Strategies for cystic fibrosis transmembrane conductance regulator inhibition: from molecular mechanisms to treatment for secretory diarrhoeas

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter. It acts as an anion-selective channel that drives osmotic fluid transport across many epithelia. In the gut, CFTR is crucial for maintaining fluid and acid-base homeostasis, and its activity is tightly controlled by multiple neuro-endocrine factors. However, microbial toxins can disrupt this intricate control mechanism and trigger protracted activation of CFTR. This results in the massive faecal water loss, metabolic acidosis and dehydration that characterize secretory diarrhoeas, a major cause of malnutrition and death of children under 5 years of age. Compounds that inhibit CFTR could improve emergency treatment of diarrhoeal disease. Drawing on recent structural and functional insight, we discuss how existing CFTR inhibitors function at the molecular and cellular level. We compare their mechanisms of action to those of inhibitors of related ABC transporters, revealing some unexpected features of drug action on CFTR. Although challenges remain, especially relating to the practical effectiveness of currently available CFTR inhibitors, we discuss how recent technological advances might help develop therapies to better address this important global health need.

Keywords: CFTR pharmacology; G907 compound; cholera; cyclic AMP; cyclic GMP; enterocyte; glibenclamide; ion-channel gating; secretory diarrhea; zosuquidar.

Fig. 1

Structural snapshots of CFTR. (A) Cartoon representation of IF CFTR conformation, based on the dephosphorylated, ATP‐free cryo‐EM model of human CFTR (PDB ID5UAK, [27]). TMD1‐NBD1, green; TMD2‐NBD2, teal. Some residues mentioned in text are shown as coloured spheres. Also highlighted is the unwound portion of TM8 (magenta). The density corresponding to the R domain, which in this conformation is located between the two TMDs, at a level below the cytosolic face of the membrane (grey band), is omitted for clarity. (B) Schematic representation of IF conformation shown in A. Numbers indicate positions of transmembrane helices. (C) Schematic representation of OF, open channel CFTR conformation. ATP is shown in yellow. The degenerate site 1 is here on the front, while canonical site 2, also occupied by ATP, is on back (not shown). Here, an open anion permeation pathway is indicated (dotted blue line), based on extensive functional evidence linking NBD dimerization to channel opening. However, we still lack structural evidence of a corresponding OF, open‐pore CFTR conformation, as in PDB ID6MSM, see D, the extracellular end of the permeation pathway is obstructed. (D) Cartoon representation of phosphorylated, ATP‐bound human E1371Q‐CFTR (6MSM, [30]). The R domain density is again not shown. In this view it lies on the back of the protein, at a level below the lasso domain (here visible as the short helix parallel to the plane of the membrane, on right of image).

Fig. 2

Location in enterocytes of ion transporters and cyclic nucleotide signalling cascades involved in enterotoxin‐induced intestinal electrolyte and fluid secretion. (A) CFTR‐mediated vectorial anion secretion in CFTR‐enriched crypt cells. The Na⁺,K⁺‐ATPase provides the driving force for basolateral Cl⁻and${\text{HCO}}_3^{-}$entryviaNa⁺‐coupled cotransport, mediated by NKCC1 and NBCe1/NBCn1, respectively. Cl⁻and${\text{HCO}}_3^{-}$exit the cellviaCFTR. In addition,${\text{HCO}}_3^{-}$also exits the cellvia ${\text{Cl}}^{-}/{\text{HCO}}_3^{-}$exchangers (DRA, PAT‐1; not shown). Transcellular, electrogenic anion secretion generates a lumen‐negative transepithelial potential difference (V_TE) that drives passive paracellular Na⁺secretion. The resulting osmotic gradient drives water movement across the tight junctions. Salt and water secretion are regulated by a plethora of neuro‐endocrine factors that control protein kinase‐mediated phosphorylation/activation of CFTR. (B) NHE3‐ and DRA‐mediated NaCl absorption in villus cells. In villus cells, the coordinated activity of NHE3 and${\text{Cl}}^{-}/{\text{HCO}}_3^{-}$exchangers (SLC26A3/DRA; and SLC26A6/PAT‐1, not shown) mediates vectorial NaCl uptake, which, in turn, promotes water absorption. cAMP‐ and cGMP‐linked signal transduction routes, that is the same pathways that activate CFTR (also present in the upper epithelium, but at comparatively low levels; see Fig. 3A), inhibit NHE3 and promote${\text{HCO}}_3^{-}$secretion. The regulation of${\text{Cl}}^{-}/{\text{HCO}}_3^{-}$exchangers is less well defined. AC, adenylyl cyclase; GC‐C, guanylyl cyclase C; H, hormone or para‐/neurocrine factor; PKA, cAMP‐dependent protein kinase, PKG2, cGMP‐dependent protein kinase 2; PDE3, phosphodiesterase 3.

Fig. 3

Immunodetection of CFTR in human small intestine and colon. Jejunal (panels A–D) and rectal (panels E and F) biopsies were fixed in performic acid, paraffine embedded and stained with the polyclonal, affinity‐purified hCFTR antibody CC24 [165]. A rabbit specific horseradish peroxidase (HRP)/3,3′‐diaminobenzidine (DAB) detection IHC Kit (Abcam, Cambridge, UK) was used to visualize CFTR protein (brown stain). (A) CFTR expression in jejunal villi showing (a) the absence of CFTR staining in the apical border of goblet cells, and (b) detection of relatively rare CFTR high expressor (CHE) cells (arrow heads). (C) High expression of CFTR protein in the luminal membrane of jejunal crypt cells. (E) CFTR staining in mid‐crypt cells of distal colon/rectum, showing the absence of CFTR in goblet cells. Panels B, D and F show the absence of CFTR immunostaining in biopsies from a homozygous F508del CF patient, confirming the high specificity of the CFTR antibody.

