Lifeng Cai

Conjugation of a Nonspecific Antiviral Sapogenin with a Specific HIV Fusion Inhibitor: A Promising Strategy for Discovering New Antiviral Therapeutics

Triterpene saponins are a major group of active components in natural products with nonspecific antiviral activities, while T20 peptide (enfuvirtide), which contains a helix zone-binding domain (HBD), is a gp41-specific HIV-1 fusion inhibitor. In this paper, we report the design, synthesis, and structure-activity relationship (SAR) of a group of hybrid molecules in which bioactive triterpene sapogenins were covalently attached to the HBD-containing peptides via click chemistry. We found that either the triterpenes or peptide part alone showed weak activity against HIV-1 Env-mediated cell-cell fusion, while the hybrids generated a strong cooperative effect. Among them, P26-BApc exhibited anti-HIV-1 activity against both T20-sensitive and -resistant HIV-1 strains and improved pharmacokinetic properties. These results suggest that this scaffold design is a promising strategy for developing new HIV-1 fusion inhibitors and possibly novel antiviral therapeutics against other viruses with class I fusion proteins.

Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development.

Design, Synthesis, and Biological Evaluation of Highly Potent Small Molecule–Peptide Conjugates as New HIV-1 Fusion Inhibitors

The small molecule fusion inhibitors N-(4-carboxy-3-hydroxyphenyl)-2,5-dimethylpyrrole (NB-2) and N-(3-carboxy-4-hydroxyphenyl)-2,5-dimethylpyrrole (A12) target a hydrophobic pocket of HIV-1 gp41 and have moderate anti-HIV-1 activity. In this paper, we report the design, synthesis, and structure-activity relationship of a group of hybrid molecules in which the pocket-binding domain segment of the C34 peptide was replaced with NB-2 and A12 derivatives. In addition, the synergistic effect between the small molecule and peptide moieties was analyzed, and lead compounds with a novel scaffold were discovered. We found that either the nonpeptide or peptide part alone showed weak activity against HIV-1-mediated cell-cell fusion, but the conjugates properly generated a strong synergistic effect. Among them, conjugates Aoc-βAla-P26 and Noc-βAla-P26 exhibited a low nanomolar IC50 in the cell-cell fusion assay and effectively inhibited T20-sensitive and -resistant HIV-1 strains. Furthermore, the new molecules exhibited better stability against proteinase K digestion than T20 and C34.

Artificial peptides conjugated with cholesterol and pocket-specific small molecules potently inhibit infection by laboratory-adapted and primary HIV-1 isolates and enfuvirtide-resistant HIV-1 strains

OBJECTIVES To develop new HIV-1 fusion inhibitors with improved antiviral activities and resistance profiles, we designed two categories of artificial peptides, each containing four heptad repeats (m4HR) conjugated with a pocket-specific small molecule (pssm) or pssm and cholesterol (chol), designated pssm-m4HR or pssm-m4HR-chol, respectively, and tested their anti-HIV-1 activity. METHODS We synthesized the artificial peptides and conjugated these peptides with pssm and chol using a standard solid-phase Fmoc protocol and a chemoselective thioether conjugation method, respectively. We tested the inhibitory activities of the peptide conjugates against HIV-1 Env-mediated cell-cell fusion and infection by laboratory-adapted and primary HIV-1 isolates, and enfuvirtide-resistant HIV-1 strains using cell-cell fusion and p24 production assays, respectively. We assessed their cytotoxicity towards MT-2 cells using the XTT assay. RESULTS We found that pssm-m4HR conjugates exhibited promising inhibitory activity against HIV-1 Env-mediated cell-cell fusion and laboratory-adapted HIV-1 replication with IC50 values at the low micromolar level, whereas the pssm-m4HR-chol conjugates exhibited dramatically increased anti-HIV-1 activities with IC50 values at the low nanomolar level. Some of the pssm-m4HR-chol conjugates (e.g. 5a and 5b) showed highly potent antiviral activity against infection by primary HIV-1 isolates and enfuvirtide-resistant HIV-1 strains. All the conjugates displayed no or low cytotoxicity towards MT-2 cells. The result of a prime/wash assay indicated pssm-m4HR-chol conjugates were strongly anchored to the membrane and sustained a potent inhibitory effect after washing. CONCLUSIONS These results suggest this scaffold design is a promising strategy for developing novel peptide conjugates with improved antiviral activity against a broad spectrum of HIV-1 strains, including those highly resistant to enfuvirtide.

Hydrophobic mutations in buried polar residues enhance HIV-1 gp41 N-terminal heptad repeat-C-terminal heptad repeat interactions and C-peptides' anti-HIV activity.

Objective:To investigate the effect of mutations in a highly conserved buried polar area on the function of HIV-1 gp41. Design:During HIV-1 entry, a six helical bundle (6-HB) formation between the C-terminal and N-terminal heptad repeat (CHR and NHR) of gp41 provides energy for virus cell membrane fusion. In 6-HB, residues at a and d (a–d) positions of CHR directly interact with NHR and are buried. They are considered critical residues for 6-HB stability and for anti-HIV-1 activity of CHR-derived peptides (C-peptides). Most of a–d residues in CHR are hydrophobic, as buried hydrophobic residues facilitate protein stability. However, HIV-1 gp41 CHR contains a highly conserved polar area with four successive buried a–d polar residues: S649/Q652/N656/E659. We mutated these buried polar residues to hydrophobic residues, either Leu or Ile, and studied its effect on the gp41 NHR–CHR interactions and anti-HIV activities of the C-peptides. Methods:We measured the C-peptide mutants’ ability to form 6-HB with NHR, thermal stability of the 6-HBs and C-peptides’ inhibitory activity against both T20-sensitive and resistant HIV-1 strains. Results:All the mutated C-peptides retained their ability to form stable 6-HB with NHR and strongly inhibited HIV-1 replication. Strikingly, S649L and E659I mutations endow C-peptide with a significantly enhanced activity against T20-resistant HIV-1 strains. Conclusion:The highly conserved buried a–d polar residues in HIV-1 gp41 CHR can be mutated as a means of developing new fusion inhibitors against drug-resistant HIV-1 strains. The concept can also be utilized to design fusion inhibitors against other viruses with similar mechanisms.