Canadian Blood Services Research and Development/Toronto General Research Institute, 67 College St., Toronto, Ontario, M5G 2M1, Canada.
Glycoconj J. 2010 Jul;27(5):515-24. doi: 10.1007/s10719-010-9297-y. Epub 2010 Jun 26.
Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb(3)/P(k)/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb(3)/P(k) (FSL-Gb(3)) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb(3), of the natural Gb(3)/P(k) molecule was synthesized. HIV-1(IIIB) (X4 virus) and HIV-1(Ba-L) (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb(3). We monitored Gb(3), CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24(gag) ELISA. Total cellular Gb(3) was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb(3) blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC(50)) of approximately 200-250 microM. FACS and TLC overlay showed that FSL-Gb(3) can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb(3) is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb(3) on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb(3) can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb(3) into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb(3), having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS.
先前的研究表明，细胞表面表达的糖鞘脂，即神经节苷脂 GD3（Gb3/ Pk/ CD77），可以抵抗 HIV-1 感染，可能是一种新发现的天然抵抗 HIV 感染的因素。我们现在研究了一种新型的、水溶性的、无毒的和完全合成的 Gb3/ Pk 类似物（FSL-Gb3）在体外抑制 HIV-1 感染的潜力。设计了一种独特的天然 Gb3/ Pk 分子的类似物 FSL-Gb3。体外评估了 FSL-Gb3 对 PHA/白细胞介素-2 激活的外周血单个核细胞（PBMC）和 Jurkat T 细胞中 HIV-1（IIIb）（X4 病毒）和 HIV-1（Ba-L）（R5 病毒）的感染，以及用 FSL-Gb3 处理后各种临床 R5 嗜性病毒对 U87.CD4.CCR5 的感染。我们通过荧光抗体细胞分选监测 Gb3、CD4 和 CXCR4 的表达，通过 p24（gag）ELISA 监测病毒复制。通过糖脂提取和薄层色谱分析总细胞 Gb3。通过对重要器官和淋巴组织的组织学评估监测体内 FSL-Gb3 的毒性。FSL-Gb3 以约 200-250 μM 的 50%抑制浓度（IC50）阻断了实验室和临床病毒株的 X4 和 R5 在激活的 PBMC 或 U87.CD4.CCR5 细胞系中的感染。FACS 和 TLC 叠加显示，FSL-Gb3 可以插入细胞质膜，并且细胞质膜吸收的 FSL-Gb3 能够抑制随后的 HIV-1 感染。FSL-Gb3 对细胞表面 CD4 或 CXCR4 的水平没有影响。因此，FSL-Gb3 可以通过两种机制抑制 HIV-1：直接抑制病毒和抑制病毒进入。在远远超过理论治疗剂量的剂量下将 FSL-Gb3 注入实验小鼠中，没有不良反应，表明其具有良好的耐受性。我们的研究结果表明，使用完全合成的、水溶性的神经节苷脂 GD3 类似物 FSL-Gb3 作为一种新的治疗方法，具有低毒性，可能用于治疗 HIV/AIDS 的系统性治疗。
