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Published as doi: 10.1096/fj.06-6979com.
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(The FASEB Journal. 2007;21:551-563.)
© 2007 FASEB

Urearetics: a small molecule screen yields nanomolar potency inhibitors of urea transporter UT-B

Marc H. Levin, Ricardo de la Fuente and A. S. Verkman1

Departments of Medicine and Physiology, Cardiovascular Research Institute, Graduate Group in Biophysics, University of California, San Francisco, California, USA

1Correspondence: 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143-0521, USA. E-mail: verkman{at}itsa.ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional studies in knockout mice indicate a critical role for urea transporters (UTs) in the urinary concentrating mechanism and in renal urea clearance. However, potent and specific urea transport blockers have not been available. Here, we used high-throughput screening to discover high-affinity, small molecule inhibitors of the UT-B urea transporter. A collection of 50,000 diverse, drug-like compounds was screened using a human erythrocyte lysis assay based on UT-B-facilitated acetamide transport. Primary screening yielded ~30 UT-B inhibitors belonging to the phenylsulfoxyoxazole, benzenesulfonanilide, phthalazinamine, and aminobenzimidazole chemical classes. Screening of ~700 structurally similar analogs gave many active compounds, the most potent of which inhibited UT-B urea transport with an EC50 of ~10 nM, and ~100% inhibition at higher concentrations. Phenylsulfoxyoxazoles and phthalazinamines also blocked rodent UT-B and had good UT-B vs. UT-A specificity. The UT-B inhibitors did not reduce aquaporin-1 (AQP1)-facilitated water transport. In AQP1-null erythrocytes, "chemical UT-B knockout" by UT-B inhibitors reduced by ~3-fold UT-B-mediated water transport, supporting an aqueous pore pathway through UT-B. UT-B inhibitors represent a new class of diuretics, "urearetics," which are predicted to increase renal water and solute clearance in water-retaining states.—Levin, M. H., de la Fuente, R., Verkman, A. S. Urearetics: a small molecule screen yields nanomolar potency inhibitors of urea transporter UT-B.


Key Words: urea transport • UT-B • kidney • diuretic • drug discovery • small-molecule discovery • aquaporin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UREA IS GENERATED as the major end product of hepatic nitrogen metabolism and is excreted primarily by the kidney. Urea and NaCl are the major solutes in the hyperosmolar renal medulla. In the antidiuretic kidney, urea is greatly concentrated with respect to plasma (up to 100-fold in humans and 250-fold in rodents; ref. 1 ) by countercurrent multiplication and exchange mechanisms. Of central importance to these mechanisms is intrarenal urea recycling, which requires facilitated urea transport by molecular urea transporters (UTs). UTs comprise two major subfamilies encoded by different genes (UT-A and UT-B, reviewed in refs. 2 , 3 ). In the kidney, a single UT-B isoform is expressed in vasa recta while several splice variant UT-A-type transporters are expressed in kidney tubule epithelia (reviewed in ref. 4 ).

Phenotype analysis of mice separately lacking vasa recta UT-B or inner medullary collecting duct UT-A1/3 implicated UT involvement in the formation of concentrated urine and in renal urea clearance (5 6 7) . The UT-B knockout mice generated by our lab manifest a urea-selective urinary concentrating defect associated with urinary hypo-osmolality and increased renal urea clearance (5) . UT-B is also expressed outside the kidney, most notably and in highest abundance in red blood cell (RBC) membranes. Loss-of-function human UT-B mutations result in greatly reduced RBC urea permeability and a mild urinary concentrating defect (8 , 9) . The mouse and human phenotypes in UT-B deficiency support the use of UT-B inhibitors as unique urea clearance-enhancing diuretics, which we call "urearetics." However, studies in genetically modified mice are potentially confounded by changes in the expression of other proteins such as membrane transporters. "Chemical knockout" by potent and specific small-molecule inhibitors would largely obviate these concerns.

The only available UT inhibitors include the nonspecific membrane intercalating agent phloretin (acting at >0.5 mM), urea analogs such as thiourea, methylurea, and dimethylurea (acting at 50–100 mM, ref. 10 ), and chemically modified urea analogs (acting irreversibly at 30–100 µM, ref. 11 ). The goal of this study was to identify potent small-molecule UT inhibitors. Since no structural information about UTs is available, nor are existing UT inhibitors useful for lead-based discovery, our strategy to identify UT inhibitors was high-throughput screening of a collection of drug-like small molecules with high chemical diversity.

Our high-throughput screening strategy, as diagrammed in Fig. 1 , was based on measurement of lysis of human RBCs after imposing a large, outwardly directed gradient of acetamide, a urea analog that is transported efficiently by UT-B. RBCs were chosen for the primary screening assay because of their 1) availability in large quantities; 2) high urea permeability resulting from strong expression of only one UT, UT-B; and 3) high aquaporin (AQP)-mediated water permeability (for a lysis-based assay). We used human RBCs for primary screening with the goal of discovering inhibitors that could be further developed as therapeutics. In the assay, imposing a large, outwardly directed gradient of acetamide causes cell swelling, which is limited by UT-B-facilitated acetamide efflux. Under appropriate conditions, UT-B inhibition slows acetamide efflux and increases cell lysis, which was assayed by near-infrared light scattering. Our cell-based lysis assay was validated and used to discover novel small-molecule UT-B inhibitors, which were then optimized by screening of chemical analogs.


