SLC26A6-selective inhibitor identified in a small-molecule screen blocks fluid absorption in small intestine.

SLC26A6 (also known as putative anion transporter 1 [PAT1]) is a Cl-/HCO3- exchanger expressed at the luminal membrane of enterocytes where it facilitates intestinal Cl- and fluid absorption. Here, high-throughput screening of 50,000 synthetic small molecules in cells expressing PAT1 and a halide-sensing fluorescent protein identified several classes of inhibitors. The most potent compound, the pyrazolo-pyrido-pyrimidinone PAT1inh-B01, fully inhibited PAT1-mediated anion exchange (IC50 ~350 nM), without inhibition of the related intestinal transporter SLC26A3 (also known as DRA). In closed midjejunal loops in mice, PAT1inh-B01 inhibited fluid absorption by 50%, which increased to >90% when coadministered with DRA inhibitor DRAinh-A270. In ileal loops, PAT1inh-B01 blocked fluid absorption by >80%, whereas DRAinh-A270 was without effect. In colonic loops, PAT1inh-B01 was without effect, whereas DRAinh-A270 completely blocked fluid absorption. In a loperamide constipation model, coadministration of PAT1inh-B01 with DRAinh-A270 increased stool output compared with DRAinh-A270 alone. These results provide functional evidence for complementary and region-specific roles of PAT1 and DRA in intestinal fluid absorption, with PAT1 as the predominant anion exchanger in mouse ileum. We believe that PAT1inh-B01 is a novel tool to study intestinal ion and fluid transport and perhaps a drug candidate for small intestinal hyposecretory disorders such as cystic fibrosis-related meconium ileus and distal intestinal obstruction syndrome.

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation.

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 µM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.

Small molecule screen yields inhibitors of Pseudomonas homoserine lactone-induced host responses.

Pseudomonas aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-κB transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-κB activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restore NF-κB-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermalC12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy.

Increased diffusional mobility of CFTR at the plasma membrane after deletion of its C-terminal PDZ binding motif.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of Madin-Darby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to approximately 90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 +/- 0.09 x 10(-10) cm2/s at 37 degrees C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-DeltaTRL and GFP-CFTR-DeltaTRA) were also mobile with D significantly increased by approximately 60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-Delta TRL, and GFP-CFTR-DeltaTRA had similar mobilities (D approximately 12 x 10(-10) cm2/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTR- DeltaTRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 +/- 0.1 x 10(-7) cm2/s. EBP50 diffusion increased by approximately 2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane but that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster.

Diffusional mobility of the cystic fibrosis transmembrane conductance regulator mutant, delta F508-CFTR, in the endoplasmic reticulum measured by photobleaching of GFP-CFTR chimeras.

Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis. The most common disease-causing mutation, DeltaF508, is retained in the endoplasmic reticulum (ER) and is unable to function as a plasma membrane chloride channel. To investigate whether the ER retention of DeltaF508-CFTR is caused by immobilization and/or aggregation, we have measured the diffusional mobility of green fluorescent protein (GFP) chimeras of wild type (wt)-CFTR and DeltaF508-CFTR by fluorescence recovery after photobleaching. GFP-labeled DeltaF508-CFTR was localized in the ER and wt-CFTR in the plasma membrane and intracellular membranes in transfected COS7 and Chinese hamster ovary K1 cells. Both chimeras localized to the ER after brefeldin A treatment. Spot photobleaching showed that CFTR diffusion (diffusion coefficient approximately 10(-9) cm(2)/s) was not significantly slowed by the DeltaF508 mutation and that nearly all wt-CFTR and DeltaF508-CFTR diffused throughout the ER without restriction. Stabilization of molecular chaperone interactions by ATP depletion produced remarkable DeltaF508-CFTR immobilization ( approximately 50%) and slowed diffusion (6.5 x 10(-10) cm(2)/s) but had little effect on wt-CFTR. Fluorescence depletion experiments revealed that the immobilized DeltaF508-CFTR in ATP-depleted cells remained in an ER pattern. The mobility of wt-CFTR and DeltaF508-CFTR was reduced by maneuvers that alter CFTR processing or interactions with molecular chaperones, including tunicamycin, geldanamycin, and lactacystin. Photobleaching of the fluorescent ER lipid diOC(4)(3) showed that neither ER restructuring nor fragmentation during these maneuvers was responsible for the slowing and immobilization of CFTR. These results suggest that (a) the ER retention of DeltaF508-CFTR is not due to restricted ER mobility, (b) the majority of DeltaF508-CFTR is not aggregated or bound to slowly moving membrane proteins, and (c) DeltaF508-CFTR may interact to a greater extent with molecular chaperones than does wt-CFTR.