A novel small-molecule thienoquinolin urea transporter inhibitor acts as a potential diuretic

Abstract

Urea transporters (UTs) are a family of membrane channel proteins that are specifically permeable to urea and play an important role in intrarenal urea recycling and in urine concentration. Using an erythrocyte osmotic lysis assay, we screened a small-molecule library for inhibitors of UT-facilitated urea transport. A novel class of thienoquinolin UT-B inhibitors were identified, of which PU-14 had potent inhibition activity on human, rabbit, rat, and mouse UT-B. The half-maximal inhibitory concentration of PU-14 on rat UT-B-mediated urea transport was ∼0.8 μmol/l, and it did not affect urea transport in mouse erythrocytes lacking UT-B but inhibited UT-A-type urea transporters, with 36% inhibition at 4 μmol/l. PU-14 showed no significant cellular toxicity at concentrations up to its solubility limit of 80 μmol/l. Subcutaneous delivery of PU-14 (at 12.5, 50, and 100 mg/kg) to rats caused an increase of urine output and a decrease of the urine urea concentration and subsequent osmolality without electrolyte disturbances and liver or renal damages. This suggests that PU-14 has a diuretic effect by urea-selective diuresis. Thus, PU-14 or its analogs might be developed as a new diuretic to increase renal fluid clearance in diseases associated with water retention without causing electrolyte imbalance. PU-14 may establish 'chemical knockout' animal models to study the physiological functions of UTs.

Phenylphthalazines as small-molecule inhibitors of urea transporter UT-B and their binding model

“…Several classes of UT-B inhibitors, including thienoquinolins, triazo lothienopyrimidines, 2,7-disubstituted fluorenones, and DMTU have been identified by high throughput screening [14,[17][18][19]34] . However, some unsatisfactory characteristics remain before these active compounds can be developed into diuretics for clinical use or toll drugs for UT-B chemical knockout mouse model.…”

“…A collection of 10 000 small, drug-like compounds with high chemical diversity was screened at 25 µmol/L in the primary screen using the mouse and human erythrocyte lysis model [14] . One compound, chemically named 2-(4-((4-(4-methoxyphenyl) phthalazin-1-yl) amino) phenoxy) acetamide ( Figure 1A) with a core structure of 1-phenylamino-4-phenylphthalazin ( Figure 1B) showed an apparent UT-B inhibitory activity in mouse erythrocytes.…”

“…Mouse or human erythrocyte urea permeability was measured by stopped-flow light scattering using an SX20 instrument (Applied Photophysics, Surrey, UK) as described previously [14] . For measurement of urea permeability, dilutions of mouse or human blood in PBS (hematocrit ~0.5%) were incubated with test compounds for 10 min and then subjected to a 250 mmol/L inwardly directed gradient of urea.…”

“…Our previous work found a novel class of thienoquinolin UT inhibitors [14,15] . The thienoquinolin UT inhibitors inhibited both UT-B-and UT-A-type urea transporters.…”

“…However, the maximum activity of thienoquinolin on mouse UT-B-mediated urea transport was only approximately 50%, which is too weak to establish a 'chemical knockout' mouse model. Using an erythrocyte lysis assay as a high-throughput screening method, several classes of small-molecule UT inhibitors have been identified with submicromolar to micromolar IC 50 s [14,[16][17][18][19][20] . However, the most potent inhibitors identified against human UT-B were substantially less potent against mouse UTs, precluding proof-of-concept studies of their urearetic efficacy in mouse models of fluid retention and uremia.…”