Discovery of novel diarylamides as orally active diuretics targeting urea transporters.
ABSTRACT: 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, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H 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 1H 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 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.
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: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:Acute heart failure (AHF) is a potentially life-threatening clinical syndrome, usually requiring hospital admission. Often the syndrome is characterized by congestion, and is associated with long hospital admissions and high risk of readmission and further healthcare expenditure. Despite a limited evidence-base, diuretics remain the first-line treatment for congestion. Loop diuretics are typically the first-line diuretic strategy with some evidence that initial treatment with continuous infusion or boluses of high-dose loop diuretic is superior to an initial lower dose strategy. In patients who have impaired responsiveness to diuretics, the addition of an oral thiazide or thiazide-like diuretic to induce sequential nephron blockade can be beneficial. The use of intravenous low-dose dopamine is no longer supported in heart failure patients with preserved systolic blood pressure and its use to assist diuresis in patients with low systolic blood pressures requires further study. Mechanical ultrafiltration has been used to treat patients with heart failure and fluid retention, but the evidence-base is not robust, and its place in clinical practice is yet to be established. Several novel pharmacological agents remain under investigation.
Project description:Despite advances in medical therapy over the past few decades, the incidence of heart failure hospitalisation continues to rise. Diuretics are the most common therapy used to treat heart failure as they relieve congestion. However, there is a lack of guidance on how to best use these medications. Guidelines support the use of diuretics at the lowest clinically effective dose but do not specify a diuretic strategy beyond that. Here we review the diuretics available for treatment, potential mechanisms of diuretic resistance and ways to address this in the ambulatory setting, and review tools that have been developed to help guide diuretic use in the treatment of chronic heart failure.
Project description:Urea transporters (UT) are a family of transmembrane proteins that specifically transport urea. UT inhibitors exert diuretic activity without affecting electrolyte balance. The purpose of this study was to discover novel UT inhibitors and determine the inhibition mechanism.The primary screening urea transporter B (UT-B) inhibitory activity was conducted in a collection of 10 000 diverse small molecules using mouse erythrocyte lysis assay. After discovering a hit with a core structure of 1-phenylamino-4-phenylphthalazin, the UT-B inhibitory activity of 160 analogs were examined with a stopped-flow light scattering assay and their structure-activity relationship (SAR) was analyzed. The inhibition mechanism was further investigated using in silico assays.A phenylphthalazine compound PU1424, chemically named 5-(4-((4-methoxyphenyl) amino) phthalazin-1-yl)-2-methylbenzene sulfonamide, showed potent UT-B inhibition activity, inhibited human and mouse UT-B-mediated urea transport with IC50 value of 0.02 and 0.69 ?mol/L, respectively, and exerted 100% UT-B inhibition at higher concentrations. The compound PU1424 did not affect membrane urea transport in mouse erythrocytes lacking UT-B. Structure-activity analysis revealed that the analogs with methoxyl group at R4 and sulfonic amide at R2 position exhibited the highest potency inhibition activity on UT-B. Furthermore, in silico assays validated that the R4 and R2 positions of the analogs bound to the UT-B binding pocket and exerted inhibition activity on UT-B.The compound PU1424 is a novel inhibitor of both human and mouse UT-B with IC50 at submicromolar ranges. Its binding site is located at the So site of the UT-B structure.
Project description:OBJECTIVE:Fluid overload is common among critically ill patients and is associated with worse outcomes. We aimed to assess the effect of diuretics on urine output, vasopressor dose, acute kidney injury (AKI) incidence, and need for renal replacement therapies (RRT) among patients who receive vasopressors. PATIENTS AND METHODS:This is a single-center retrospective study of all adult patients admitted to the intensive care unit between January 2006 and December 2016 and received >6 hours of vasopressor therapy and at least one concomitant dose of diuretic. We excluded patients from cardiac care units. Hourly urine output and vasopressor dose for 6 hours before and after the first dose of diuretic therapy was compared. Rates of AKI development and RRT initiation were assessed with a propensity-matched cohort of patients who received vasopressors but did not receive diuretics. RESULTS:There was an increasing trend of prescribing diuretics in patients receiving vasopressors over the course of the study. We included 939 patients with median (IQR) age of 68(57, 78) years old and 400 (43%) female. The average hourly urine output during the first six hours following time zero in comparison with average hourly urine output during the six hours prior to time zero was significantly higher in diuretic group in comparison with patients who did not receive diuretics [81 (95% CI 73-89) ml/h vs. 42 (95% CI 39-45) ml/h, respectively; p<0.001]. After propensity matching, the rate of AKI within 7 days of exposure and the need for RRT were similar between the study and matched control patients (66 (15.6%) vs. 83 (19.6%), p = 0.11, and 34 (8.0%) vs. 37 (8.7%), p = 0.69, respectively). Mortality, however, was higher in the group that received diuretics. Ninety-day mortality was 191 (45.2%) in the exposed group VS 156 (36.9%) p = .009. CONCLUSIONS:While the use of diuretic therapy in critically ill patients receiving vasopressor infusions augmented urine output, it was not associated with higher vasopressor requirements, AKI incidence, and need for renal replacement therapy.
