Figure 3. The antiviral activity of LJ001 is dependent on its ability to generate singlet oxygen (¹O₂).

(A) LJ001, or control LJ025, was added to a solution of DMA and kept under light. After 6 h, DMA conversion was detected by ¹H-NMR (DMA∶oxiDMA = 3.1 ppm:2.1 ppm (methyl peak)). Reactions were performed in CDCl₃ using 1 equivalent of each reagent. CDCl₃ was saturated with oxygen by bubbling O₂ through the solvent for 30 min and the reaction was kept under O₂ gas atmosphere, except for Ar where oxygen was exchanged with argon by freeze/thaw method. Data represents the mean ± SD of duplicate experiments. (B) HIV-1_IIIB, Herpes Simplex Virus-1 (HSV) or Newcastle disease virus (NDV) were incubated with 0.25 µM of LJ001 in the presence of 50 µM α-tocopherol or DMA, or 100 mM NaN₃. Infectivity was determined as described in Materials and Methods, and results presented as infection relative to untreated virus (100%). HIV: mean ± SD of duplicate measurements, representative of three independent experiments. HSV and NDV: results representative of three independent experiments. #: NaN₃ was toxic to TZM-Bl cells used to assay HIV entry. (C) HSV was incubated with 5, 50 or 500 nM of LJ001 and exposed to white light for 2, 5, 10, 20, 40 or 80 min. Infectivity was determined as described in Materials and Methods, and results presented as infection relative to untreated virus (100%) at a given time, to account for loss of infectivity over time, and as a function of time of light exposure. Data are representative of two independent experiments. (D) HIV-1_IIIB, HSV or NDV were treated in the dark with 1 µM of LJ001, and subsequently either exposed to a white light source or left in the dark, for 10 min, before infection of cells in the dark. Relative infectivity was determined as in (B). LJ001-treated viruses exposed to light had >99% reduction in infectivity. Data represents the mean ± SD of two independent experiments.