Figure 1
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  		Figure 1. Erythrocyte osmotic lysis assay for UT-B inhibitor discovery. A) Human RBCs expressing water and urea channels (AQP1 and UT-B) are preloaded with urea or a urea analog, such as acetamide. After replacement of the external buffer with urea/acetamide-free isosmolar solution, water entry results in cell swelling, which is limited by UT-B-mediated urea/acetamide efflux. Under optimized assay conditions, UT-B-facilitated urea/acetamide prevents osmotic lysis (top), whereas UT-B inhibition impairs urea/acetamide exit resulting in substantial lysis (bottom). B) Biphasic cell volume changes in the lysis assay. Increased RBC volume beyond a threshold results in lysis. The dashed curve shows the hypothetical time course of RBC volume if lysis had not occurred.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of human and mouse blood
Human venous blood obtained from a single donor (M.H.L.) was collected into Vacutainers coated with sodium heparin (Becton-Dickinson, Franklin Lakes, NJ, USA), stored at 4°C, and used within 48 h of collection. Human procedures were approved by the University of California, San Francisco Committee on Human Research. Whole mouse blood was collected from 8–12 wk-old (25–35 g) wild-type (WT), AQP1-null, or UT-B-null mice (5 , 12) in a CD1 genetic background by orbital puncture after subcutaneous injection with sodium heparin (150 USP units). Animal protocols were approved by the University of California, San Francisco Committee on Animal Research.

Instrumentation and compounds for high-throughput screening
Screening was carried out using a Beckman Coulter (Fullerton, CA, USA) integrated system consisting of a 3-meter robotic arm, microplate carousel, liquid handling work station with parallel 96-well solution mixing and transfer (Biomek FX, Beckman Coulter), plate sealer, and two fluorescence plate readers (FLUOstar Optima; BMG LABTECH Gmbh; Durham, NC, USA), each equipped with a 710 ± 5 nm absorption filter (Chroma, Rockingham, VT, USA). Primary screening was done using a collection of 50,000 diverse, drug-like compounds (>90% with molecular size 250–500 Da) from a commercial source (ChemDiv Inc., San Diego, CA, USA). 96-well plates containing four compounds per well (each 2.5 mM) were prepared for screening and stored frozen in DMSO. Plates with one compound per well (at 10 mM in DMSO) were stored separately and used later to identify and characterize active compounds.

Screening procedures
At the time of the assay, whole human blood was diluted to a hematocrit of 1% in hyperosmolar PBS containing 1.25 M acetamide and 5 mM glucose (1550 mOsm, measured using freezing point depression osmometry; Precision Systems, Natick, MA, USA). Identical assay results were obtained when washed/centrifuged RBCs were used instead of whole blood. RBC suspensions were maintained at room temperature for up to 2 h by periodic pipette mixing. 99 µl from a reservoir containing the RBC suspension was added to each well of a 96-well round-bottom microplate (FALCON, Becton Dickinson), to which test compounds were added (1 µl, 25 µM final compound concentration, 1% final DMSO concentration). After 6 min incubation, 20 µl of the RBC suspension was added rapidly to each well of a 96-well black-walled plate (Costar, Corning, NY, USA) containing 180 µl isosmolar buffer (PBS containing 1% DMSO) in each well. Vigorous mixing was achieved by repeated pipetting.

RBC lysis was quantified from a single time point measurement of absorbance at 710 nm wavelength (13 , 14) , made within 5 min after hypo-osmolar shock. Absorbance values were stable for at least 1 h. Each assay plate contained eight negative no-lysis controls (isotonic buffer; PBS+1.25 M acetamide with 1% DMSO) and eight positive full-lysis controls (distilled H20 with 1% DMSO) that were mixed with DMSO vehicle-treated blood. The statistical Z' factor, indicating "goodness of the assay" (15) , was computed using data from test plates as defined by Z' = 1 – 3·[ (SDpos + SDneg)/(Apos – Aneg)], where SDi and Ai are the standard deviation and mean absorbance values for positive (pos) and negative (neg) controls. The percentage of RBC lysis in each test well of a given plate was calculated using control values from the same plate as % lysis = 100%·(Aneg – Atest)/(Aneg – Apos), where Atest is the absorbance value from a test well. During assay optimization, some test wells were incubated with the nonspecific UT-B inhibitor phloretin (0.7 mM, dissolved at 100x in DMSO stock solution) as an additional positive control. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.

Stopped-flow measurement of RBC urea and water permeability
RBC urea and water permeabilities were assayed by stopped-flow light scattering using a Hi-Tech Sf-51 instrument (Wiltshire, UK). To measure urea permeability, dilutions of whole blood (human or mouse) in PBS (hematocrit ~0.5%) were incubated with test compounds for 5 min, then subjected to a 250 mM inwardly directed gradient of urea. After an initial osmotic shrinking phase, the kinetics of increasing cell volume caused by urea influx was measured as the time course of 90° scattered light intensity at 530 nm, with increasing cell volume resulting in reduced scattered light intensity. As a positive control, 0.7 mM phloretin was added to the RBC suspension prior to stopped-flow experiments. Measurements of water permeability were carried out similarly, using sucrose (cell-impermeant) instead of urea to establish a 250 mM osmotic gradient. As a positive control, HgCl2 (0.3 mM) was added to the RBC suspension prior to stopped-flow measurements. Osmotic water permeability coefficients (Pf) were computed from light scattering data as described (16) .

