基于I类包膜病毒膜融合机制的广谱抗病毒多肽设计

The aim of the applied project is to discover and identify broad-spectrum viral fusion inhibitors against enveloped viruses with class I fusion proteins. It is known that all the enveloped viruses with class I fusion proteins, e.g., HIV, SARS-CoV, MERS-CoV, Ebola virus and influenza viruses, catalyze their virus-cell fusion via a common mechanism. Class I fusion proteins are composed of a surface subunit and a transmembrane subunit. The ectodomain of the transmembrane subunit containing functional domains, e.g., fusion peptide (FP), N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), is directly involved in virus-cell membrane fusion. The NHR and CHR form a coiled coil six helix bundle (6HB) conformation during the fusion process, and the energy released from the 6HB formation brings the viral and host cell membranes into close apposition to facilitate fusion. Any chemical entity that disrupts 6HB formation has the potential to inhibit the fusion process, thereby blocking virus entry into the target cells. The peptides derived from the CHR region (C-peptides), as well as their derivatives, which specifically interact with the viral NHR to prevent fusogenic core formation, have been shown to be potent inhibitors of virus fusion. The coiled coil 6HB is known as the fusion central core of all the class I enveloped viruses. In addition, C-peptide derivatives and artificial peptides in α-helical conformation, even though they are non-homologous with naturally occurring CHR sequences, can inhibit virus-target cell fusion by forming heterogeneous 6HBs with their NHR counterparts. Following the logic described above, we hypothesize broad-spectrum fusion inhibitory peptides against class I enveloped viruses membrane fusion may be designed by using artificial peptides with α-helical conformation conjugate with bioactive small molecule. This hypothesis is supported by our preliminary results. Our work will not only shed new light on the design of broad-spectrum antiviral therapeutics, but also provide prevention and control measures for emerging and re-emerging epidemic diseases.

I类包膜病毒与靶细胞膜间的融合机制具有以下共性：（1）病毒融合蛋白由表面亚基和跨膜亚基组成；（2）跨膜亚基含有N末端重复序列（NHR）和C末端重复序列（CHR）等功能区；（3）NHR与CHR相互作用形成六股α螺旋束（6HB）促使病毒与靶细胞融合，进而完成感染。源自CHR区域的多肽（C肽）及其衍生物能够与NHR结合，阻止6HB形成，从而抑制膜融合。基于I类包膜病毒融合过程中共同经历6HB的高级结构特征和C肽在一定程度上的序列非特异性，我们假设：以α螺旋肽为结构基础，模拟天然CHR活性构象，靶向NHR，并缀合与NHR有广泛作用的活性小分子，由此设计具有广谱活性的I类包膜病毒融合抑制剂。该假设已获前期工作初步验证。本项目的立项和完成将为广谱抗病毒药物的设计和发现提供创新性思路并打下理论和实践基础，同时为快速应对病毒性传染病的爆发提供应急防控手段。

I型包膜病毒具有相似的病毒–宿主细胞膜融合机制：在膜融合时，病毒包膜蛋白会变构形成六股α螺旋束(6HB)结构。本项目以I型包膜病毒融合机制的共性和6HB的本质特征为基础，通过将易于形成α螺旋的人工多肽与具有质膜“锚定”能力的棕榈酸分子共价缀合，由此设计针对该类病毒的广谱抗病毒药物。通过构建化合物库，结合基于MERS-CoV S蛋白介导的细胞-细胞融合模型、基于HIV-1 Env蛋白介导的细胞-细胞融合模型、H1N1型活病毒和H3N2型活病毒感染MDCK细胞模型进行生物学活性评价，得到了一系列对上述病毒均有抑制活性的人工α螺旋多肽–棕榈酸缀合物，并发现了药物候选化合物IIQ。结果表明，IIQ可以显著抑制MERS-CoV与靶细胞的融合，其EC50为0.11 μM；此外，IIQ还可以有效抑制HIV-1与宿主细胞的融合（EC50为3.63 μM）；在H1N1型和H3N2型活病毒感染MDCK细胞模型中，随着IIQ浓度的增加，病毒的毒性呈现下降趋势，其EC50值分别为1.73和0.70 μM。鉴于多肽药物易在体内酶解这一瓶颈问题，我们进一步研究了IIQ的药代动力学性质，结果表明IIQ的体内半衰期长达6.6小时。作用机制研究表明，IIQ可以与MERS-CoV、HIV-1、H3N2等病毒融合蛋白的NHR结构域相互作用。IIQ以病毒相关蛋白作为靶标，且作用于病毒与靶细胞的早期膜融合阶段，能够有效保护宿主细胞的功能以及活性，避免病毒进入细胞后整合到人类基因组，因此化合物IIQ显示出更加重要的临床开发意义。综上所述，本项目的完成，为研发创新、有知识产权的广谱抗病毒药物打下了基础。对于突发的已知或未知高危病毒感染，本研究设计的广谱抗病毒多肽可作为应急防治手段，用于控制病毒蔓延，消除公众在突发疫情时的过度恐慌，维持社会稳定；同时对维护国家安全、保障打赢未来战争也具有重要意义。