Abstract
Broad-spectrum antivirals are urgently needed to counter emerging viral threats. Targeting the viral envelope, an essential, conserved, and host-derived structure, offers a promising strategy with a low risk of resistance. Here, we report the in silico design and experimental characterization of P1.6, a 24-mer peptide generated using an evolutionary molecular dynamics (Evo-MD) platform and optimized to sense and exploit lipid packing defects in viral membranes. Among nine Evo-MD-derived candidates, P1.6 showed the strongest membrane-disruptive activity and inhibited HIV-1, Zika virus, and herpes simplex viruses with IC(50) values ranging from ∼0.06 to 3.5 µm. P1.6 efficiently disrupted virus-like liposomes without causing cytotoxicity or hemolysis at antiviral concentrations. All-atom MD simulations predicted a predominantly α-helical solution structure with a central kink and flexible termini. Upon membrane engagement, this kink was largely lost, yielding a more continuous and stabilized helix. ATR-FTIR spectroscopy confirmed the membrane-induced increase in helicity. Coarse-grained MD simulations further demonstrated that P1.6 stabilizes transient membrane pores, while electron microscopy of treated HIV-1 particles revealed extensive envelope rupture and capsid release. Together, these results establish P1.6 as a potent membrane-active antiviral lead and highlight the utility of Evo-MD-guided peptide design to target conserved biophysical vulnerabilities in viral envelopes.