Inhibitor optimization by analysis of structure-activity relationship (SAR)
Approximately 700 commercially available analogs (ChemDiv Inc. and Asinex; Moscow, Russia) of active compounds identified in the primary screen were tested against human and mouse UT-B using the RBC lysis assay. Dose-response experiments were performed for some compounds (with human and/or mouse blood) by the lysis assay. EC50 was calculated by nonlinear regression to the equation: % lysis = % lysismin + (% lysismax.[inh]H)/(EC50H + [inh]H), where [inh] is inhibitor concentration and H is the Hill coefficient.

Determination of EC50 for UT-B inhibition from stopped-flow measurements
EC50 for inhibition of RBC urea transport was determined independently by comparing stopped-flow light scattering curves to a model of cell shrinking-swelling. For stopped-flow experiments, a 100 mM gradient of urea (for human RBCs) or N-methylurea (for mouse RBCs) was used to minimize competition effects (apparent urea and N-methylurea affinities at 23°C are ~200 and 100 mM, respectively; ref. 10 ). N-methylurea, with >2-fold slower RBC permeability than urea, was used in mouse studies to better resolve overlapping water and urea transport kinetics.

The two coupled differential equations describing water efflux and solute influx in response to externally added urea or methylurea were numerically integrated using the forward Euler method ({Delta}t=0.01 s) to model the biphasic changes in cell volume observed experimentally. Computations performed using a smaller time step ({Delta}t=0.001 s) gave similar results, confirming an adequacy of the 0.01 s time step. Water flux, Jv (in cm3/s), across erythrocyte membranes is Jv = –Pf·S·vw·[(Ie – Ic(i)) + (Ue – Uc(i)) ]; solute flux, Js (in mol/s), is: Ps·S·(Ue – Uc(i)). Permeability coefficients (Pf and Ps) are expressed in units of cm/s, cell surface area (S) in cm2, extracellular (e) and cellular (c) concentrations of impermeant (I) and urea/methylurea (U) solute in mol/cm3, and vw is 18 mol/cm3. Initial conditions were Ie = Ic(0) = 2.9 x 10–4 mol/cm3, Ue = 10–4 mol/cm3, and Uc(0) = 0. For each time step, a new cell volume (normalized to the initial size; V(i+1)/V(0)) and a new cell-permeant concentration (U(i+1)) were calculated from V(i+1)/V(0) = V(i)/V(0) – {Delta}t.Pf·(S/V(0))·vw·[ Ie (1 – V(i)/V(0)) + (Ue – Uc(i)) ] and Uc(i+1) = Uc(i)/V(0) – {Delta}t·Us·(S/V(0))·vw· (Ue – Uc(i)). Normalized cell volume was assumed to be inversely proportional to scattered light intensity. The product of Pf and the surface area-to-volume ratio (S/V(0)) was determined to be 3.4 x 102 s–1 and 8.5 x 102 s–1 for human and mouse erythrocytes, respectively, from water permeability measurements. Ps was varied to reproduce experimental data, and EC50 was computed using nonlinear regression (as above) of Ps vs. [inh] data. In some experiments, to determine the sidedness of inhibitor action, compounds were added only to the urea-containing solution (at concentrations 2-fold higher than their EC50) before mixing with RBCs in stopped-flow measurements. To assay for reversibility, compounds (at concentrations 4-fold higher than their EC50) were added to RBCs for 10 min, then washed by centrifugation prior to stopped-flow measurements.

Assay of UT-A1-facilitated urea transport
MDCK cells stably transfected with rat UT-A1 (MDCK-UT-A1, ref. 17 ) were generously provided by Dr. Jeffrey Sands. Cells were grown in Dulbecco’s modified Eagle medium with bicarbonate and supplemented with 10% FBS, 25 mM HEPES buffer, penicillin G (100 U/ml), streptomycin (100 µg/ml), and hygromycin (500 µg/ml). For assay of urea flux, cells were grown on 12 mm collagen-coated Transwell inserts (0.4 µm pore size; Costar) as described (17 , 18) . Inserts were incubated in hygromycin-free medium for 1 h in a 5% CO2 tissue culture incubator (37°C), then 2 x 105 cells/cm2 were loaded onto each insert. Cells were used after culture for 4 days in hygromycin-free medium, at which time they formed tight monolayers (transepithelial resistance 500–600 {Omega} cm2).