Project description:The effectiveness of the clinical strategy of empiric potassium supplementation in reducing the frequency of adverse clinical outcomes in patients receiving loop diuretics is unknown. We sought to examine the association between empiric potassium supplementation and 1) all-cause death and 2) outpatient-originating sudden cardiac death (SD) and ventricular arrhythmia (VA) among new starters of loop diuretics, stratified on initial loop diuretic dose.We conducted a one-to-one propensity score-matched cohort study using 1999-2007 US Medicaid claims from five states. Empiric potassium supplementation was defined as a potassium prescription on the day of or the day after the initial loop diuretic prescription. Death, the primary outcome, was ascertained from the Social Security Administration Death Master File; SD/VA, the secondary outcome, from incident, first-listed emergency department or principal inpatient SD/VA discharge diagnoses (positive predictive value = 85%).We identified 654,060 persons who met eligibility criteria and initiated therapy with a loop diuretic, 27% of whom received empiric potassium supplementation (N = 179,436) and 73% of whom did not (N = 474,624). The matched hazard ratio for empiric potassium supplementation was 0.93 (95% confidence interval, 0.89-0.98, p = 0.003) for all-cause death. Stratifying on initial furosemide dose, hazard ratios for empiric potassium supplementation with furosemide < 40 and ? 40 milligrams/day were 0.93 (0.86-1.00, p = 0.050) and 0.84 (0.79-0.89, p < 0.0001). The matched hazard ratio for empiric potassium supplementation was 1.02 (0.83-1.24, p = 0.879) for SD/VA.Empiric potassium supplementation upon initiation of a loop diuretic appears to be associated with improved survival, with a greater apparent benefit seen with higher diuretic dose. If confirmed, these findings support the use of empiric potassium supplementation upon initiation of a loop diuretic.
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:Background In acute decompensated heart failure, guidelines recommend increasing loop diuretic dose or adding a thiazide diuretic when diuresis is inadequate. We set out to determine the adverse events associated with a diuretic strategy relying on metolazone or high-dose loop diuretics. Methods and Results Patients admitted to 3 hospitals using a common electronic medical record with a heart failure discharge diagnosis who received intravenous loop diuretics were studied in a propensity-adjusted analysis of all-cause mortality. Secondary outcomes included hyponatremia (sodium <135 mE q/L), hypokalemia (potassium <3.5 mE q/L) and worsening renal function (a ?20% decrease in estimated glomerular filtration rate). Of 13 898 admissions, 1048 (7.5%) used adjuvant metolazone. Metolazone was strongly associated with hyponatremia, hypokalemia, and worsening renal function ( P<0.0001 for all) with minimal effect attenuation following covariate and propensity adjustment. Metolazone remained associated with increased mortality after multivariate and propensity adjustment (hazard ratio=1.20, 95% confidence interval 1.04-1.39, P=0.01). High-dose loop diuretics were associated with hypokalemia and hyponatremia ( P<0.002) but only worsening renal function retained significance ( P<0.001) after propensity adjustment. High-dose loop diuretics were not associated with reduced survival after multivariate and propensity adjustment (hazard ratio=0.97 per 100 mg of IV furosemide, 95% confidence interval 0.90-1.06, P=0.52). Conclusions During acute decompensated heart failure, metolazone was independently associated with hypokalemia, hyponatremia, worsening renal function and increased mortality after controlling for the propensity to receive metolazone and baseline characteristics. However, under the same experimental conditions, high-dose loop diuretics were not associated with hypokalemia, hyponatremia, or reduced survival. The current findings suggest that until randomized control trial data prove otherwise, uptitration of loop diuretics may be a preferred strategy over routine early addition of thiazide type diuretics when diuresis is inadequate.
Project description:Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors.