Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats.
ABSTRACT: 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??M. 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.
Project description:Urea transporter (UT) proteins, including UT-A in kidney tubule epithelia and UT-B in vasa recta microvessels, facilitate urinary concentrating function. A screen for UT-A inhibitors was developed in MDCK cells expressing UT-A1, water channel aquaporin-1, and YFP-H148Q/V163S. An inwardly directed urea gradient produces cell shrinking followed by UT-A1-dependent swelling, which was monitored by YFP-H148Q/V163S fluorescence. Screening of ~90,000 synthetic small molecules yielded four classes of UT-A1 inhibitors with low micromolar half-maximal inhibitory concentration that fully and reversibly inhibited urea transport by a noncompetitive mechanism. Structure-activity analysis of >400 analogs revealed UT-A1-selective and UT-A1/UT-B nonselective inhibitors. Docking computations based on homology models of UT-A1 suggested inhibitor binding sites. UT-A inhibitors may be useful as diuretics ("urearetics") with a mechanism of action that may be effective in fluid-retaining conditions in which conventional salt transport-blocking diuretics have limited efficacy.
Project description: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, ?-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 ?M. 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 ?-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.
Project description:Urea transporters (UTs) are transmembrane proteins selectively permeable to urea and play an important role in urine concentration. UT-knockout mice exhibit the urea-selective urine-concentrating defect, without affecting electrolyte balance, suggesting that UT-B inhibitors have the potential to be developed as novel diuretics. In this study, we characterized a novel compound 5-ethyl-2-methyl-3-amino-6-methylthieno[2,3-b]pyridine-2,5-dicarboxylate (CB-20) with UT inhibitory activity as novel diuretics with excellent pharmacological properties. This compound was discovered based on high-throughput virtual screening combined with the erythrocyte osmotic lysis assay. Selectivity of UT inhibitors was assayed using transwell chambers. Diuretic activity of the compound was examined in rats and mice using metabolic cages. Pharmacokinetic parameters were detected in rats using LC-MS/MS. Molecular docking was employed to predict the potential binding modes for the CB-20 with human UT-B. This compound dose-dependently inhibited UT-facilitated urea transport with IC50 values at low micromolar levels. It exhibited nearly equal inhibitory activity on both UT-A1 and UT-B. After subcutaneous administration of CB-20, the animals showed polyuria, without electrolyte imbalance and abnormal metabolism. CB-20 possessed a good absorption and rapid clearance in rat plasma. Administration of CB-20 for 5 days did not cause significant morphological abnormality in kidney or liver tissues of rats. Molecular docking showed that CB-20 was positioned near several residues in human UT-B, including Leu364, Val367, and so on. This study provides proof of evidence for the prominent diuretic activity of CB-20 by specifically inhibiting UTs. CB-20 or thienopyridine analogs may be developed as novel diuretics.
Project description:Urea transporter A (UT-A) isoforms encoded by the Slc14a2 gene are expressed in kidney tubule epithelial cells, where they facilitate urinary concentration. UT-A1 inhibition is predicted to produce a unique salt-sparing diuretic action in edema and hyponatremia. Here we report the discovery of 1,2,4-triazoloquinoxalines and the analysis of 37 synthesized analogues. The most potent compound, 8ay, containing 1,2,4-triazolo[4,3- a]quinoxaline-substituted benzenesulfonamide linked by an aryl ether, rapidly and reversibly inhibited UT-A1 urea transport by a noncompetitive mechanism with IC50 ? 150 nM; the IC50 was ?2 ?M for the related urea transporter UT-B encoded by the Slc14a1 gene. Molecular modeling suggested a putative binding site on the UT-A1 cytoplasmic domain. In vitro metabolism showing quinoxaline ring oxidation prompted the synthesis of metabolically stable 7,8-difluoroquinoxaline analogue 8bl, which when administered to rats produced marked diuresis and reduced urinary osmolality. 8bl has substantially improved UT-A1 inhibition potency and metabolic stability compared with prior compounds.
Project description:Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, <i>N</i>-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (<b>1H</b>), with excellent <i>in vitro</i> UT inhibitory activity at the submicromolar level<i>.</i> The half maximal inhibitory concentrations of <b>1H</b> against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 ?mol/L, respectively. Further investigation suggested that 8 ?mol/L <b>1H</b> more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of <b>1H</b> at a dose of 100 mg/kg showed superior diuretic effect <i>in vivo</i> without causing electrolyte imbalance in rats. Additionally, <b>1H</b> did not exhibit apparent toxicity <i>in vivo</i> and <i>in vitro</i>, and possessed favorable pharmacokinetic characteristics. <b>1H</b> shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.
