Multivalent Virucidal Antivirals: Synthesis, Mechanisms of Action, and Immune-stimulating Effects

Viruses initiate invasion by binding to cell surface glycoproteins. Materials mimicking the carbohydrate motifs of these glycoproteins, such as heparan sulfate (HS) and sialic acid (SA) can block viral attachment and inhibit the infection. Multivalent display of virus-binding motifs on the scaffold enhances binding affinity, and virucidal activity is important to prolong viral inhibition under diluted conditions. The first work investigates scaffold structure and its relationship with antiviral/virucidal activity, focusing on HS and SA ligands. Structurally-defined virucidal compounds with various lengths, numbers and hydrophobicity of the linkers and cores properties, were firstly synthesized. We found that benzene core with six 11-methyelene chains, ended with sulfates (B6C11S) displayed the lowest EC50 (nM to µM range), highest selectivity index and virucidal activity against HSV-2, SARS-CoV-2, H1N1. B6C11S stands out from previously developed virucidal materials due to its remarkable virucidal activity against SARS-CoV-2. Syrian hamster study demonstrates the in vivo efficacy of B6C11S against SARS-CoV-2. By modifying the SA on the benzene alkyl scaffold, we obtained virucidal compound against SA-binding H1N1 influenza. These findings establish benzene with multiple alkyl chains as a versatile scaffold with virucidal potential. The scaffold enables future integration of novel virus-binding ligands for developing potential antiviral agents against current and emerging viruses.The second study reveals the anti-influenza and virucidal mechanism of CD-6'SLN, a sialic acid-modified cyclodextrin. Previous research focused solely on simulating the interaction between CD-6'SLN and the influenza hemagglutinin (HA)-a natural receptor for sialic acid 6'SLN. However, it lacks experimental evidence and remains uncertain whether other targets exist. This is particularly relevant given the potential interaction between the hydrophobic undecyl linker of CD-6'SLN and the viral envelope. We confirmed that CD-6'SLN inhibits the influenza virus through an extracellular mechanism by interacting with HA, but not with neuraminidase, despite the latter also having a binding pocket for the SA. We found that CD-6'SLN interacts with the viral envelope as it elicits the release of a fluorophore embedded in the membrane. Two similar compounds were designed to test the effect of CD-6'SLN and of the undecyl moiety that links the CD to 6'SLN. Neither showed membrane interaction or virucidal effect, confirming that both components are essential to the membrane interaction and virucidal action. These data suggest that CD-6'SLN's antiviral activity relies on a dual action involving both the HA and the viral envelope, with the latter being crucial for its the virucidal activity.The third work explores the possibility of using a biological functional scaffold for developing virucidal antivirals. An immune-stimulating scaffold Pam3CSK4 was modified with several HA-binding peptides. Multivalent peptide display enhances potency against H1N1 with an EC50 of 20 nM, and the hydrophobic tails of Pam3CSK4 enable virucidal activity. Importantly, peptide chemical modification maintained the ability of the scaffold to stimulate dendritic cell maturation. This multivalent antiviral platform demonstrates a unique immune activation effect compared to other anti-influenza treatments, offering synergistic effects with modified antiviral ligands to expedite viral elimination.