蟾蜍甾烯类化合物结构-代谢稳定性关系研究

Bufadienolides are an important group of polyhydroxy C-24 steroids, characterised by the presence of a six-membered lactone (a-pyrone) ring located at position C-17β. They possess significant cardiotonic and antitumor effects, are critical consitituents in many famous complicated formulas of TCMs such as Liushen Pill, Shexiang Baoxin Pill and Kyushin. However, the metabolic enzyme mediated clearance and inactivation of bufadienolides make a great influence on the in vivo process and anti-tumor effects of bufadienolides. Therefore, disclosure the structure-metabolic stability relationship of bufadienolides including structure features of bufadienolides as well as selectivity and the catalytic efficacy of metabolic enzyme are crucial important for understanding metabolic mechanism as well as rational designation candidates of bufadienolides. Based on our previous work, this project will investigate the interaction of bufadienolides and key human metabolic enzyme by construct a rational modification bufadienolides substrate bank, thereafter, to explore the differences of enzyme specificity and selectivity as well as catalysis behavior of bufadienolides by using qualitative and quantitative methods. In addition, computational biology tools including homology modeling, molecular docking and molecular dynamic calculation will be employed to discovery the significant structure feature on bufadienolides as well as the enzyme binding sites and key amino acid residues on the key metabolic enzyme, disclosure the structure-metabolic stability relationship and the mechanism of different catalytic behaviors from both small molecule and macro-molecule approaches, ultimately provides the academic evidence and guidance for the rational development of bufadienolides.

蟾蜍甾烯是一类24碳多环甾体化合物，以碳17连有六元不饱和内酯环为显著特征。该类化合物具有抗肿瘤、强心等多种药理活性，是中药复方如麝香保心丸、六神丸等的重要药效成分。蟾蜍甾烯在体内被多酶迅速代谢失活，严重影响了其的体内进程和药效发挥。因此，揭示结构-代谢稳定性关系对于深入了解其代谢清除机制、理性设计具有适宜代谢稳定性的候选药物等具有重要意义。本项目拟通过对蟾蜍甾烯类化合物的理性修饰及系列衍生构建底物库，系统开展其代谢行为研究，着重从定性和定量两方面剖析底物结构差异对代谢酶选择性和催化效率的影响。并在此基础上，借助分子对接/动力学模拟等计算生物学手段从三维构象上揭示影响蟾蜍甾烯差异性代谢的关键结构特征和影响代谢酶识别、催化的关键位点/结构域，最终从小/大分子双层面揭示蟾蜍甾烯类化合物结构-代谢稳定性关系及代谢酶差异性催化关键影响因素和机制，为蟾蜍甾烯类化合物的理性开发提供理论依据和指导。

蟾蜍甾烯类化合物是中药蟾酥中的主要药效成分，是麝香保心丸、六神丸等著名中药复方的重要活性成分。时至今日，蟾蜍甾烯的代谢路径均尚未被很好的研究与揭示。本课题以该类化合物在人体和实验动物体内的主要代谢路径、关键代谢酶等为主要研究内容，以进一步揭示蟾蜍甾烯结构代谢规律。研究发现：1）细胞色素P450介导的羟化反应和磺酸转移酶介导的磺酸结合反应分别被鉴定为蟾蜍甾烯系列化合物在人体中代谢清除的I相和II相代谢路径。蟾蜍甾烯的5位和1位碳是该类化合物的主要羟化位点，催化这一代谢反应的代谢酶被归属为细胞色素P450 3As。此外，蟾蜍甾烯的3位羟基是该类化合物的主要磺酸结合位点，催化这一代谢反应的代谢酶被归属为磺酸转移酶2A1。而蟾蜍甾烯3位羟基构型是决定该类化合物能否发生磺酸结合反应的关键。2）蟾蜍甾烯类化合物结构母核上的取代基可显著影响自身与代谢酶的相互作用，因此，这类化合物在人体内的代谢行为千差万别。研究发现，蟾蜍甾烯结构差异可显著影响蟾蜍甾烯与代谢酶CYP3As和SULT2A1的结合作用，影响代谢稳定性。蟾蜍甾烯-I相代谢稳定性关系：母核连有羟基（尤其是C11位）代谢稳定性好；16位乙酰基的引入一定程度上提高了代谢稳定性；14位羟基取代一定程度上优于14、15位环氧环化合物的代谢稳定性。研究蟾蜍甾烯-II相代谢稳定性关系时发现：亲水取代基（如C11、C14、C16位羟基）会显著削弱蟾蜍甾烯与SULT2A1的亲和力，而C5位羟基作为例外则会显著增强与SULT2A1的亲和力；C19位醛基会阻碍磺酸结合反应；C16位乙酰基会降低与SULT2A1的亲和力，影响代谢稳定性。3）蟾蜍甾烯在不同种属实验动物中代谢轮廓与人较为相似，但小鼠和狗肝S9缺少磺酸结合代谢路径。CYP3As和SULT2A1被分别鉴定为不同种属动物肝脏中介导I相和II相代谢的主要代谢酶。然而，不同种属间肝脏制备物对蟾毒灵和脂蟾毒配基的催化效率间存在显著差异。相关研究结果为蟾蜍甾烯类新药的研发以及含该类成分的中药复方在临床的安全合理使用提供研究基础和理论依据。