Project description:Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.
Project description:Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.
Project description:Kidney urea transporters are targets for development of small-molecule inhibitors with action as salt-sparing diuretics. A cell-based, functional high-throughput screen identified 2,7-bisacetamido fluorenone 3 as a novel inhibitor of urea transporters UT-A1 and UT-B. Here, we synthesized twenty-two 2,7-disubstituted fluorenone analogs by acylation. Structure-activity relationship analysis revealed: (a) the carbonyl moiety at C9 is required for UT inhibition; (b) steric limitation on C2, 7-substituents; and (c) the importance of a crescent-shape structure. The most potent fluorenones inhibited UT-A1 and UT-B urea transport with IC50 ~ 1 ?M. Analysis of in vitro metabolic stability in hepatic microsomes indicated metabolism of 2,7-disubstituted fluorenones by reductase and subsequent elimination. Computational docking to a homology model of UT-A1 suggested UT inhibitor binding to the UT cytoplasmic domain at a site that does not overlap with the putative urea binding site.
Project description:The urea channel Slc14a2 (or UT-A1) mediates vasopressin-regulated urea transport across the inner medullary collecting duct (IMCD). Previously, UT-A1 was found to present in a high molecular weight complex, suggesting UT-A1 is involved in certain protein-protein interactions. The present study sought to identify the proteins that interact with UT-A1 in this complex for a better understanding of how UT-A1 is regulated. Rat IMCD suspensions were treated with or without V2 receptor agonist, dDAVP, followed by in-cell crosslinking using BSOCOES and detergent solubilization. Immunoprecipitation using Dynabeads coated with UT-A1 specific antibody successfully pulled down the UT-A1 proteins. In-gel digestion protocol was carried out to prepare samples for liquid chromatographic mass spectrometry analysis of tryptic peptides using a Velos-Orbitrap mass spectrometer. The peptides passing stringent spectral quality thresholds were quantified (label-free) to identify those with (UTA-1 antibody/preimmune IgG) >4. A total of 128 UT-A1 interacting proteins were identified. Gene Ontology analysis maps the distribution of these proteins throughout major cell compartments: endoplasmic reticulum, Golgi, endosomes, cytosol and plasma membrane. Among them are four protein kinases (Cdc42bpb, Phkb, Camk2d, Mtor) that play roles in vasopressin-regulated phosphorylation of UT-A1. Non-label quantification was also performed to determine the stoichiometry of UT-A3 with UT-A1, the result does not support an oligomeric complex formation of UT-A1/A3. In conclusion, we have provided a refined list of UT-A1 binding proteins which can be useful for further analysis of the vasopressin signaling pathway in regulation of UT-A1 in IMCD.
Project description:The UT-A1 urea transporter plays an important role in the urinary concentration mechanism. However, the molecular mechanisms regarding UT-A1 trafficking, endocytosis, and degradation are still unclear. In this study, we identified the small GTPase Rab14 as a binding partner to the C terminus of UT-A1 in a yeast 2-hybrid assay. Interestingly, UT-A1 binding is preferential for the GDP-bound inactive form of Rab14. Coinjection of Rab14 in Xenopus oocytes results in a decrease of UT-A1 urea transport activity, suggesting that Rab14 acts as a negative regulator of UT-A1. We subsequently found that Rab14 reduces the cell membrane expression of UT-A1, as evidenced by cell surface biotinylation. This effect is blocked by chlorpromazine, an inhibitor of the clathrin-mediated endocytic pathway, but not by filipin, an inhibitor of the caveolin-mediated endocytic pathway. In kidney, Rab14 is mainly expressed in IMCD epithelial cells with a pattern identical to UT-A1 expression. Consistent with its role in participating in clathrin-mediated endocytosis, Rab14 localizes in nonlipid raft microdomains and codistributes with Rab5, a marker of the clathrin-mediated endocytic pathway. Taken together, our study suggests that Rab14, as a novel UT-A1 partner, may have an important regulatory function for UT-A1 urea transport activity in the kidney inner medulla.