Background: Human immunodeficiency virus type-1 (HIV-1) infection is the reason for the epidemic of acquired immunodeficiency syndrome (AIDS). The development of HIV-1 fusion inhibitors has gained increasing attention as they were found to be effective in the early stage of HIV-1. DNA G-quadruplex-based inhibitors have been found to interact with HIV-1 envelope glycoprotein, showing anti–HIV-1 fusion activity. C-peptide-derived molecules with Met-Thr terminal also showed potent anti-fusion activity; the Met-Thr dipeptide adopted a hook-like structure (termed MT hook) in the hydrophobic pocket to “anchor” inhibitors to the N-terminal heptad repeat (NHR) of HIV-1 envelope glycoprotein gp41.

Objective: Our work was aimed to conjugate MT hooks to the 5'-terminal ends of DNA quadruplex- based inhibitor and demonstrate its biophysical characterization and anti–HIV-1 fusion activity.

Methods: A 6-aminohexanol phosphonamidite was utilized in solid synthesis for the conjunction of oligodeoxynucleotide and MT dipeptide. Hydrophobic groups were introduced by a nucleoside analogue from the base site. Circular dichroism spectrum and native polyacrylamide gel electrophoresis were used to confirm the helix formation. A cell-cell fusion assay was carried out to test the anti-fusion activity.

Results: The conjugate G1 showed improved anti-cell-cell fusion activity than quadruplex without MT hook. The MT hook did not affect the oligodeoxynucleotide (ODN) G-quadruplex assembly. It was also proved that G1 could effectively interfere with endogenous 6-helical bundle (6HB) formation between the N-terminal heptad repeat N36 (NHR) and the C-terminal heptad repeat C34 (CHR) during virus fusion course.

Conclusion: In this work, a conjugate of DNA-oligopeptide was successfully synthesized. The conjugation of MT hook did improve the anti-fusion activity of DNA G-quadruplex-based inhibitors. Our results can provide information regarding structure-activity relationships of DNA helix-based inhibitors and a reference for the follow-up experimental studies.

背景：人类免疫缺陷病毒 1 型 (HIV-1) 感染是获得性免疫缺陷综合征 (AIDS) 流行的原因。HIV-1融合抑制剂的开发越来越受到关注，因为它们被发现在HIV-1早期有效。已发现基于 DNA G-四链体的抑制剂与 HIV-1 包膜糖蛋白相互作用，显示出抗 HIV-1 融合活性。具有 Met-Thr 末端的 C 肽衍生分子也显示出有效的抗融合活性；Met-Thr 二肽在疏水口袋中采用钩状结构（称为 MT 钩）将抑制剂“锚定”到 HIV-1 包膜糖蛋白 gp41 的 N 末端七肽重复序列 (NHR)。

目的：我们的工作旨在将 MT 钩连接到基于 DNA 四链体的抑制剂的 5' 末端，并证明其生物物理特性和抗 HIV-1 融合活性。

方法：利用6-氨基己醇亚膦酰胺进行寡脱氧核苷酸与MT二肽结合的固体合成。通过核苷类似物从碱基引入疏水基团。圆二色光谱和天然聚丙烯酰胺凝胶电泳用于确认螺旋的形成。进行细胞-细胞融合试验以测试抗融合活性。

结果：缀合物G1显示出比没有MT钩的四联体更高的抗细胞-细胞融合活性。MT 钩不影响寡脱氧核苷酸 (ODN) G-四链体组装。还证明了在病毒融合过程中，G1可以有效干扰N端七肽重复N36（NHR）和C端七肽重复C34（CHR）之间的内源性6螺旋束（6HB）形成。

结论：本工作成功合成了一种DNA-寡肽偶联物。MT 钩的结合确实提高了基于 DNA G-四链体的抑制剂的抗融合活性。我们的研究结果可以提供有关基于 DNA 螺旋的抑制剂的构效关系的信息，并为后续实验研究提供参考。