酶学及热力学研究：天然产物抗HIV机制及作用特点

Human immunodeficiency virus infects vital cells in the human immune system leading to acquired immunodeficiency syndrome (AIDS) which could not be cured. The clinical therapeutics obstacle was HIV drug resistance, which was caused by HIV mutating and reproducing itself in the presence of antiretroviral drugs, leading to treatment failure. Therefore, keeping development of novel anti-HIV drugs is highly required. .Reverse transcription is a unique characteristic of HIV which can be blocked by non-nucleoside reverse transcriptase inhibitors. NNRTIs are important components for the combination of antiretroviral (ARV) drugs for the standard antiretroviral therapy..We have found abstracts from Machilus exhibited inhibitory effects on HIV replication. Structural modification and SAR analysis study showed that aryl tetrahydronaphthalene derivatives inhibited HIV reverse transcription process (compound RM-1 has be most potent activity which was relative to Nevirapine. Furthermore, this compound had good bioactivities on NNRTIs-resistant HIV replication as well..This study will focus on the mechanisms and pharmacological characteristics of compound RM-1, which represents aryl tetrahydronaphthalene derivatives. The research will include the effects of the compound on RT's multi-catalytic activities; the effects on drug-resistant reverse transcriptase activities; effects on RT catalytic kinetics; and the thermodynamic signatures with RT. The compound-RT interaction sites will be analyzed by work station calculating based on our experimental results. This research will theoretically and substantially contribute to novel NNRTIs development.

本项目用酶学和热力学方法研究一类有效的天然物质抗HIV的作用机理和特征。为了克服HIV对逆转录酶抑制剂的耐药性，本项目发现天然活性成分及其改构物芳基四氢萘化合物RM-1不仅抑制野生型HIV-1逆转录的活性与奈韦拉平相当，且对耐药病毒也有高抑制活性（RM-1可抑制8种耐药毒株中的6株；而奈韦拉平全部耐药）。.在此基础上，本项目以RM-1为模型化合物，研究这类化合物的药理作用机制和特点，包括1.解析对逆转录酶多重催化的抑制环节；2.用等温滴定量热法（ITC）研究有代表性化合物热力学特征，分析抑制剂和酶结合的自由能(ΔG)，焓(ΔH)，熵(-TΔS)和Ki（EC50）相关性，从微观和深层次上揭示抑制剂与酶的结合内涵；3.结合分子模拟方法，从结构生物学和构效关系的角度解析化合物与野生和耐药毒株逆转录酶结合模式的异同，为设计和研究新型NNRTIs提供理论和结构依据。

非核苷类逆转录酶抑制剂是高效抗逆转录病毒疗法的重要组成部分。由于艾滋病患者长期用药，体内出现耐药病毒，因此需要持续研发新型抗耐药HIV-1药物。本项目旨在通过应用酶动力学及热力学方法研究润楠提取物RM-1抗HIV-1的作用机制和作用特点。研究发现RM-1为HIV-1逆转录酶抑制剂，对HIV-1 RTK103N、RTL100I/K103N、RTG190A和RTK103N/G190A耐药突变酶均具有抑制活性，而 RTY181C、RTK103N/Y181C、RTY188L、RTY181C/G190A和RTK103N/Y181C/G190A耐药酶对RM-1产生耐药，耐药倍数分别为22、18、18、11和19倍，但明显优于阳性药物奈韦拉平。机制研究显示RM-1抑制逆转录酶的RNA依赖DNA聚合酶活性，而对RNaseH活性无显著影响。分子对接结果显示RM-1位于由逆转录酶Pro95, Leu100, Lys103, Lys101, Val106, Tyr181, Leu234, Trp229 和Pro236残基形成的疏水性口袋中，RM-1与RT间没有形成氢键，而是四氢化萘环与Tyr181芳环形成π-π相互作用。动力学研究显示RM-1与RT结合速度常数(kon)为1.08 × 104 M-1S-1，解离速度常数为(koff)为 6.8 × 10-3 S-1，解离常数(KD)为 6.3 × 10-7M；我们以奈韦拉平为阳性药物，其结合速度常数(kon)为7.55 × 103 M-1S-1，解离速度常数为(koff)为2.33× 10-3 S-1，解离常数(KD)为 3.08 × 10-7M。该结果表提示RM-1与逆转录酶的结合时间较奈韦拉平长，有利于其药效。多个不同作用机制的抗HIV-1药物联合应用是艾滋病人临床标准治疗方案，因此研究化合物与现有一线药物联合使用的药效学是其作用特点的重要指标，本项目考察了RM-1与TDF+3TC及AZT+3TC两种组合方式，结果显示，RM-1与上述两种组合方式联合使用均为协同作用，与NVP和EFV相比，对TDF和3TC的剂量降低倍数更显著，而对AZT+3TC组合中的两种NRTI剂量降低倍数与NVP和EFV相当。通过项目的实施，RM-1抗HIV-1机制和特点已明确，本研究为开发天然物质来源的抗艾滋病药物提供了物质基础。

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