Advanced Molecular Tweezers Effectively Target Membranes Lacking Choline Headgroups for Broad-Spectrum Antiviral Efficacy.

Broad-spectrum antivirals are urgently required to counter present and emerging viral threats. It has previously been shown that the parental molecular tweezers CLR01 and CLR05 disrupt viral envelopes by complexing choline headgroups and that ester-functionalized "advanced" tweezers display markedly enhanced antiviral potency. Here, we determine the molecular basis of this improved activity. Using liposome leakage assays, giant unilamellar vesicles, NMR, Langmuir film balance experiments, and atomistic simulations, we demonstrate that advanced tweezers not only encapsulate choline-containing lipids but also engage lipids lacking choline headgroups via transient and conserved hydrophobic insertion events. These interactions preferentially destabilize membranes enriched in sphingomyelin, unsaturated acyl chains, or inverted cone-shaped lipids and are especially effective against small, highly curved particles resembling viral particles, explaining their broad antiviral activity for enveloped viruses. Our findings reveal a dual mechanism of action, choline binding and hydrophobic insertion, that underpins the broad-spectrum antiviral activity of advanced molecular tweezers and establish them as a promising new class of membrane-targeting antivirals for prophylactic and therapeutic use.