Probing the Binding of Torasemide to Pepsin and Trypsin by Spectroscopic and Molecular Docking Methods
WANG Yi-run1, FANG Qing1, GUO Chen-hui1, LIU Ying1,2*
1. College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China 2. Beijing Engineering Research Center of Food Environment and Public Health, Minzu University of China, Beijing 100081, China
Abstract：Torasemide (TOR) belongs to the pyridine sulfonylurea class of loop diuretics and is widely and effectively used in the treatment of hypertension, heart failure, chronic renal failure and liver disease. One of the adverse reactions caused by TOR was a slight gastrointestinal discomfort in the course of treatment. However, the molecular interactions of TOR with digestive proteases (trypsin and pepsin) rarely reported. The attempt of this paper was to completely investigate the binding characteristics between TOR and trypsin or pepsin at different temperatures under imitated physiological conditions by fluorescence spectroscopy, UV-vis absorption, circular dichroism (CD) and molecular modeling technique. The inner filter effect of all fluorescence data in the paper was eliminated to get accurate binding parameters. It was found that the fluorescence quenching of trypsin and pepsin by TOR was a static quenching type. The Stern-Volmer quenching constants (KSV) of TOR-pepisn and TOR-trypsin were inversely correlated with temperatures. The binding of TOR changed the conformational structures and internal micro-environment of pepsin and trypsin by UV-vis absorption, synchronous fluorescence, three dimensional (3D) fluorescence and circular dichroism (CD) spectroscopy. The results showed the polarity around Tyr residues of pepsin or trypsin was changed more obviously than that around Trp residues, the TOR alters the secondary structure of trypsin and pepsin and reduces the β-sheet content of protein, which may affect its physiological function. The molecular docking results showed that TOR inserted into the active site of pepsin to interact with the catalytic residues Asp32 and Asp215, and caused a decrease in pepsin activity. TOR bound into the primary substrate-binding pocket (S1 binding pocket) of trypsin by hydrophobic forces and affected the function of trypsin by increasing its catalytic activity. Our results offer insights for the binding and toxicity mechanism of TOR with pepsin and trypsin in vivo, which provides important information for using the TOR safely.
王艺润1，方 庆1，郭晨辉1，刘 颖1,2* . 光谱法和分子对接模拟技术研究托拉塞米与胃蛋白酶和胰蛋白酶的相互作用 [J]. 光谱学与光谱分析, 2016, 36(10): 3414-3421.
WANG Yi-run1, FANG Qing1, GUO Chen-hui1, LIU Ying1,2* . Probing the Binding of Torasemide to Pepsin and Trypsin by Spectroscopic and Molecular Docking Methods. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2016, 36(10): 3414-3421.
[1] Shen L L, Xu H, He Z D, et al. Spectrochim. Acta A，2015, 135: 256. [2] Zeng H J, Liang H L, You J, et al. Luminescence，2014, 29: 715. [3] Liu Y Y, Zhang G W, Liao Y J, et al. Spectrochim. Acta A，2015, 151: 498. [4] Arumugam S, Remya S, Miyashita S, et al. Exp. Mol. Pathol.，2014, 97: 137. [5] Li C X, Liu S P, Hu X L, et al. Acta Chimica Sinica，2011, 69: 1408. [6] Hu T Y, Liu Y. J. Pharm. Biomed. Anal.，2015, 107: 325. [7] Zeng H J, Yang R, Liang H L, et al. Spectrochim. Acta A，2015, 151: 576. [8] Chai J, Xu Q F, Liu R T, et al. Spectrochim. Acta A，2013, 105: 200. [9] Hu X X, Yu Z H, Liu R T. Spectrochim. Acta A，2013, 108: 50. [10] Wang Y Q, Zhang H M, Kang Y J, et al. J. Mol. Struct.，2016, 1107: 91. [11] Song H, Chen C Y, Zhao S L, et al. J. Food. Drug. Anal.，2015, 23: 234. [12] Song W, Yu Z H, Liu R T, et al. Spectrochim. Acta A，2015, 137: 286.
