Tools and assays
In June 2024 the following cascade was established for the continued exploration of the Green site. Antiviral activity and toxicity will be tested in parallel with protein interaction in the picogreen assay and aggregation studies. Compounds that pass the cut-offs will be moved to more advanced testing.
Details provided by the individual research groups involved.
Huh7 cells were used to study the antiviral activity using a luciferase NanoGlow kit that indirectly measures the function of RdRp. Cells were seeded in a 96-well plate, and the next day the cells were infected with DENV viral material; test compounds were also added at this time (serial dilution). The cells were incubated for 48h after which the nanoluciferase signal was read using the kit and a luminescence plate reader. Dose-response curves were read at 50% inhibition of viral activity.
HEK293 cells were used to study antiviral replication using a focus forming assay to quantify viral concentration in the sample (measuring number of virions). Cells were seeded in a 96-well plate, and the next day the cells were infected with DENV viral material; test compounds were also added at this time (serial dilution). The cells were incubated for 72h after which the supernatant in each well was analyzed using the focus forming assay. Dose-response curves were read at 90% inhibition of viral activity.
Cytotoxicity was tested on Huh7 cells using a Cell Titer Glo kit. Cells were prepared as for the nLuc EC50 assay described above but without infecting the cells with viral material; cells were seeded in a 96-well plate and the next day test compounds were added to the wells (serial dilution), and the cells were incubated for 48h. The glo kit was added to each well and analyzed with a plate reader.
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Aggregation was measured using Dynamic Light Scattering (DLS). Compounds were serially diluted from DMSO stocks using filtered buffer to a final DMSO concentration was 2%. The resulting samples were then distributed into 384-well plates, with each well containing 20 μl. The sample plate was centrifuged before loading into DynaPro DLS Plate Reader III. Buffer with 2% DMSO was used as a control. Normalized intensity (kCnt/s) and laser power (%) were used as indicators for estimating compound aggregation and solubility.
Laser power dropping below 100% is considered to indicate aggregation. A recent article highlighted the possible false positives that an aggregating compound can generate for viral targets and ways to confirm if aggregation is the cause of activity. Further reading in this book.
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