HIV-1 Antiviral Activity Evaluation

The antiviral activity of investigated compounds was evaluated by measuring the IC₅₀ values against the HIV-1 wild-type reference strain NL4-3 in a TZM-bl cell line-based phenotypic assay named BiCycle Assay [14 (link)]. The method includes a first round of infection in H9 cells at 0.08 MOI in the presence of the serial dilution of compounds in a 96-well plate. In each plate the reference compound, the mock control (uninfected cells) and the virus control were included. After 72 h, 50 µl of supernatants from each well were used to infect the TZM-bl cell line, which allows the quantitative analysis of HIV-1 infection by measuring the expression of the luciferase gene integrated in the genome of the cells under the control of the HIV-1 LTR promoter. After 48 h, dose–response curves were generated by measuring reporter gene expression in each well by using Bright-Glo Luciferase Assay (Promega) through the GloMax^® Discover Multimode Microplate Reader (Promega). Relative luminescence units measured in each well were elaborated with the GraphPad PRISM software version 9 to calculate IC₅₀ values.

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Cesarini S., Vicenti I., Poggialini F., Filippi S., Mancin E., Fiaschi L., De Marchi E., Giammarino F., Vagaggini C., Bizzarri B.M., Saladino R., Dreassi E., Zazzi M, & Botta L. (2024). Serendipitous Identification of Azine Anticancer Agents Using a Privileged Scaffold Morphing Strategy. Molecules, 29(7), 1452.

Publication 2024

Bright-Glo Luciferase Assay GloMax® Discover Multimode Microplate Reader PRISM software version 9

Corresponding Organization :

Other organizations : Università degli Studi della Tuscia, University of Siena

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Variable analysis

independent variables

Serial dilution of compounds

dependent variables

IC50 values against the HIV-1 wild-type reference strain NL4-3

control variables

Virus control
Mock control (uninfected cells)

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