Appendix
Supplementary Data SD1: Studies with different compounds
SD1.1. Inhibition of dengue virus infection in BHK-21 cells by different compounds
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Percentage of DENV NS3-positive cells (mean ± SD)
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Compoundb 6.25 µM 12.5 µM 25 µM 50 µM
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3 98 ± 14 87 ± 15 41 ± 11 18 ± 3
4 111 ± 10 111 ± 15 108 ± 3 114 ± 14
6 93 ± 3 101 ± 8 93 ± 12 84 ± 6
7 97 ± 16 103 ± 6 86 ± 10 92 ± 4
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BHK-21 cells were infected with DENV at an MOI of 0.1. Two hours later, cells were treated with the indicated compound or with 0.5% DMSO. Forty-six hours later, the cells were harvested, stained with anti-DENV NS3 antibody and FITC-labeled secondary antibody, and analyzed by flow cytometry. The percentage of positive cells for the mock control (0.5% DMSO) was (60±1)%, and the value was used for normalization of the data for compound-treated cells. The data are derived from three independent experiments.
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SD1.2. Structure of different compounds used in this study
Compound 4 Compound 6 Compound 7
Supplementary Data SD2: Effect of compound 3 on DENV and JEV infection
BHK-21 cells were infected with DENV-2 strain 16681 (MOI 0.5) or JEV strain RP-9 (MOI 0.1). Two hours later, cells were treated with the indicated concentrations of compound 3 or with 0.5% DMSO. Twenty-four hours later, the cells were stained with rabbit anti-flavivirus NS5 antibody followed by FITC-labeled secondary antibody. The cells stained with anti-NS5 antibody and their phase contrast image are shown. It is notable that DENV NS5 is located in the nucleus, whereas JEV NS5 is located in the cytoplasm.
Supplementary Data SD3: Studies with compound 3 analogues
SD3.1. Structure of compound 3-3 and 3-4 and replicon cell-based assay
Luciferase expression in BHK-D2-Fluc-SGR-Neo-1 cells treated with the indicated compound at 0-100 µM for 24 h before they were subjected to luciferase activity assay (filled circles). In parallel, each compound was tested for cell toxicity in naïve BHK-21 cells by MTT cell proliferation assay four days after treatment with the compound (open circles). Data represent the mean ± SD of at least three independent experiments.
SD3.2. Inhibition of dengue virus infection in BHK-21 cells by compounds 3-3 and 3-4
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Percentage of DENV NS3-positive cells (mean ± SD)
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Compoundb 6.25 µM 12.5 µM 25 µM 50 µM
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3 98 ± 14 87 ± 15 41 ± 11 18 ± 3
3-3 104 ± 12 100 ± 1 82 ± 4 21 ± 5
3-4 84 ± 5 80 ± 8 52 ± 18 44 ± 10
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BHK-21 cells were infected with DENV at an MOI of 0.1. Two hours later, cells were treated with the indicated compound or with 0.5% DMSO. Forty-six hours later, the cells were harvested, stained with anti-DENV NS3 antibody and FITC-labeled secondary antibody, and analyzed by flow cytometry. The percentage of positive cells for the mock control (0.5% DMSO) was (60±1)%, and the value was used for normalization of the data of compound-treated cells. The data are derived from three independent experiments.
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