UT-A1-facilitated urea flux in the basolateral-to-apical direction across unstimulated and forskolin-stimulated MDCK-UT-A1 cell layers was measured in response to a 15 mM urea gradient. Experiments were carried out in 12-well plates in which PBS, containing either DMSO vehicle or forskolin, with or without UT-B inhibitor, was added to both the apical-facing (0.2 ml) and basal-facing (1 ml) surfaces of cells on the porous filters. Cultures were incubated in the absence of urea for 30 min at 37°C, then the basal-facing solution was replaced by PBS (containing the same components) with 15 mM urea. Samples (5 µl) of apical fluid were collected at specified times during incubation at 37°C for assay of urea concentration using a commercial kit based on chromogenic urea complexation at 520 nm wavelength (QuantichromTM Urea Assay Kit, BioAssay Systems, Hayward, CA, USA). Forskolin (10 µM), with or without UT-B transport inhibitors, was added from 1000x DMSO stock solutions (0.2% final DMSO). Inhibition of UT-A1-mediated transport was computed as % inhibition = 100%·(Aforsk–Atest)/(Aforsk–Aphlor). Aforsk and Aphlor are averaged absorbance values (at 520 nm) for cultures treated with forskolin and forskolin + phloretin, respectively, and Atest are values from cultures treated with forskolin + test compound.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Assay development and validation
An RBC lysis assay was developed for identification of small-molecule UT-B inhibitors. As shown in Fig. 1A and explained in the opening paragraphs of this paper, UT-B inhibition was assayed by increased RBC lysis when urea- or acetamide-loaded RBCs were rapidly diluted into PBS. Conditions were optimized for high-throughput screening to give a robust assay with high sensitivity and a low false-positive rate. Absorbance at 710 nm was measured as a readout of RBC lysis to minimize interference by test compounds and hemoglobin. Urea and a panel of small urea-like solutes (formamide, N-methylurea, acetamide, proprianamide, butyramide, and isobutyramide) were evaluated as the loading solute based on their UT-B transport kinetics. Acetamide was selected because its equilibration in RBCs was ~2-fold slower than water, which is optimal in an osmotic lysis assay, and because >95% of its transport in RBCs is UT-B-dependent (as determined by stopped-flow light scattering; data not shown).

The optimal acetamide loading concentration was determined to identify UT-B inhibitors in an automated 96-well format. Figure 2 A shows RBC lysis, measured by absorbance at 710 nm (O.D.710), as a function of the acetamide concentration used to load RBCs before mixing with acetamide-free buffer. Greater lysis, seen as reduced O.D.710, was found with increasing acetamide concentration, as expected. Fifty percent lysis was seen at ~1.6 M acetamide under control conditions (open circles) and at ~1.1 M when UT-B-facilitated acetamide transport was inhibited by phloretin (filled circles). We chose to use 1.25 M acetamide (dashed vertical line) for the assay to best distinguish between control vs. inhibited UT-B. Other technical considerations addressed during assay optimization included maintenance of RBC viability and uniform suspension, mixing conditions (rates, volumes, and pipette tip locations in wells), and incubation time/temperature. The goodness of the optimized assay was evaluated by screening plates containing positive and negative controls (0 and 100% lysis), which gave a good statistical Z' factor of 0.57 for the screen (Fig. 2B ).


Figure 2
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  		Figure 2. Identification of UT-B inhibitors by high-throughput screening. A) Effect of acetamide concentration on RBC osmotic lysis. Human RBC suspensions, loaded with indicated concentrations of acetamide, were diluted in acetamide-free buffer in the absence (open circles) or presence (filled circles) of 0.7 mM phloretin. RBC lysis was assayed by absorbance at 710 nm (O.D.710) (±SE, 4 wells per condition). The dashed line indicates the condition chosen for high-throughput screening. B) Frequency histogram of O.D.710 for positive and negative controls from eight 96-well plates, with Z' value shown. C) Frequency histogram of percent erythrocyte lysis for the primary screen in (12,500 test wells with 4 compounds per well; 50,000 compounds total), with the dashed line representing the criteria chosen to define hits. D) Left: Urea permeability measured from the kinetics of light scattering in response to a 250 mM inwardly directed urea gradient in the absence of inhibitor (control) or in the presence of 0.7 mM phloretin (positive control) or 5 µM of indicated compounds. Right: Osmotic water permeability of human RBCs measured by light scattering in response to a 250 mM inwardly directed sucrose gradient in the absence or presence of 0.3 mM HgCl2 (positive control) or 25 µM of UT-B inhibitors.

Identification and optimization of small-molecule UT-B inhibitors
A collection of 50,000 small, drug-like compounds with high chemical diversity was screened at 25 µM. Figure 2C shows the frequency histogram of O.D.710 values for all test compounds in the primary screen. Most compounds showed no significant UT-B inhibition based on little (<30%) RBC lysis. Approximately 100 compounds producing > 75% lysis were selected for further evaluation.

After confirming active compounds using the 96-well plate RBC lysis assay, bona fide urea transport inhibition was confirmed by stopped-flow light scattering from the kinetics of urea influx (RBC swelling) in response to an inwardly directed urea gradient. Rapid mixing of an RBC suspension with a hyperosmolar solution containing excess 250 mM urea produced rapid cell shrinking due to osmotic water efflux, followed by cell swelling as urea (and water) influx occurred. Thirty-two compounds in four distinct chemical structural classes were identified, which at 5 µM produced substantial (>95%) inhibition of UT-B-facilitated urea transport. Other compounds, with either much lower or no activity in the stopped-flow assay, may have had apparent UT-B inhibitory activity in the primary screen because of RBC toxicity and consequent partial lysis. Original stopped-flow urea transport data for one representative compound (at 5 µM) of each class is shown in Fig. 2D (left). Tracings from control (no inhibitors) and phloretin-treated RBCs are provided for comparison. The new compounds at 5 µM inhibited UT-B-facilitated urea transport in human RBCs by > 95%, which is as good as or better than that with 0.7 mM phloretin. Figure 2D (right) shows that none of the UT-B inhibitors, tested at an even higher concentration of 25 µM, inhibited RBC osmotic water permeability as measured by cell shrinking in response to a sucrose gradient. Curves from negative control (no inhibitor) and positive control (HgCl2 water transport inhibitor) are provided for comparison.

