School of Life Sciences, Tsinghua University, Key Laboratory for Protein Sciences of MOE, Beijing 100084, China.
J Antimicrob Chemother. 2013 Nov;68(11):2533-44. doi: 10.1093/jac/dkt230. Epub 2013 Jun 21.
We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy.
Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay.
At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity.
The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.
我们先前基于 HIV-1 gp41 的 N-七肽重复（NHR）序列构建了三聚体卷曲螺旋 N28Fd，作为有前途的 HIV-1 进入抑制剂。在此，我们试图通过在 N28Fd 的 N 端添加链间二硫键来构建稳定的三聚体卷曲螺旋 ccN28Fd，以改善其物理化学性质和抗 HIV-1 功效。
应用分子生物学技术构建 ccN28Fd。使用圆二色性和沉降速度分析分别确定其二级结构和热稳定性以及聚合状态。通过 p24 或荧光素酶表达评估抗 HIV-1 活性。通过 XTT 测定评估其细胞毒性。
在低 pH 值下，ccN28Fd 和 N28Fd 分别处于三聚体和单体构象。ccN28Fd 表现出更高的热稳定性和对 HIV-1 IIIB（X4）和 Bal（R5）株更强的抗病毒活性。ccN28Fd 有效抑制广泛的原发性 HIV-1 分离株和恩夫韦肽耐药株的感染，并阻断细胞间 HIV-1 的传播。ccN28Fd 与核苷逆转录酶抑制剂为基础的杀微生物剂替诺福韦联合使用具有协同抗 HIV-1 活性。ccN28Fd 在 pH 值为 7.2 时对蛋白酶 K 和 pH 值为 1.5 时对胃蛋白酶具有高度抗性，其抗 HIV-1 活性不受羟乙基纤维素凝胶、精液或阴道液模拟物的存在显著影响。它没有明显的体外细胞毒性。
工程化的 ccN28Fd 在低 pH 值环境下保持高度稳定，表现出强大而广泛的抗 HIV-1 活性，表明其作为有效和安全的阴道杀微生物剂预防 HIV 性传播具有良好的开发潜力。
