A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats.

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.

Structure guided development of potent piperazine-derived hydroxamic acid inhibitors targeting falcilysin.

The protozoan parasite Plasmodium falciparum causes the most severe form of human malaria and is estimated to kill 400,000 people a year. The parasite infects and replicates in host red blood cells (RBCs), where it expresses an array of proteases to carry out multiple essential processes. We are investigating the function of falcilysin (FLN), a protease known to be required for parasite development in the RBC. We previously developed a piperazine-based hydroxamic acid scaffold to generate the first inhibitors of FLN, and the current study reports the optimization of the lead compound from that series. A range of substituents were tested at the N1 and N4 positions of the piperazine core, and inhibitors with significantly improved potency against purified FLN and cultured P. falciparum were identified. Computational studies were also performed to understand the mode of binding for these compounds, and predicted a binding model consistent with the biochemical data and the distinctive SAR observed at both the N1 and N4 positions.

Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

Interstitial cells of Cajal, which express the calcium-activated chloride channel transmembrane member 16A (TMEM16A), are an important determinant of gastrointestinal (GI) motility. We previously identified the acylaminocycloalkylthiophene class of TMEM16A inhibitors, which, following medicinal chemistry, gave analog 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide (TMinh-23) with 30 nM half-maximal inhibitory concentration. Here, we tested the efficacy of TMinh-23 for inhibition of GI motility in mice. In isolated murine gastric antrum, TMinh-23 strongly inhibited spontaneous and carbachol-stimulated rhythmic contractions. Pharmacokinetic analysis showed predicted therapeutic concentrations of TMinh-23 for at least 4 h following a single oral or intraperitoneal dose at 10 mg/kg. Gastric emptying, as assessed following an oral bolus of phenol red or independently by [99mTc]-diethylenetriamine pentaacetic acid scintigraphy, was reduced by TMinh-23 by ∼60% at 20 min. Interestingly, there was little effect of TMinh-23 on baseline whole-gut transit time or time to diarrhea induced by castor oil. Consequent to the delay in gastric emptying, TMinh-23 administration significantly reduced the elevation in blood sugar in mice following an oral but not intraperitoneal glucose load. These results provide pharmacological evidence for involvement of TMEM16A in gastric emptying and suggest the utility of TMEM16A inhibition in disorders of accelerated gastric emptying, such as dumping syndrome, and potentially for improving glucose tolerance in diabetes mellitus/metabolic syndrome and enhancing satiety in obesity.-Cil, O., Anderson, M. O., Yen, R., Kelleher, B., Huynh, T. L., Seo, Y., Nilsen, S. P., Turner, J. R., Verkman, A. S. Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

Correction: X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of Pseudomonas aeruginosa Homoserine Lactone-Mediated Apoptosis.

[This corrects the article DOI: 10.1371/journal.ppat.1003576.].

Aquaporin-Targeted Therapeutics: State-of-the-Field.

Drugs targeting aquaporins have broad potential clinical applications, including cancer, obesity, edema, glaucoma, skin diseases and others. The astrocyte water channel aquaporin-4 is a particularly compelling target because of its role of brain water movement, neuroexcitation and glia scarring, and because it is the target of pathogenic autoantibodies in the neuroinflammatory demyelinating disease neuromyelitis optica . There has been considerable interest in the identification of small molecule inhibitors of aquaporins, with various candidates emerging from testing of known ion transport inhibitors, as well as compound screening and computational chemistry. However, in general, the activity of reported aquaporin inhibitors has not been confirmed on retesting, which may be due to technical problems in water transport assays used in the original identification studies, and the challenges in modulating the activity of small, compact, pore-containing membrane proteins. We review here the state of the field of aquaporin-modulating small molecules and biologics, and the challenges and opportunities in moving forward.

Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1.

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.

Structure-activity analysis of thiourea analogs as inhibitors of UT-A and UT-B urea transporters.

Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC50 of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC50 of ~0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC50 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC50>15 vs. 2.8 mM).

Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1.

Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca(2+) activated Cl(-) channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16Ainh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16Ainh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16Ainh-01 was tolerated, with most compounds showing no activity at 10 µM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16Ainh-01 with 2-thiophene, had IC50 ∼1 µM for inhibition of TMEM16A chloride conductance.

Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP.

We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP.

Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats.

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics ('urearetics') with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10-20 µM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2-3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 h, and a urine concentration of 20-40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of syndrome of inappropriate antidiuretic hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Global identification of peptidase specificity by multiplex substrate profiling.

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

X-box binding protein 1 (XBP1s) is a critical determinant of Pseudomonas aeruginosa homoserine lactone-mediated apoptosis.

Pseudomonas aeruginosa infections are associated with high mortality rates and occur in diverse conditions including pneumonias, cystic fibrosis and neutropenia. Quorum sensing, mediated by small molecules including N-(3-oxo-dodecanoyl) homoserine lactone (C12), regulates P. aeruginosa growth and virulence. In addition, host cell recognition of C12 initiates multiple signalling responses including cell death. To gain insight into mechanisms of C12-mediated cytotoxicity, we studied the role of endoplasmic reticulum stress in host cell responses to C12. Dramatic protection against C12-mediated cell death was observed in cells that do not produce the X-box binding protein 1 transcription factor (XBP1s). The leucine zipper and transcriptional activation motifs of XBP1s were sufficient to restore C12-induced caspase activation in XBP1s-deficient cells, although this polypeptide was not transcriptionally active. The XBP1s polypeptide also regulated caspase activation in cells stimulated with N-(3-oxo-tetradecanoyl) homoserine lactone (C14), produced by Yersinia enterolitica and Burkholderia pseudomallei, and enhanced homoserine lactone-mediated caspase activation in the presence of endogenous XBP1s. In C12-tolerant cells, responses to C12 including phosphorylation of p38 MAPK and eukaryotic initiation factor 2α were conserved, suggesting that C12 cytotoxicity is not heavily dependent on these pathways. In summary, this study reveals a novel and unconventional role for XBP1s in regulating host cell cytotoxic responses to bacterial acyl homoserine lactones.

Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors.

Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ∼1 µM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by ∼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.

Small-molecule inhibitors of urea transporters.

Urea transporter (UT) proteins, which include isoforms of UT-A in kidney tubule epithelia and UT-B in vasa recta endothelia and erythrocytes, facilitate urinary concentrating function. Inhibitors of urea transporter function have potential clinical applications as sodium-sparing diuretics, or 'urearetics,' in edema from different etiologies, such as congestive heart failure and cirrhosis, as well as in syndrome of inappropriate antidiuretic hormone (SIADH). High-throughput screening of drug-like small molecules has identified UT-A and UT-B inhibitors with nanomolar potency. Inhibitors have been identified with different UT-A versus UT-B selectivity profiles and putative binding sites on UT proteins. Studies in rodent models support the utility of UT inhibitors in reducing urinary concentration, though testing in clinically relevant animal models of edema has not yet been done.

Substituted 4-methylcoumarin inhibitors of SLC26A3 (DRA) for treatment of constipation and hyperoxaluria.