We next screened ~700 commercially available analogs of compounds from the four chemical classes in order to establish structure-activity relationships (SAR) and to potentially identify compounds with improved UT-B inhibitory potency. Analogs were screened at 25 µM. Concentration-inhibition data were obtained for those compounds with >75% apparent UT-B inhibition by the RBC lysis assay. Figure 3 A shows chemical structures of potent compounds from each of the phenylsulfoxyoxazole, benzenesulfonanilide, phthalazinamine, and aminobenzimidazole classes. These structures are unrelated to phloretin or urea analog inhibitors. Figure 3B shows concentration-inhibition data with apparent EC50 values (in nM): ureainh-101, 30; ureainh-201, 300; ureainh-302, 100; and ureainh-404, 400. However, the apparent EC50 values in this acetamide-based RBC lysis assay are not true EC50 values because of nonlinearity between acetamide permeability and percentage RBC lysis, and possible acetamide-inhibitor competition.


Figure 3
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  		Figure 3. Nanomolar potency UT-B inhibitors identified from the primary high-throughput screen and assay of analogs. A) Chemical structures of UT-B inhibitors (ureainh), with one compound per class shown. B) Dose inhibition data for inhibitors as in panel A, determined by the lysis assay in human RBCs (±SD) and fit to calculate EC50 (solid lines) as described in Materials and Methods.

To quantify urea transport inhibition directly, RBC urea transport was measured by stopped-flow light scattering using a nonsaturating concentration of extracellular urea (to avoid possible competition effects). Figure 4 A shows representative data for inhibition of RBC urea transport by ureainh-101 and ureainh-302. Urea permeability coefficients (Ps) were determined from light-scattering curves by numerical integration of the flux equations for coupled RBC water/urea transport (see Materials and Methods). An example of computed concentration-inhibition data is plotted in Fig. 4B . The deduced EC50 values from stopped-flow measurements were in general agreement with those determined in the lysis assay. As expected, the computations indicated that 50% UT-B inhibition produces only a subtle (~2-fold slowing) change in the light-scattering curve, whereas the more obvious visual evidence for slowed kinetics is seen at >95% inhibition. In addition, these computations indicated that many of the new inhibitors produced >99% UT-B inhibition.


Figure 4
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  		Figure 4. Stopped-flow measurements of urea transport in human RBCs. A) Concentration/inhibition curves for indicated compounds (structures shown in Fig. 3 ) determined by light scattering in response to a 100 mM inwardly directed urea gradient. RBCs were incubated for 5 min with compounds at indicated concentrations prior to stopped-flow measurements. B) Numerically simulated inhibitor concentration dependence used to determine EC50 from stopped-flow experiments as in panel A (see Materials and Methods for details). The inverse of normalized cell volume, Vo/V(t), is plotted to approximate light-scattering data at indicated percentages of urea transport inhibition. C) Membrane sidedness of UT-B inhibition. Experiments performed as in panel A, except that inhibitors (0.1 µM ureainh-101 and 0.05–0.2 µM ureainh-302) added only to the urea-containing solution (250 mM urea+PBS) where indicated. D) Reversibility of UT-B inhibition. Where indicated, inhibitors (0.1 µM ureainh-101 and 0.4 µM ureainh-302) were washed out after 10 min incubation, prior to stopped-flow measurements.

To determine the sidedness of UT-B inhibitor action, RBCs were exposed externally to ureainh-101 and ureainh-302 at final concentrations of 0.1 and 0.2 µM, respectively (several times their EC50), just at the time of stopped-flow experiments (inhibitor inclusion only in urea-containing solution). Whereas ureainh-101 did not inhibit urea transport under these conditions, suggesting an intracellular site of action, ureainh-302 had a sizable effect (Fig. 4C ). The inhibition of urea permeability by externally added ureainh-302 was concentration dependent. To test reversibility of inhibition, RBCs were preincubated with ureainh-101 or ureainh-302 for 10 min (at 0.1 and 0.4 µM, respectively), producing >95% transport inhibition. After washing, urea transport was comparable to that in noninhibitor-exposed RBCs (Fig. 4D ), indicating reversible inhibition.

Structure-activity analysis of UT-B inhibitors
UT-B inhibitory potencies for the most active compounds of each chemical class are summarized in Tables 1 –4. The conclusions from SAR analysis are summarized in Fig. 5 . Class 1 compounds consisted primarily of phenylsulfoxyoxazoles, but also included several phenylsulfoxyimidazoles (ureainh-130–132). In highly active compounds, unsubstituted thioglycoamide was present as R1 (ureainh-101–119). Compounds with reduced activity often had amino groups such as mono/dialkylated amines (ureainh-120–123), n-morpholino (ureainh-124–125), and hexahydro-1-H-azepine-1-yl (ureainh-126–128) as R1, whereas activity was lost with R1 as thioglycoamides or mono/dialkylated amides. The best compounds (EC50 <100 nM) contained 2-thiopene or phenyl rings as R2, though 2-furan also gave submicromolar potency. Methyl or halo substitutions at the 4-position of the phenyl ring as R2 reduced activity whereas compounds with 3/di/trisubstituted phenyl rings as R2 were inactive. For R3 substitutions, halo and methyl groups conferred substantial activity compared to unsubstituted analogs.