Fig. 4

Activation of CFTR by MsbA inhibitors. Acute treatment with selective quinoline MsbA inhibitors G592, G593, G247 and G907 [98] results in an increase in cellular anion conductance. Functional analysis of wild‐type YFP‐CFTR expressed in HEK293 cells using an image‐based fluorescence microscopy assay [101, 102]. YFP‐CFTR is expressed from a pIRES2‐mCherry‐YFPCFTR plasmid, in which YFP‐CFTR and a soluble mCherry red fluorescent protein are translated from a single bicistronic mRNA. (A) Mean normalized CFTR conductance (G _CFTR_normalized) and cell membrane potential (V _M) were estimated by fitting YFP quenching time course (see B), following extracellular addition of iodide. Before iodide addition, a 230 s pretreatment allowed CFTR activation to reach a steady state. Bars represent different pretreatment: vehicle control (DMSO, dark grey bar), 0.3 µmforskolin (light grey bar), 0.3 µmforskolin + 10 µmG592 (blue bar), 0.3 µmforskolin + 10 µmG593 (red bar), 0.3 µmforskolin + 10 µmG247 (yellow bar) or with 0.3 µmforskolin + 10 µmG907 (black bar) (n ≥ 5, as indicated by solid circles, each representing one measurement obtained on an independent plate). To account for possible differences in transfection efficiency between wells, CFTR conductance is normalized using the mean mCherry fluorescence measured within cells [102]. Data from wells belonging to the same 96‐well plate were paired, and pairedt‐tests were used to determine statistical significance of comparisons (**P < 0.01; ****P < 0.0001). (B) Time course of YFP quenching following addition of extracellular iodide. Observed relative fluorescence values are shown as yellow symbols, while solid lines are fits. For each compound, graphs compare quenching time curve following pretreatment with vehicle control (squares, dark grey line), 0.3 µmforskolin alone (triangles, light grey line) and 0.3 µmforskolin + 10 µmcompound (circles, coloured line). Compound colour‐coding as in A.

Fig. 5

Mechanism of action of CFTR and ABC transporter inhibitors. (A) Gating of phosphorylated CFTR channels is driven by ATPase cycles. In the absence of drugs, for phosphorylated, ATP‐gated wild‐type CFTR channels, opening is coupled to formation of a tight NBD1/NBD2 dimer; while channel closing is triggered by ATP hydrolysis at site 2 (hidden in this view). In physiological conditions, degenerate site 1 is likely not as open as seen in5UAKand as depicted here [166]. (B) G907 binds between TM4,5, 6 (and/or 10, 11, 12). While it inhibits MsbA, it increases anion conductance of CFTR‐expressing cells. Drug binding to CFTR is here hypothesized to alter position of coupling helices, bringing them closer together, and forcing a dislocation of ball‐and‐socket joint with NBD1. Conformational changes at the extracellular end of the inner vestibule result in an opening of the permeation pathway. (C) Glibenclamide is an open‐pore channel blocker, whose binding site is within the membrane electric field. It accesses its binding site from the cytosol, and apparent affinity is increased by hyperpolarization. (D) At low micromolar concentrations, GlyH‐101 is a nonabsorbable open‐pore channel blocker, which acts from the luminal side of the membrane. Its binding is favoured at depolarized membrane potential. (E) BPO‐27 competes with ATP for binding at canonical site 2, preventing the IF‐to‐OF transition, and therefore channel opening. (F) Binding of CFTR_inh‐172 triggers a conformational change that leads to channel closure (without requiring dissociation of NBDs). Here, this conformational change is shown to close the cytosolic portal between TM4 and TM6.

Fig. 6

Different potency of GlyH‐101 analogue iOWH032 in human rectal biopsies as compared to 2D rectal colonoids originating from the same individual. (A) Rectal biopsy specimens obtained from healthy individuals were mounted in Ussing chambers and short‐circuit currents (I _sc), representing CFTR‐mediated anion secretion, were assessed as described in detail elsewhere [81, 82]. iOWH032, a GlyH‐101 analogue [40], was added only to the luminal bath; the cAMP agonist forskolin (F; 10 µm) and 3‐isobutyl‐1‐methylxanthine (I; 100 µm) were added to both the luminal and basolateral bath. (B) Undifferentiated colonoid monolayers derived from the rectal biopsies shown in panel A were grown on Transwell filters and subsequently mounted in Ussing chambers.I _scmeasurements were performed as described for panel A. (C) iOWH‐032 mediated inhibition of the cAMP‐dependentI _scresponse in colon tissue (Tis.) and organoids (Org.), as assessed at the maximal iOWH032 concentration tested in panels A and B. Horizontal bars depict mean ± SD of 6 (Tis.) or 5 (Org.) experiments. Data were statistically evaluated by ANOVA. The improved efficacy of iOWH032 in colonoid monolayers as compared to the rectal biopsies may have multiple causes: (a) the flat structure of the monolayer, preventing convective washout of the inhibitor as occurs in intestinal crypts [122]; (b) the lack of goblet cells and therefore of a mucus barrier in undifferentiated intestinal organoids [167]; (c) CFTR is rate‐limiting for the forskolin/cAMP‐induced anion secretory current in colonoids but not in rectal biopsies from healthy individuals [38].