基于病毒表面融合蛋白的生物活性构象设计MERS冠状病毒融合抑制剂

The aim of the applied project is to discover and identify highly potent Middle East respiratory syndrome coronavirus (MERS-CoV) fusion inhibitors. It is known that all the enveloped viruses with class I fusion proteins, e.g., MERS-CoV and HIV, catalyze their virus-cell membrane fusion via a common mechanism. MERS-CoV spike proteins are composed of a surface subunit S1 and a transmembrane subunit S2. 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. The peptides derived from the MERS-CoV S2 CHR region (C-peptides), which specifically interact with the viral NHR to prevent fusogenic 6HB formation, have been shown to be potent MERS-CoV fusion inhibitors. However, they can only inhibit MERS-CoV replication and its spike protein-mediated cell-cell fusion at micromolar concentrations. This project will construct MERS-CoV CHR-based fusion inhibitors with promising inhibitory activity, via taking advantage of peptide stapling techniques and introducing an exogenously artificial heptad repeat peptide template to the N- and C-terminus of the CHR helices. This design is supported by our preliminary results. Our work will not only shed new light on the design of MERS-CoV fusion inhibitors to treat patients with MERS-CoV infection and combat the potential pandemic, but also provide prevention and control measures for emerging and re-emerging similar viral infectious diseases.

衍生于MERS-CoV融合蛋白CHR结构域的多肽 (C肽) 可以阻止病毒–宿主细胞膜的融合，从而抑制病毒感染。鉴于目前MERS-CoV大流行的潜在可能和临床上特异性药物或疫苗的缺乏，在现有基础上进一步提高C肽的抗病毒效果，既有科学意义，又有临床价值。MERS-CoV和HIV具有相似的病毒–宿主细胞膜融合机制。HIV融合抑制多肽的设计是申请人的重要研究领域。结合申请人在研发抗HIV多肽中的成功经验，本课题拟基于多肽装订策略和人工α螺旋肽片段嵌合策略，在稳定C肽自身α螺旋活性构象的同时强化C肽与靶标的相互作用，由此发现高效低毒的抗MERS-CoV多肽。该设计思想已获前期工作初步验证。实施本课题，将为发现特异性药物以应对潜在的MERS疫情提供创新性思路；同时为其它具有相似融合机制的烈性病毒融合抑制多肽的研发打下理论和实践基础。

2012年，中东呼吸综合征冠状病毒（MERS-CoV）暴发。研发可以有效抑制MERS-CoV感染的特异性药物，用于临床治疗以及预防MERS-CoV潜在的大流行可能，成为当务之急。MERS-CoV和人类免疫缺陷病毒（HIV）均属于I型包膜病毒，具有相似的病毒–宿主细胞间的膜融合机制。在融合过程中，病毒融合蛋白的HR1结构域和HR2结构域进行分子内折叠形成六股螺旋束（six-helix bundle, 6HB）结构。HIV融合抑制多肽的研发经验使我们充分认识到，6HB的本质是一种蛋白−蛋白相互作用；HR1和HR2结构域的结合存在一定程度的序列非特异性。本项目以I型包膜病毒膜融合机制的共性和6HB的本质特征为基础，创造性地提出：MERS-CoV 6HB中残基一一对应的、与HR2特异结合的HR1三股螺旋束结构可以被HIV融合蛋白的HR1三股螺旋束替代；该三股螺旋多肽便可作为MERS-CoV融合抑制剂。通过构建化合物库，结合基于MERS-CoV S蛋白介导的细胞-细胞融合模型和MERS-CoV假病毒感染模型，我们得到了一系列对MERS-CoV具有抑制活性的三股α螺旋多肽，并发现了药物候选化合物N3G。结果表明，N3G可以显著抑制MERS-CoV与靶细胞膜的融合，其EC50为0.34 μM，与阳性对照HR2P-M2多肽相当。通过ex vivo活性评价发现，N3G具有相较于HR2P-M2更长的体内暴露时间；该实验也进一步确证了N3G在体内的抗病毒活性。作用机制研究表明，N3G可以与MERS-CoV融合蛋白的HR2结构域相互作用。综上所述，我们成功建立了基于多肽高级结构的抗病毒药物设计思想，并成功设计合成了具有稳定三股α螺旋结构的高效MERS-CoV融合抑制剂。本项目的完成为预防和控制MERS-CoV的大规模暴发、消除公众对于病毒蔓延所引发的恐慌，提供行之有效的策略。