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

托拉塞米(TOR)属于吡啶磺酰脲类袢利尿剂，被广泛有效地用于高血压，心脏衰竭，慢性肾功能衰竭和肝脏疾病的治疗。TOR在治疗过程中易引起的不良反应之一为轻微肠胃不适。然而，TOR与消化蛋白酶(胰蛋白酶和胃蛋白酶)分子间的相互作用鲜有报道。在模拟生理条件下，采用荧光光谱、紫外-可见吸收光谱、圆二色谱和分子对接技术研究了不同温度下托拉塞米(Torasemide, TOR)与胃蛋白酶(Pepsin)和胰蛋白酶(Trypsin)间的相互作用。所有荧光数据均进行了内滤光校正以获得更准确的结合参数。结果表明，TOR-Pepsin和TOR-Trypsin体系的猝灭常数(K_SV)均与温度呈负相关，说明TOR与Pepsin及Trypsin之间的作用机制均为静态荧光猝灭。利用紫外-可见吸收光谱、同步荧光光谱、3D荧光光谱和圆二色光谱法考查了TOR对Trypsin和Pepsin构象的影响。结果发现胃蛋白酶或胰蛋白酶中酪氨酸残基的极性改变较色氨基更明显，TOR可改变色氨酸残基的微环境并降低Trypsin和Pepsin中β-折叠结构，进而可能影响其生理功能。分子对接结果表明，TOR与Pepsin的结合位点位于由Asp-32和Asp-215组成的活性中心周围，从而抑制Pepsin活性。而TOR通过疏水作用力结合在Trypsin的口袋型底物结合位点(S1口袋)，促进底物进入酶活性中心，最终表现为Trypsin活性升高。该研究探讨了TOR与胃蛋白酶和胰蛋白酶的结合作用和毒性机制，为TOR的安全使用提供重要依据。

Probing the Binding of Torasemide to Pepsin and Trypsin by Spectroscopic and Molecular Docking Methods

Torasemide (TOR)belongs to the pyridine sulfonylurea class of loop diuretics and is widely and ef-fectively 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 absorp-tion,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,syn-chronous 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 theβ-sheet con-tent 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 tryp-sin by hydrophobic forces and affected the function of trypsin by increasing its catalytic activity.Our results of-fer insights for the binding and toxicity mechanism of TOR with pepsin and trypsin invivo,which provides im-portant information for using the TOR safely.