Synthesis, Biophysical Characterization, and Anti-HIV-1 Fusion Activity of DNA Quadruplex-based Inhibitors with Dipeptide MT Hook Conjugation.

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.

DNA Quadruplex-Based Inhibitor With Flexible Fragments at the 3' Terminal Shows Enhanced Anti-HIV-1 Fusion Activity.

Chemically optimizing the molecular structure of aptamers may enhance properties such as biological activity or metabolic stability. DNA quadruplex-based HIV-1 fusion inhibitors were found to interact with HIV-1 surface glycoprotein in aptamer mode. In this work, a series of quadruplex-based HIV-1 fusion inhibitors with flexible oligodeoxynucleotide fragments at the 3' terminal was discovered. The flexible extension did not greatly influence quadruplex formation at the 5'-end. Increasing the length of the flexible fragment may increase antifusion activity. Compared with a traditional inhibitor, d(5'TGGGAG3')4, these novel inhibitors showed enhanced interaction with HIV-1 glycoproteins gp120 and gp41, which increased inhibition of 6-helical bundle formation during the course of virus fusion. These inhibitors also showed improved stability, compared with natural oligodeoxynucleotide. This work may inform the design of anti-HIV-1 DNA helix-based inhibitors with new structures or mechanisms.

A novel multivalent DNA helix-based inhibitor showed enhanced anti-HIV-1 fusion activity.

DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates, but further research is required to enhance their efficiency. The trimeric structure of the HIV-1 envelope glycoprotein provides a structural basis for multivalent drug design. In this work, a "multi-domain" strategy was adopted for design of an oligodeoxynucleotide with assembly, linkage, and activity domains. Built on the self-assembly of higher-order nucleic acid structure, a novel category of multivalent DNA helix-based HIV-1 fusion inhibitor could be easily obtained by a simple annealing course in solution buffer, with no other chemical synthesis for multivalent connection. An optimized multivalent molecule, M4, showed significantly higher anti-HIV-1 fusion activity than did corresponding monovalent inhibitors. Examination of the underlying mechanism indicated that M4 could interact with HIV-1 glycoproteins gp120 and gp41, thereby inhibiting 6HB formation in the fusion course. M4 also showed anti-RDDP and anti-RNase H activity of reverse transcriptase. Besides, these assembled molecules showed improved in vitro metabolic stability in liver homogenate, kidney homogenate, and rat plasma. Moreover, little acute toxicity was observed. Our findings aid in the structural design and understanding of the mechanisms of DNA helix-based HIV-1 inhibitors. This study also provides a general strategy based on a new structural paradigm for the design of other multivalent nucleic acid drugs.

Synthesis, biophysical characterization, and anti-HIV-1 fusion activity of DNA helix-based inhibitors with a p-benzyloxyphenyl substituent at the 5'-nucleobase site.

DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates. Introduction of hydrophobic groups to a nucleobase provides an opportunity to design inhibitors with novel structures and mechanisms of action. In this work, two novel nucleoside analogues (1 and 2) were synthesized and incorporated into four DNA duplex- and quadruplex-based inhibitors. All the molecules showed anti-HIV-1 fusion activity. The effect of the p-benzyloxyphenyl group and the attached linker on the helix formation and thermal stability were fully compared and discussed. Surface plasmon resonance analysis further indicated that inhibitors with the same DNA helix may still have variable reaction targets, mainly attributed to the different hydrophobic modifications.