Acyclovir

Vero cells were seeded on a µClear black 96-well plate at a density of 25,000 cells/well with MEM supplemented with 10% FBS. After 24 h of incubation at 37 °C with 5% CO₂, cells were infected with 10-fold serial dilutions of HSV-1 F. After 2 h of adsorption, cells were labeled with 1 µg/mL Hoechst 33342 for 10 min, washed once with 1X PBS supplemented with 1% FBS and incubated for 96 h at 37 °C-5% CO₂ with MEM AutoMod^TM supplemented with 2% FBS, 1% carboxymethylcellulose, 2.5 µg/mL of propidium iodide, and different rising concentrations of acyclovir (Sigma, 0–3000 ng/mL). Images of the whole well were acquired at 96 h post-infection with a Lionheart™ FX automated microscope and viral plaques were confirmed by crystal violet staining. Automated viral plaques and dead cells counts were performed by image analysis with the software CellProfiler 4.0, using a customized pipeline called “Plaques&CytotoxicityAnalysis.cpproj” (Supplementary Material). The 50% inhibitory concentration (IC₅₀), defined as the concentrations of antiviral required to reduce virus titers by 50%, as well as the 50% cytotoxic concentration (CC₅₀), defined as the concentration of antiviral that reduces cell viability by 50%, were calculated using non-linear regression with the software GraphPad Prism 8.0. (GraphPad Software).

Cells were treated for 48 h beginning at day 2 of differentiation with DMSO (vehicle control, ATCC), aceclidine (Sigma-Aldrich), acyclovir (Sigma-Aldrich), alloxazine (Sigma-Aldrich), carbadox (Sigma-Aldrich), felodipine (Sigma-Aldrich), GW5074 (Sigma-Aldrich), isoproterenol (Sigma-Aldrich), isradipine (Sigma-Aldrich), lacidipine (Sigma-Aldrich), nafadotride (Tocris Bioscience), nandrolone (Sigma-Aldrich), nifedipine (Sigma-Aldrich), nilvadipine (Sigma-Aldrich), alloxazine (Sigma-Aldrich) diluted in cell-type specific differentiation media at doses listed in figures.

The cytotoxicity of compounds was evaluated by using the neutral red dye-uptake method as previously described [19 (link),20 (link)]. Briefly, the test compounds 1–12, along with 13 (a + b) and acyclovir (Sigma-Aldrich, Germany), used as a positive control were diluted in 0.1% dimethyl sulfoxide (DMSO). Stock solutions were prepared at a concentration of 420 μg/mL using deionized water and then sterilized. Vero cell monolayers cultivated in 96-well microtiter plates with two-fold serial dilutions of the test compounds or the positive control were incubated for 84 h at 37 °C in 5% CO₂ atmosphere. After incubation, the morphological alterations of the treated cells were determined using an inverted optical microscope (Leitz, Berlin, Germany) and the maximum non-toxic concentrations (MNTC) were determined. The CC₅₀ values (concentrations of each drug required to reduce the cell viability by 50%) of compounds 1–12, along with 13 (a + b) and acyclovir were calculated as compared with the untreated control cells. The cytotoxicity of compounds 1–2, 5–6, and 12, active in further anti-HSV tests, was evaluated in duplicate in at least three independent experiments to obtain statistically relevant data for final calculations.

For HSV-1 infection, Vero cells were pretreated with 100 µM Acyclovir (Cat #: A4669; Sigma-Aldrich, St. Louis, MO, USA) for 1 h, followed by infection. Vero cells were treated with 100 µg/mL Cycloheximide (Cat #: 66819, Acros Organics, Geel, Belgium) post-infection to inhibit viral de novo protein synthesis and viral proliferation, respectively. Vero cells were post-treated with 10 mM of NaF (Cat #: 470302-536, Avantor, Radnor Township, PA, USA) to prevent glycolysis. To examine viral entry without transcription and translation, Vero cells were infected with an ultraviolet (UV)-inactivated virus. Spectroline UV Crosslinker Select Series (Model XLE-1000, No. 1010-1) operating on the optimal crosslink setting with the 17-Syn⁺/GFP virus was used to prepare UV-inactivated viruses. Similar to infected samples, UV-inactivated virus was prepared at a multiplicity of infection of 1 and harvested at 10 and 24 hpi.

PSSD was provided by the Marine Biomedical Research Institute of Qingdao (Qingdao, China). Acyclovir was purchased from Sigma Aldrich (St. Louis, MO, USA). HSV-2 strain 333 was obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences. Vero cells were cultured in minimum essential medium (MEM) supplemented with 10% FBS (ExCell Bio, Shanghai, China), penicillin (100 U/mL), and streptomycin (100 μg/mL). HeLa cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (ExCell Bio, China), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO₂. The anti-HSV-2 ICP27 antibody was purchased from Santa Cruz (Santa Cruz, CA, USA).