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  		Table 1. Structure/activity analysis of phenylsulfoxyoxazolesa


Figure 5
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  		Figure 5. Structure-activity analysis of UT-B inhibitors: phenylsulfoxyoxazoles, benzenesulfonanilides, phthalazinamines and aminobenzimidazoles. General structures, with substitution preferences indicated. See Tables 1 2 3 4 for UT-B inhibition activities.

Most active benezesulfonanilides of Class 2 contained either 2,5-dimethoxy groups (ureainh-201–205) or a fused 1,4-dioxane ring at the 3,4 positions (ureainh-207–216) of the aniline phenyl, with the former producing greater UT-B inhibition. Compounds with other mono/disubstitutions at R1/R2, including 3-methoxy, 4-amino, and 3,4-dimethoxy, were inactive. The benzene sulfonamide phenyl ring tolerated a range of mono-substitutions as R3, including bromo, fluoro, thiomethyl, methoxy, and acetyl (ureainh-203–205, 209), but not methoxy or methyl functions. However, methoxy and methyl groups were tolerated as disubstitutions as R3/R5 (ureainh-201, 208) and to a lesser extent as R3/R4 (ureainh-212, 214).

SAR of Class 3 phthalazinamines indicated preferred mono-substitution at the 4 position of the 4-phenyl ring, especially with carboxamide or methoxy functions (ureainh-301–307, 312). Other acceptable 4-phenyl mono-substitutions included 4-methyl, 4-hydroxy, 4-diethylcarboxamide, and 4-n-hydroethylcarboxamide (ureainh-308–311, 314–317). Active phthalazinamines containing disubstitutions on this phenyl ring combined methyl at the 3-position, with a variety of unsubstituted and mono/dialkylated sulfanoyls at the 4-position (ureainh-321–331). Alkylation of 1-amino resulted in complete loss of activity, whereas replacement by oxygen reduced activity only partially (ureainh-332, 333). Inhibitory activity was also lost when the 1-amino group was substituted by phenylmethyl rather than phenyl. Analogs were active when the n-phenyl-1-amino moiety was substituted at the 4-position, particularly with methyl and methoxy groups and mono/dialkylated carboxamides (ureainh-301–304); also well tolerated were hydroxy, sulfanoyl, glycoamide, and n-methyl-glycoamide substitutions (ureainh-306, 307, 311, 312).

For the Class 4 aminobenzimidazoles, no substitutions were allowed at R1 except for methyl, which reduced activity (ureainh-404 vs. 420). The 2-hydroxyphenylmethyl group was necessary for activity, with activity eliminated by replacement of hydroxy with methoxy or substituted sulfonyl amino groups. The loss of activity on hydroxy-methylation could be due to disruption of hydrogen bond donor effects. Additional substitutions at the benzyl function, such as 5-bromo, 5-chloro, and 5-methyl, increased activity (ureainh-401–406, 414–417), though compounds without 5-position substitutions were also active (ureainh-407–413). In contrast, compounds with substituents at the 3-, 4-, or 6-positions were generally inactive. Alkylation of the imidazole nitrogen (R3) was favorable, especially with ethyl, n-propyl, isopropyl, and 2-propenyl groups (ureainh-401–406, 409–411, 414–416). Several bulky alkyl chains carrying substituted amino functions were also active (ureainh-407, 408).

UT-B inhibitor efficacy on rodent UTs
To identify UT-B inhibitors for application to mouse tissues, we screened the inhibitors of human UT-B for activity against mouse UT-B. Whereas many highly active phenylsulfoxyoxazoles and phthalazinamines (Classes 1 and 3) against human UT-B were active against mouse UT-B by lysis assay, no benzenesulfonanilides or aminobenzimidazoles (Classes 2 or 4) were active even at 25 µM, which was surprising given the 85% sequence identity of human and mouse UT-B (5) . Similar UT-B inhibitory potencies were measured in mouse and rat RBCs (data not shown), as expected from their closely related sequences.

As with human RBCs, EC50 for the most potent compounds with activity in the mouse RBC lysis assay was determined by stopped-flow light scattering. Representative curves for two UT-B inhibitors (ureainh-101 and ureainh-302) are shown in Fig. 6 A. For these studies methylurea was used as the transported solute instead of urea because its transport is slower, allowing better estimation of EC50 values. Concentration-inhibition data indicated EC50 ~ 200 nM for mouse UT-B for the most potent Class 1 and 3 compounds. These compounds, when tested at 25 µM, did not affect urea transport in RBCs from UT-B-null mice (data not shown).


