Our hypothesis predicts that as a photosensitizer, LJ001′s antiviral activity should also be dependent on light. Indeed, the antiviral activity of LJ001 was dependent on both its concentration and the time-of-exposure to white light. For example, doubling the time of light exposure achieved the same viral inhibitory selleck chemicals effect at ten-fold lower concentrations (Figure 3C, compare 50 and 500 nM curves). Importantly, LJ001′s antiviral activity was absent when no visible light source was used (Figure 3D). Since LJ001 membrane intercalation is dictated by its lipophilic properties and not the presence of light, this latter observation underscores our previous observations  that, at the active concentrations used, membrane insertion itself does not account for the antiviral activity of LJ001.
Finally, to provide independent confirmation of the type II photosensitizing properties of LJ001, we subjected a solution of LJ001 in CD2Cl2 under ambient conditions to flash excitation, and observed the characteristic 1O2 emission in the near-infrared (Figure S5). Figure 3 The antiviral activity of LJ001 is dependent on its ability to generate singlet oxygen (1O2). The effect of LJ001 on the biophysical properties of model versus cellular membranes We propose that after insertion into the viral membrane, light activation of LJ001 triggers the generation of 1O2 that oxidizes the unsaturated chains of fatty acids composing the phospholipids of the viral membrane.
In further support of our model, we showed that LJ001 (and LJ025) efficiently partitions into model lipid membranes mimicking the lipid packing density, fluidity, and composition of viral (HIV-like) or cell (POPC) membranes (Figure 4A and Table S1). Indeed, when lipid membranes were non-limiting (>50-fold molar excess of lipid), over 85% of LJ001 or LJ025 were protected from the water-soluble quencher (acrylamide), and thus, completely buried in the lipid bilayer (Figure S6). 1O2-mediated oxidation of unsaturated phospholipids proceeds by a ��singlet oxygen ene�� reaction, resulting in a cis-to-trans isomerization of a double bond in the unsaturated fatty acids and the presence of a polar group (hydroperoxy- or hydroxy-) in the hydrophobic core of the lipid bilayer (Figure S7, first and second panel).
Cis-to-trans isomerization allows for closer packing of the fatty acid acyl chains in the lipid bilayer, which could result in a tighter positive curvature, while lipid oxidation results in clustering of the oxidized lipids into microdomains, reducing exposure of the polar groups to the hydrophobic acyl chains in the lipid Dacomitinib bilayer core (Figure S7, third and fourth panel) . The latter effectively reduces membrane average fluidity (and/or increases rigidity), as lipid species are now not as freely diffusible.