A Small Molecule Screen Identifies Selective Inhibitors of Urea Transporter UT-A

• UT-A1 screen assay was established using fluorescent, UT-A1-expressing MDCK cells
• 4 UT-A1 inhibitor classes were identified with different UT-A1/UT-B selectivity
• Functional studies showed a reversible, noncompetitive binding mechanism
• Computational studies suggested inhibitor binding in the UT-A1 hydrophobic pore

SummaryUrea 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.

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