Figure 6
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  		Figure 6. Activity of UT-B inhibitors against rodent UT-B and UT-A1. A) Dose-inhibition relationships for ureainh-101 (left) and ureainh-302 (right) against mouse UT-B. Stopped-flow light scattering measurements using RBCs from WT mice in response to a 100 mM inwardly directed gradient of N-methylurea. B) UT-A1-mediated urea flux in stably transfected MDCK cells. Cells were treated (open circles and open triangles) or not treated (filled circles) with 10 µM forskolin. Where indicated, phloretin (0.7 mM) was present (open triangles) (±SE, 3 filters per condition). Dashed line indicates the time chosen (15 min) to evaluate UT-A1 inhibition in panel C. C) Concentration-dependent inhibition of mouse UT-B (triangles, dashed lines) and rat UT-A1 (circles, solid lines) by ureainh-101 (filled symbols) and ureainh-302 (open symbols), determined from data as in panels A, B.

Because of significant sequence similarities between UT-B and UT-A isoforms, we measured UT-A inhibition using available MDCK cells expressing rat UT-A1. Compounds with inhibition activity against mouse UT-B were tested. MDCK-UT-A1-expressing cells were grown on collagen-coated porous filters until they were electrically tight, at which time 15 mM urea was introduced into the buffer bathing the basolateral cell surface. Figure 6B shows the kinetics of urea appearance in the apical solution. As reported earlier, UT-A1-facilitated urea transport was strongly increased by the cAMP agonist forskolin and inhibited by phloretin (17) . Concentration-inhibition data were obtained at a 15 min time point, when urea accumulation in the apical bathing solution is approximately linear. Ureainh-101 was more active (EC50~1.2 µM) against rat UT-A1 than ureainh-302 (EC50~15 µM) (Fig. 6C ). For comparison, concentration-inhibition data are shown for mouse UT-B, indicating good selectivity of these compounds for UT-B over UT-A1. Neither ureainh-201 nor ureainh-404 significantly inhibited rat UT-A1 at 25 µM (not shown).

UT-B chemical knockout in RBCs reveals UT-B-facilitated water transport
Compounds ureainh-101 and ureainh-302, which have good inhibitory potency against mouse UT-B, were used to test the hypothesis that UT-B contains a pore that conducts water in response to an osmotic gradient (see Discussion). Osmotic water permeability was measured by stopped-flow light scattering in RBCs from WT and AQP1-null mice as shown in Fig. 7 A. We reasoned that the low water permeability of AQP1-null RBCs would make it possible to see UT-B-facilitated water transport if it was present. Water permeability coefficients are summarized in Fig. 7B . The UT-B inhibitors phloretin, ureainh-101, and ureainh-302 had little effect on water transport in RBCs from WT mice, as expected, since AQP1 provides the principal route for water transport. Phloretin at 0.7 mM produced a small but significant reduction in Pf that is likely due to its nonspecific effects on membrane fluidity. AQP1-null RBCs had > 5-fold reduced Pf than WT RBCs. As seen in Fig. 7B , ureainh-101 and ureainh-302 further inhibited water permeability in AQP1-null RBCs, providing strong evidence for UT-B-facilitated water transport.


Figure 7
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  		Figure 7. UT-B-facilitated water transport demonstrated by chemical UT-B knockout. Osmotic water permeability was measured from the time course of RBC volume in response to a 250 mM inwardly directed sucrose gradient. A) Representative traces of mouse RBC water permeability done at 10°C, with genotypes and conditions indicated. Inhibitors ureainh-201 and ureainh-302 were used at 25 µM. B) Osmotic water permeability coefficients (Pf) from experiments as in panel A (±SE, 3–7 curves per group of RBCs pooled from 4 mice per genotype). *P < 0.01 compared with no inhibitor; #P < 0.01 compared with WT (no inhibitor).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report here the discovery of potent, small-molecule inhibitors of the UT-B urea transporter. The primary goal of this study was to identify small molecules with strong activity against human UT-B to initiate development of a new type of clinically useful diuretic—a urearetic. A secondary goal was to identify those compounds with activity against rodent UT-B to enable chemical knockout studies in animal tissues. We developed a lysis-based high-throughput screening assay that relied on opposing water and urea transport processes in RBCs. Our assay allowed a simple static readout of percentage RBC lysis, which provided an accurate surrogate of the complex and difficult-to-measure kinetics of RBC water/solute transport. The high "hit" rate in the primary screen and the ease in identification of analogs with submicromolar potency indicate the "druggability" of urea transporters.

Diuretics are used widely to increase renal salt and water clearance in a variety of conditions associated with total body fluid overload, such as congestive heart failure and cirrhosis, as well in normovolemic states such as hypertension and syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Most diuretics are inhibitors of salt absorption by kidney tubules, such as furosemide block of Na+/K+/2Cl– cotransport in the thick ascending limb of Henle and thiazide block of Na+/Cl– cotransport in the distal tubule. Recently, a new type of diuretic, which has been called an "aquaretic," was introduced to increase renal water clearance in hyponatremia associated with fluid overload or SIADH (reviewed in refs. 19 ,20 ). Several vasopressin-2 receptor (V2R) antagonist aquaretics are in use, and aquaporin inhibitor aquaretics are under development. We propose here the concept of a urearetic as a third type of diuretic, which targets renal urea clearance mechanisms. Since urea is of at least equal importance to NaCl in the renal countercurrent mechanism for urinary concentration (1 , 21) , possible indications of urearetics would include increasing solute clearance and free water excretion in states of fluid overload, hypertension, and perhaps in chronic renal insufficiency.

