Dual-acting stapled peptides target both HIV-1 entry and assembly

Area of Science:

• Biochemistry
• Virology
• Medicinal Chemistry

Background:

• Previously developed cell-penetrating peptide NYAD-1, derived from CAI using i,i + 4 hydrocarbon stapling, binds HIV-1 capsid protein (CA) and disrupts virus particle formation.
• NYAD-1 exhibits potent anti-HIV-1 activity in cell culture.

Purpose of the Study:

• To explore i,i + 7 hydrocarbon-stapled peptides for enhanced antiviral activity and to delineate their mechanism of action against HIV-1.
• To identify novel inhibitors targeting HIV-1 assembly and infectivity.

Main Methods:

• Synthesis and characterization of i,i + 7 hydrocarbon-stapled peptides.
• Nuclear Magnetic Resonance (NMR) and Isothermal Titration Calorimetry (ITC) for binding studies.
• Cell-based assays to evaluate virus particle formation, Gag processing, and infectivity.
• Resistance selection studies to identify viral mutations.

Main Results:

• Identified three potent i,i + 7 stapled peptide inhibitors (NYAD-36, -66, -67) that bind HIV-1 CA and disrupt mature-like particle formation.
• These peptides exhibit alpha-helical structures and cell penetration, though less efficient than i,i + 4 peptides.
• The i,i + 7 peptides impair Gag precursor processing and reduce HIV-1 infectivity, with resistance emerging due to mutations in gp120 (V120Q, A327P).

Conclusions:

• The i,i + 7 stapled peptides unexpectedly target both HIV-1 capsid protein (CA) and the V3 loop of gp120.
• Dual-targeting efficacy depends on cell penetration and net charge.
• This dual-targeting mechanism provides a basis for developing more potent anti-HIV-1 agents.