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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(KSV)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。
Toramide belongs to the class of pyridosulfonylurea loop diuretics and is widely used in the treatment of hypertension, heart failure, chronic renal failure and liver diseases. One of the most common side effects of TOR during treatment is mild gastrointestinal upset. However, little is known about the interaction between TOR and digestive proteases (trypsin and pepsin) molecules. Under simulated physiological conditions, the relationship between Torasemide (TOR) and Pepsin and trypsin was studied by fluorescence spectroscopy, UV-Vis absorption spectroscopy, circular dichroism and molecular docking Interaction. All fluorescence data was internally filtered to obtain more accurate binding parameters. The results showed that the quenching constants (KSV) of TOR-Pepsin and TOR-Trypsin systems were negatively correlated with temperature, indicating that the mechanism of action between TOR and Pepsin and Trypsin is static fluorescence quenching. The effects of TOR on the conformations of Trypsin and Pepsin were investigated by UV-Vis, Simultaneous Fluorescence, 3D Fluorescence and Circular Dichroism Spectroscopy. It was found that the change of tyrosine residues in pepsin or trypsin was more pronounced than that of the tryptophan. TOR changed the tryptophan residue microenvironment and decreased the β-sheet structure in Trypsin and Pepsin, which may affect the physiology Features. The molecular docking results showed that the binding site of TOR and Pepsin is located around the active center composed of Asp-32 and Asp-215, thereby inhibiting Pepsin activity. TOR, however, binds to Trypsin’s pocket-type substrate binding site (S1 pocket) through hydrophobic interaction, promoting substrate entry into the enzyme’s active site, ultimately exhibiting an increase in Trypsin activity. This study explored the TOR and pepsin and trypsin binding and toxicity mechanisms for the safe use of TOR provide an important basis.