (10 mg/kg ip) treatment for 5 days starting on day 0. *P < 0.05 compared with day 0. Adapted with permission from Ref. .

Figure 5.

Small molecule inhibitor of chloride/bicarbonate exchanger SLC26A3 (DRA) expressed in colonic epithelium. A: DRA_inh-270 concentration-dependent inhibition of SLC26A3 anion exchange. Structure shown in inset. B: DRA_inh-270 normalized stool water content in a loperamide-induced mouse model of constipation (means ± SE, 4–6 mice per group, **P < 0.01 compared with loperamide with no inhibitor). Adapted with permission from Ref. .

Figure A1.

Chemical structures of selected chloride transport modulators.