双靶点HIV新型融合抑制剂的设计合成与生物学评价

HIV envelope glycoprotein transmembrane subunit gp41 plays a critical role in the virus and target cell membranes fusion. Fusion inhibitors, targeting the gp41, represent an emerging new class of potent antiretroviral agents that are urgently needed to suppress HIV in the face of growing resistance to reverse transcriptase and protease inhibitors. The representative drug T20, a 36-mer peptide derived from the HIV-1 gp41, was successfully licensed for clinical use in 2003, which is the milestone in the history of anti-HIV-1 drugs. However, drug resistance and adverse reactions during long-term medication make it essential to continue developing novel anti-HIV drugs with new mechanisms of action. ..The gp41 ectodomain contains five essential regions for fusion: a fusion peptide (FP), two heptad repeats (CHR and NHR), a membrane proximal region (MPR) and a transmembrane domain (TM). In this study, based on the structural features of HR1 and FP, we design a series of dual-target novel peptide fusion inhibitors, which were expected to possess intramolecular synergistic effect. The objective peptides are mainly composed of two peptide fragments. One fragment, mimic CHR structural, can interact with the NHR to inhibit the formation of six-helix bundle (6-HB); another peptide fragment interacts with FP, so that to anchor the host cell membrane, and increases the local inhibitor concentration close to the cell membrane. Our preliminary results showed that the dual-target fusion inhibitors showed high synergistic activity and resulted in 2-3 orders of potent increase compared with the single-acting peptide fragments. Furthermore, we also carried on an initial study on the mechanism of synergistic action by using different biophysical and biochemical methods...Because HIV-1-cell membrane fusion process has not completely clarified yet, our study will shed new light on fully understanding the HIV-1 fusion mechanism, as well as obtaining a novel fusion inhibitor.

HIV 融合抑制剂是近年来抗HIV 药物研究的热点之一，本课题拟在课题组相关研究基础上，根据HIV 跨膜糖蛋白gp41中HR1 区域和FP 区域的结构功能特征，设计一系列"双靶位"的新型肽类融合抑制剂,在一个分子中实现不同功能区的协同作用。此类多肽主要由两个肽片断组成,其中一个片断模拟HR2 结构特征，可与HR1 区域亲和作用,抑制6-HB形成;另一肽片断则与FP区域结合,使抑制剂分子锚定于宿主细胞膜中,从而增加膜附近的局部抑制剂浓度。初步研究结果表明：基于双靶点设计的全新抑制剂表现出较高的协同作用，其中活性最高的比相应单作用位点前体高出2－3 个数量级，已跨入已见报道的最高活性的HIV 融合抑制剂之列。.由于HIV-1 病毒融合过程涉及多种功能蛋白和细胞分子的参与，目前对于gp41 在细胞融合过程中的介导作用仍未彻底阐明，因此本研究是融合机制的一个切入点，同时获得全新结结构融合抑制剂。

在HIV-1病毒与靶细胞膜融合过程中，跨膜糖蛋白gp41的融合肽（FP）首先插入宿主细胞膜中，然后，gp41 N端的NHR和C端的CHR形成六股螺旋束核心结构，拉近病毒包膜和靶细胞膜的距离，从而实现融合。因此，FP和NHR区域都可以作为融合抑制剂作用的重要靶标。本研究根据gp41 中NHR 区域和FP 区域的结构功能特征，设计并合成了一系列双靶位的新型肽类融合抑制剂,在一个分子中实现不同功能区的协同作用。此类多肽主要由两个肽片断组成,其中一个片断模拟CHR 结构特征，可与NHR 区域亲和作用, 抑制6-HB 形成;另一肽片断则与FP 区域结合,使抑制剂分子锚定于宿主细胞膜中,从而增加膜附近的局部抑制剂浓度。抑制HIV-1 IIIB感染和包膜介导的细胞-细胞融合实验结果表明：基于双靶点设计的全新抑制剂表现出较高的协同作用，其中活性最高的比相应单作用位点前体高出2－3 个数量级，进一步的作用机制研究表明，该类多肽可以抑制六螺旋的形成并且可以与同时包含NHR和FP区域的多肽N70相互作用。由于HIV-1 病毒融合过程涉及多种功能蛋白和细胞分子的参与，目前对于gp41 在细胞融合过程中的介导作用仍未彻底阐明，因此本研究为进一步探讨和阐明gp41与细胞膜的融合机制提供了宝贵的线索，并对设计新的HIV及其他含1类膜融合蛋白的包膜病毒的融合抑制剂提供了新的方法。