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. 2010 Mar 12;15(3):1690-704.
doi: 10.3390/molecules15031690.

HTS-driven Discovery of New Chemotypes With West Nile Virus Inhibitory Activity

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Free PMC article

HTS-driven Discovery of New Chemotypes With West Nile Virus Inhibitory Activity

Dong Hoon Chung et al. Molecules. .
Free PMC article

Abstract

West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a high throughput screen to discover small molecule probes that inhibit virus infection of Vero E6 cells. A primary screen of a 13,001 compound library at a 10 microM final concentration was conducted using the 384-well format. Z' values ranged from 0.54-0.83 with a median of 0.74. Average S/B was 17 and S/N for each plate ranged from 10.8 to 23.9. Twenty-six compounds showed a dose response in the HT screen and were further evaluated in a time of addition assay and in a titer reduction assay. Seven compounds showed potential as small molecule probes directed at WNV. The hit rate from the primary screen was 0.185% (24 compounds out of 13,001 compounds) and from the secondary screens was 0.053% (7 out of 13,001 compounds) respectively.

Figures

Figure 1
Figure 1
Assay optimization for assay media, incubation period, and MOI. (A) Vero E6 cells were infected with WNV at 0.05 MOI using the 96-well plate format and developed on successive days. Virus cultured in MEM-E produced a higher CPE between day 3 and day 5 than in DMEM. (B) Assay robustness with Z´ factor and inhibition profiles using MPA (positive control) and different amounts of virus in a 384-well plate format. Z´ factor was higher than 0.75 in a 0.01~1 MOI range. The inhibitory activity of MPA was greater at the lower MOI.
Figure 2
Figure 2
Assay performance and robustness and % inhibition profiles of compounds in the primary HTS. (A) The average luminescence readings for cell control, virus control and control drug, MPA at 5 μg/mL of 47 plates depicted with standard deviations (B) Assay robustness depicted with Z′ values with a median of 0.74. (C) Percent inhibition ranging from 79% to -10% with a mean of 0.70%.
Figure 3
Figure 3
Examples of changes in EC50 at different times post-infection. Dose response assay in 384-well format was executed for each time point and the EC50 calculated as described in the text. (A) Compounds failed to exhibit significant change in EC50 within 18 h post-infection. Meanwhile, the other compounds showed sudden increase in EC50 between 8 and 18 h post-infection (B). * EC50 calculated was higher than 60 μM. For graphical purpose, the lines were extended beyond border of graph.
Figure 4
Figure 4
Dose response anti-NS2b-NS3pro activity of SRI-19093. IC50, denoted in the figure, was measured as 0.44 μM. Standard Curves, Four Parameter Logistic Curve was used for plotting regression curves and calculating IC50 values.
Figure 5
Figure 5
Titer reduction assay in a dose response format. Virus was grown in the presence of compound and then harvested 48 h post-infection. Virus titer was measured as TCID50/mL. Each data point represents the mean from experiments performed in duplicate.
Figure 6
Figure 6
Schematic diagram of discovery for anti-WNV probes from a 13,001 compounds library. The numbers represent the number of compounds tested or categorized.

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