title: Phenylphthalazines as small-molecule inhibitors of urea transporter UT-B and their binding model.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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 μmol/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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing 100191, China.|Department of Anatomy, Laboratory of Stem and Tissue Engineering, Basic Medical College, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Ran
authors.affiliation: Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing 100191, China.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: School of Medicine, College of Medicine, China Medical University Hospital, Taichung 40402, Taiwan, China.
authors.forename: Weng-Ieong
authors.initials: WI
authors.lastname: Tou
authors.affiliation: Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing 100191, China.
authors.forename: Tian-Luo
authors.initials: TL
authors.lastname: Lei
authors.affiliation: Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing 100191, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Human Genetic Center, Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan, China.|Research Center for Chinese Medicine & Acupuncture, China Medical University, Taichung 40402, Taiwan, China.|Department of Biomedical Informatics, Asia University, Taichung 41354, Taiwan, China.
authors.forename: Calvin Yu-Chian
authors.initials: CY
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing 100191, China.
authors.forename: Bao-Xue
authors.initials: BX
authors.lastname: Yang
mesh_terms: D000818:Animals; D004912:Erythrocytes / Q000187:drug effects; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D062105:Molecular Docking Simulation; D010793:Phthalazines / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: C000621617:5-(4-((4-methoxyphenyl)amino)phthalazin-1-yl)-2-methylbenzene sulfonamide; D026901:Membrane Transport Proteins; D010793:Phthalazines; D054852:Small Molecule Libraries; D013449:Sulfonamides; C452857:urea transporter B, mouse