Linker-Extended Native Cyanovirin-N Facilitates PEGylation and Potently Inhibits HIV-1 by Targeting the Glycan Ligand

Cyanovirin-N (CVN) potently inhibits human immunodeficiency virus type 1 (HIV-1) infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN) was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser)3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD)-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.蓝藻抗病毒蛋白N（Cyanovirin-N, CVN）能够强效抑制1型人类免疫缺陷病毒（human immunodeficiency virus type 1, HIV-1）的感染，但其细胞毒性与免疫原性均阻碍了该蛋白开发为可行的治疗药物。分子对接分析表明，反向平行的CVN二聚体与寡糖靶点相互作用时涉及多达12个残基，其中亮氨酸-1（Leu-1）是最为关键的热点残基。这一发现为N端聚乙二醇化CVN丧失抗HIV-1活性的现象提供了合理解释。因此，研究人员设计了连接肽-CVN（linker-CVN, LCVN），作为在N端引入柔性亲水连接肽(Gly4Ser)3的CVN衍生物。对LCVN的N端α-氨基进行聚乙二醇（polyethylene glycol, PEG）修饰，得到10 K 聚乙二醇醛基（PEG-aldehyde, ALD）-LCVN（即10 K PEG-ALD-LCVN）。LCVN与10 K PEG-ALD-LCVN均保留了CVN对高甘露糖型N-聚糖的结合特异性与亲和力。此外，LCVN在HaCaT角质形成细胞系与MT-4 T淋巴细胞系中展现出显著的抗HIV-1活性，同时细胞毒性有所减弱。10 K PEG-ALD-LCVN同样可有效灭活HIV-1，其细胞毒性显著降低，且在体外具有显著的细胞间融合抑制活性。经连接肽修饰的CVN及其单聚乙二醇化衍生物被认定为抗HIV-1药物开发的极具潜力的候选对象。该衍生化策略为无需引入点突变即可实现生物候选药物的聚乙二醇化修饰提供了可行模型。