Synergistic effects of peptide mimics against enveloped viruses

The global health impact of viruses highlights the urgent need for innovative antiviral strategies. This study investigated the synergistic potential of two anthranilamide-based peptide mimics (RK610 and RK758) in combination, and one peptide mimic (RK610) in combination with the cationic peptides Mel4 and melimine, against murine hepatitis virus (a coronavirus; MHV-1), influenza virus (H1N1), and Herpes simplex virus (HSV-1). Checkerboard assays demonstrated RK610+RK758 had synergy against MHV-1 and H1N1 (∑FICI values of 0.14 and 0.5, respectively), while RK610+Mel4 showed potent synergy against HSV-1 (∑FICI = 0.18). Co-treatment outperformed sequential application. Transmission electron microscopy confirmed structural damage to virions, while cytotoxicity assays indicated that all tested combinations were non-toxic in MDCK, A9, and Vero cells, except for melimine and RK610+melimine with A9 cells. Biophysical analyses using DOPC (100 %) and DOPC: POPS (70:30) lipids provided mechanistic ideas into peptide-mimic interactions with lipid envelopes. Tethered bilayer lipid membranes (tBLMs) in conjunction with electrical impedance spectroscopy revealed that both peptide mimics and their combinations reduced membrane conductance, regardless of lipid composition. Quartz crystal microbalance with dissipation monitoring (QCM-D) revealed that RK610 and RK758 induced mass addition at the outer layer, significantly increasing with POPS. In DOPC, RK610 increased the surface pressure in a Langmuir-Blodgett trough while RK758 reduced it. The 610 + 758 and 610+Mel4 combinations raised maximum pressures. In DOPC + POPS, RK758 destabilized the monolayer (35 mN/m, no plateau), whereas combinations restored stability. These findings highlight peptide mimics as broad-spectrum antiviral agents that show synergy to target viral envelopes, paving the way for safer and low-resistance therapeutics.