Many active phenylsulfoxyoxazole, benzenesulfonanilide, phthalazinamine, and aminobenzimidazole compounds were identified in the primary and analog screens, none of which have been reported previously to have significant biological activity (SciFinder database search). The most potent compounds of each class had submicromolar potency for inhibition of human UT-B as measured by the RBC lysis assay (Fig. 3) . The phenylsulfoxyoxazole class contained the most potent UT-B inhibitors, with several compounds identified in the primary screen having EC50 between 20 and 100 nM. The EC50 obtained by this indirect measure of acetamide transit were in general agreement with EC50 measured for the most potent compounds by direct stopped-flow measurement of urea permeability (Fig. 6A , B). Analysis of a series of chemical analogs of each class indicated the structural requirements for UT-B inhibition and provided many potent UT-B inhibitors for optimization of ADME (absorption, distribution, metabolism, excretion) properties for development as possible diuretics.

Membrane sidedness-of-action and reversibility were studied for the most potent phenylsulfoxyoxazoles and phthalazinamines. External addition of the phthalazinamine inhibitor just at the time of stopped-flow experiments produced strong UT-B inhibition, suggesting an external site of action for class 3 compounds. It is unlikely that inhibition was produced by action at an intracellular site, which would require permeation of this relatively polar compound (CLogP 4.8) in less than 1 s. In contrast, the lack of inhibition of external added phenylsulfoxyoxazole suggests an intracellular site of action, but very slow binding to an external site, while unlikely, cannot be ruled out. Inhibition by both the phenylsulfoxyoxazoles and phthalazinamines was fully reversed upon washout.

An important application of UT-B inhibitors is in the analysis of UT-B functions in animal tissues by UT-B chemical knockout. Here we used UT-B inhibitors and mouse erythrocytes to clarify the possibility of UT-B-facilitated water transport. Our lab first reported UT-B-facilitated osmotic water transport from measurements of urea permeability and reflection coefficient in UT-B-expressing Xenopus oocytes (22) . A subsequent study confirmed our original data, but concluded from oocyte studies that UT-B-facilitated water transport is a phenomenon seen only at supraphysiological UT-B expression levels, and thus likely to be insignificant in mammalian physiology (5 , 23) . A more recent study from our lab comparing RBCs from AQP1-null and AQP1/UT-B double knockout mice provided evidence for UT-B-facilitated osmotic water transport (24) , though the data could not rule out compensatory effects that might occur with AQP1/UT-B gene deletions. The ~3-fold reduced osmotic water permeability measured in AQP1-deficient erythrocytes on specific chemical inhibition of UT-B (Fig. 7) supports the conclusions of Yang et al. (24) that UT-B provides an efficient, bona fide route for water transport.

After characterization of inhibitor pharmacokinetics and ADME properties, acute inactivation of renal medulla UT-B in animal models should reveal the role of UT-B in normal kidney physiology and as a possible drug target in states of volume overload and azotemia. Chemical knockout of renal UT-B will be particularly useful because up-regulation of UT-A and AQP expression has been found in UT-B knockout mice (25) , which might influence their urinary concentrating ability and confound conclusions about the role of UT-B. Compared to UT-B knockout mice, UT-A1/3-deficient mice have a more severe urinary concentrating defect, with ~65% reduced urine osmolarity (6) . Given the important contribution of UT-A to urinary concentration in the medullary collecting duct, either a UT-A-selective inhibitor or a nonselective UT inhibitor would be most desirable for urearetic application. The UT-A1 isoform evaluated here contains both UT-A2 and UT-A3 amino acid sequences (2) . As such, similar UT-A2 and UT-B sequences and predicted membrane topologies (62% identity in the rat, ref. 26 ) likely account for the measured cross-reactivity of UT-B inhibitors toward UT-A1. Nevertheless, the most potent of the "rodent-active" inhibitors had a high degree of selectivity, with >10-fold greater potency for rodent UT-B vs. UT-A1 (Fig. 6C ). Class 3 phthalazinamines had even greater specificity than class 1 phenylsulfoxyoxazoles. UT-B inhibitors should thus enable selective chemical knockout of UT-B in the renal medulla to define the role of UT-B in the urinary concentrating mechanism.


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  		Table 2. Structure-activity analysis of benzenesulfonanilidesa


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  		Table 3. Structure-activity analysis of phthalazinaminea


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  		Table 4. Structure-activity analysis of aminobenzimidazolesa


   ACKNOWLEDGMENTS
 
We thank Dr. Nitin Sonawane for advice on analog selection and SAR analysis, Dr. Jung Kyung Kim for help with computer modeling, Dr. Baoxue Yang for valuable advice, Drs. Shannon Sullivan and Dennis Nielson for venipuncture, and Liman Qian for mouse breeding and genotype analysis. This study was supported by National Institutes of Health (NIH) grants DK35124, EB00415, HL73856, HL59198, and EY13574, Research and Translational Core Center grant DK72517, and Research Development Program and Drug Discovery grants from the Cystic Fibrosis Foundation. M.H.L. was a student supported in part by an NIH Medical Scientist Training Grant.

Received for publication August 24, 2006. Accepted for publication August 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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