SLC26A3, also known as downregulated in adenoma (DRA), is an anion (Cl-, HCO3- and oxalate) exchanger in the luminal membrane of intestinal epithelial cells. Loss of DRA function in mice and humans causes congenital chloride-losing diarrhea and reduces urinary excretion of oxalate, a major constituent of kidney stones. Thus, inhibition of DRA is a potential treatment approach for constipation and calcium oxalate kidney stones. High-throughput screening previously identified 4,8-dimethylcoumarins (4a-4c) as DRA inhibitors, with lead candidate 4b having an IC50 of 40-50 nM for DRA inhibition. Here, we explored the effects of varying substituents at the 8-position, and replacing 8-methyl by 5-methyl (4e-4h). A focused library of 17 substituted compounds (4d-4t) was synthesized with good yield and purity. Compounds were tested for DRA inhibition potency using Fischer rat thyroid cells stably expressing DRA and a halide-sensitive YFP. Structure-activity analysis revealed that 8-bromo- (4m-4p) and 8-fluoro-coumarins (4q-4t) were slightly less potent than the corresponding 8-chloro analogs, demonstrating that the size of methyl or chloro substituents at the coumarin 8 position affects the potency. An analog containing 8-chlorocoumarin (4k) had ∼2-fold improved potency (IC50 25 nM) compared with the original lead candidate 4b. 5,8-Dimethylcoumarins were active against DRA, but with much lower potency than 4,8-disubstituted coumarins. In mice, orally administered 4k at 10 mg kg-1 reduced constipation and normalized stool water content in a loperamide-induced constipation model with comparable efficacy to 4b. Pharmacokinetic analysis of orally administered 4k at 10 mg kg-1 in mice indicated serum levels of >10 µM for at least six hours after single dose. This study expands SAR knowledge of 4,8-disubstituted coumarin inhibitors of DRA as novel drug candidates for constipation and kidney stones.

DRA involvement in linaclotide stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

A small-molecule screen yields idiotype-specific blockers of neuromyelitis optica immunoglobulin G binding to aquaporin-4.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to AQP4 on astrocytes. A screen was developed to identify inhibitors of NMO-IgG-dependent, complement-dependent cytotoxicity. Screening of 50,000 synthetic small molecules was done using CHO cells expressing human AQP4 and a human NMO recombinant monoclonal antibody (rAb-53). The screen yielded pyrano[2,3-c]pyrazoles that blocked rAb-53 binding to AQP4 and prevented cytotoxicity in cell culture and spinal cord slice models of NMO. Structure-activity analysis of 82 analogs yielded a blocker with IC(50) ∼ 6 µm. Analysis of the blocker mechanism indicated idiotype specificity, as (i) pyrano[2,3-c]pyrazoles did not prevent AQP4 binding or cytotoxicity of other NMO-IgGs, and (ii) surface plasmon resonance showed specific rAb-53 binding. Antibody structure modeling and docking suggested a putative binding site near the complementarity-determining regions. Small molecules with idiotype-specific antibody targeting may be useful as research tools and therapeutics.

Small-molecule inhibitors of NMO-IgG binding to aquaporin-4 reduce astrocyte cytotoxicity in neuromyelitis optica.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of spinal cord and optic nerve caused by pathogenic autoantibodies (NMO-IgG) against astrocyte aquaporin-4 (AQP4). We developed a high-throughput screen to identify blockers of NMO-IgG binding to human AQP4 using a human recombinant monoclonal NMO-IgG and transfected Fisher rat thyroid cells stably expressing human M23-AQP4. Screening of ∼60,000 compounds yielded the antiviral arbidol, the flavonoid tamarixetin, and several plant-derived berbamine alkaloids, each of which blocked NMO-IgG binding to AQP4 without affecting AQP4 expression, array assembly, or water permeability. The compounds inhibited NMO-IgG binding to AQP4 in NMO patient sera and blocked NMO-IgG-dependent complement- and cell-mediated cytotoxicity with IC(50) down to ∼5 µM. Docking computations identified putative sites of blocker binding at the extracellular surface of AQP4. The blockers did not affect complement-dependent cytotoxicity caused by anti-GD3 antibody binding to ganglioside GD3. The blockers reduced by >80% the severity of NMO lesions in an ex vivo spinal cord slice culture model of NMO and in mice in vivo. Our results provide proof of concept for a small-molecule blocker strategy to reduce NMO pathology. Small-molecule blockers may also be useful for other autoimmune diseases caused by binding of pathogenic autoantibodies to defined targets.