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

作　　者：Jian-hua RAN Min LI Weng-leong TOU Tian-luo LEI Hong ZHOU Calvin Yu-Chian CHEN Bao-xue YANG 

机构地区：[1]Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing 100191, China [2]Department of Anatomy, Laboratory of Stem and Tissue Engineering, Basic Medical College, Chongqing Medical University, Chongqing 400016, China [3]School of Medicine, College of Medicine, China Medical University Hospital, Taichung 40402, Taiwan, China [4]Human Genetic Center, Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan, China [5]Research Center for Chinese Medicine & Acupuncture, China Medical University, Taichung 40402, Taiwan, China [6]Department of Biomedical Informatics, Asia University, Taichung 41354, Taiwan, China

出　　处：《Acta Pharmacologica Sinica》2016年第7期973-983,共11页中国药理学报（英文版）

摘　　要：Aim： 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 10000 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-l-yl）-2-methylbenzene sulfonamide, showed potent UT-B inhibition activity, inhibited human and mouse UT-B-mediated urea transport with IC5o value of 0.02 and 0.69 IJmol/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. Conclusion： The compound PU1424 is a novel inhibitor of both human and mouse UT-B with ICso at submicromolar ranges. Its binding site is located at the So site of the UT-B structure.Aim： 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 10000 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-l-yl）-2-methylbenzene sulfonamide, showed potent UT-B inhibition activity, inhibited human and mouse UT-B-mediated urea transport with IC5o value of 0.02 and 0.69 IJmol/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. Conclusion： The compound PU1424 is a novel inhibitor of both human and mouse UT-B with ICso at submicromolar ranges. Its binding site is located at the So site of the UT-B structure.

关 键 词：urea transport UT-B small-molecule inhibitor phenylphthalazines drug discovery stopped-flow light scattering in silico 

分 类 号：TQ441.41[化学工程—化学肥料工业] S852.65[农业科学—基础兽医学]