Cristina Esteva-Font

Aquaporin-1 gene deletion reduces breast tumor growth and lung metastasis in tumor-producing MMTV-PyVT mice

Aquaporin 1 (AQP1) is a plasma membrane water‐transporting protein expressed strongly in tumor microvascular endothelia. We previously reported impaired angiogenesis in implanted tumors in AQP1‐deficient mice and reduced migration of AQP1‐deficient endothelial cells in vitro. Here, we investigated the consequences of AQP1 deficiency in mice that spontaneously develop well‐differentiated, luminal‐type breast adenomas with lung metastases [mouse mammary tumor virus‐driven polyoma virus middle T oncogene (MMTV‐PyVT)]. AQP1+/+ MMTV‐PyVT mice developed large breast tumors with total tumor mass 3.5 ± 0.5 g and volume 265 ± 36 mm3 (se, 11 mice) at age 98 d. Tumor mass (1.6±0.2 g) and volume (131±15 mm3, 12 mice) were greatly reduced in AQP1–/– MMTV‐PyVT mice (P<0.005). CD31 immunofluorescence showed abnormal microvascular anatomy in tumors of AQP1–/– MMTV‐PyVT mice, with reduced vessel density. HIF‐1α expression was increased in tumors in AQP1–/– MMTV‐PyVT mice. The number of lung metastases (5± 1/mouse) was much lower than in AQP1+/+ MMTV‐PyVT mice (31±8/mouse, P<0.005). These results implicate AQP1 as an important determinant of tumor angiogenesis and, hence, as a potential drug target for adjuvant therapy of solid tumors.—Esteva‐Font, C., Jin, B.‐J., Verkman, A. S. Aquaporin‐1 gene deletion reduces breast tumor growth and lung metastasis in tumor‐producing MMTV‐PyVT mice. FASEB J. 28, 1446–1453 (2014). www.fasebj.org

Urea transporter proteins as targets for small-molecule diuretics

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.

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 UTA. 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.—Esteva‐Font, C., Cil, O., Phuan, P.‐W., Su, T., Lee, S., Anderson, M. O., Verkman, A. S. Diuresis and reduced urinary osmolality in rats produced by small‐molecule UT‐A‐ selective urea transport inhibitors. FASEB J. 28, 3878‐3890 (2014). www.fasebj.org

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.

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 hours, 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.

Discovery, synthesis and structure–activity analysis of symmetrical 2,7-disubstituted fluorenones as urea transporter inhibitors

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 μM. 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.

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).

O-071 Salt-sparing Diuretic Action Of A Urea Analogue Inhibitor Of Urea Transporters Ut-a And Ut-b In Rats

Background and aims Urea is end metabolite of protein metabolism and is crucial for generation of hypertonic renal medulla. Urea transport to medullary interstitium is facilitated by urea transporters (UT-A and UT-B). UT inhibitors have potential use as a novel class of salt-sparing diuretics. Methods UT inhibitor effect of urea analogue dimethylthiourea (DMTU) was investigated and characterised in cell-based assays. Kidney function tests were investigated in both 1-day and 7-days DMTU-treated (500 mg/kg ip initially, then 125 mg/kg ip every 12h) rats. The effect of DMTU on maximum urine concentrating function was investigated in low (6%) and normal (20%) protein-fed rats. Results DMTU non-competitively inhibited UT-A and UT-B with IC50 of ~3 mM. Following 500 mg/kg ip injection, plasma DMTU concentration was initially 10 mM (plasma elimination t1/2 ~10 h) and urine DMTU concentration was >20 mM for 12 h. DMTU-treated rats showed reversible, sustained reduction in urine osmolality (>60%) and 3-fold increased daily urine output. DMTU increased renal electrolyte-free water excretion without altering solute excretion. DMTU impaired maximum urinary concentrating function only in normal protein-fed rats. Methylurea, a non-UT inhibitor urea analogue, had no effect on either urine volume or osmolality. DMTU-treated rats had greater diuresis and much reduced urinary salt loss compared to that of furosemide-treated rats. Conclusions These results establish a rat model of sustained UT inhibition and demonstrate remarkable diuretic efficacy of UT inhibition. Prominent effect of UT inhibitors on net renal water excretion implies a novel therapeutic strategy for treatment of oedema in hypervolemic diseases.