Supporting Information

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Fig. 6. Flow-chart for assays and infection experiments. (A) For the primary assay, cells were infected with DENV at an moi of 1 incubation with virus for 60 min (37°C, 5% CO₂) with gentle rocking. Following this, they were washed to remove excess virus. Culture medium containing the test compounds at set concentrations were then added to the cells. Following 3 days (37°C, 5% CO₂), cells were fixed and then stained for the presence of DENV Envelope protein (Env). (B) To evaluate compounds for the ability to inhibit postviral entry processes ("postinoculation"), cells were infected with DENV at moi 1 and then treated with test compound as described above. On day 3 postinfection, culture supernatants were harvested and viral titers were quantified by plaque assay. (C) To evaluate compounds for the ability to inhibit processes that occur early in the viral life cycle ("preinoculation"), cells were preincubated with compound for 2 h (37°C, 5% CO₂). Following this, three washes were performed to remove excess compound. Cells were then infected with DENV as described above. On day 3, postinfection, culture supernatants were harvested and viral titers were quantified by plaque assay. (D) In plaque assays, test samples were used to generate a dilution series that was then used to infect cells for 1 h (37°C, 5% CO₂) with gentle rocking. After washes to remove excess virus, the cell monolayers were covered with a carboxymethylcellulose (CMC) overlay and incubated at 37°C (5% CO₂) for 5 days. Cells were then fixed and stained with crystal violet, and plaques were quantified to determine titers (per ml of supernatant) in the test samples.

Fig. 7. Anti-DENV activity of AZD0530. (A) AZD0530, a Src-selective kinase inhibitor, decreases the productive viral titer in DENV2-infected C6/36 cells, Vero cells, and Huh7 cells in a dose-dependent manner. (B) DENV viral envelope protein accumulates in AZD0530-treated Vero cells. DENV-infected Vero cell monolayers were treated with 2.5 µM AZD0530 or DMSO. Noninfected cells are indicated by arrowheads; the accumulation of viral envelope protein in the perinuclear region is indicated by arrows. Cell nuclei are stained blue with DAPI.

Table 2. List of cellular protein kinases targeted by the inhibitors screened in this study

Table 3. Structure and specificity of inhibitors examined in secondary screening efforts

SI Materials and Methods

Cell lines, Virus and Antibodies. All work with infectious dengue virus was performed in a biosafety level 2+ laboratory using protocols approved by the Harvard Committee on Microbiological Safety. The human hepatoma Huh-7 cell line and green African monkey kidney (Vero) cells were cultured in DMEM medium with 10% FBS at 37ºC with 5% CO₂. C6/36 cells, a continuous mosquito cell line derived from Aedes albopictus (Diptera: Culicidae) embryonic tissue was grown in L-15 medium containing 10 % FBS at 28 ºC. Both Huh-7 and Vero cells were purchased from ATCC; C6/36 cells were a kind gift from Lee Gerhke (Massachusetts Institute of Technology, Cambridge, MA). The four serotypes of DENV (DENV1, Hawaii strain; DENV2, New Guinea C strain; DENV3, H87 strain; and DENV4, H241 strain) and Modoc virus (M544 strain) used in this study were propagated in C6/36 cells throughout this study. DENV serotypes 1, 3, and 4, and Modoc virus were purchased from ATCC (Manassas, VA). DENV2 and poliovirus type 1 were provided by Lee Gerhke (Massachusetts Institute of Technology) and Marylynn V. Yates (University of California, Riverside, CA), respectively. Poliovirus type 1 was cultured in Vero cells. Quantification of the viruses was performed with Vero cells using a plaque assay as described in the following section. Monoclonal anti-DENV E protein antibody (US Biologicals, Swampscott, MA), polyclonal anti-DENV E protein antibody (Abcam, Cambridge, MA), polyclonal anti-Modoc virus antibody (ATCC, Manassas, VA), and monoclonal anti-poliovirus type 1 antibody (Chemicon, Temecula, CA) were used for immunofluorescence detection of virus infection in cells. Antibodies against c-Src and GAPDH, respectively, were purchased from Abcam (Cambridge, MA).

The cells were first washed once with 50 µl of PBS (Cellgro, Herndon, VA) per well in the 384-well format. The cells were then fixed with 50 µl of cold absolute methanol (Sigma, St. Louis, MO) per well in the 384-well format for 15 min at -20ºC. The subsequent cell washing steps were carried out using an automated 384-well format plate washer (EMBLA, Molecular Devices, Sunnyvale, CA). The cells were washed and rehydrated with PBS (Cellgro, Herndon, VA) by pipetting 50 µl of PBS per well in the 384-well format for 10 min. To prevent nonspecific binding of antibodies, a blocking step using 1% BSA (Sigma, St. Louis, MO) in PBS for 1 h at room temperature was conducted. Thirty microliters of a 1:5,000 diluted anti-DENV2 virus envelope antibody (US Biologicals, Swampscott, MA) was added to each well, followed by incubation of the plate for 1 h at 37 ºC. Following this, the cells were washed three times with PBS (Cellgro, Herndon, VA) with 0.05% Trition-X 100 (Sigma, St. Louis, MO) per well in the 384-well format (10 minutes/wash). Goat anti-murine monoclonal-FITC antibody (Jackson Immunoresearch, WestGrove, PA) diluted 1:5,000 was then added to each well. Incubation was carried out for 1 h at 37 ºC. After the incubation, the cells were again washed three times with PBS (Cellgro, Herndon, VA) with 0.05% Trition-X 100 (Sigma, St. Louis, MO). The cells were then counter-stained with DAPI (100 nM; Molecular Probes) prior to collection of image data by the immunofluorescence microscopy with the excitation wavelength (495 nm) and emission wavelength (520 nm) for FITC and the excitation wavelength (358 nm) and emission wavelength (461 nm) for DAPI.

1 − (3 Ã— S.D.DMSO + 3 Ã— S.D.MPA)/(meanDMSO − meanMPA).

Infectious Virus Plaque Assay.

For the immunoblotting of proteins, the gel was removed from the electrophoresis set and soaked in transfer buffer (25mM Tris-base with 20% methanol, pH 8.5). The 0.45 mm nylon membrane (Osmonics, Trevose, PA) and filter paper (BioRad, Hercules, CA) were presoaked in transfer buffer. The separation gel was placed on the nylon membrane and sandwiched between two pieces of filter papers. The transfer was carried out using BIORAD semi-dry transfer system (BioRad, Hercules, CA) at 15 mA for 30 minutes. After the blotting period, the marker lane was cut out from the membrane. The remaining blot was blocked in PBS (Cellgro, Herndon, VA) containing 5% skim milk (BioRad, Hercules, CA) for one hour to saturate the protein binding sites on the nlyon membrane. The blot was then washed thrice with TBST (25 mM Tris•HCl, pH 8.0, 125 mm NaCl, 0.1% Tween 20). Primary antibody (rabbit polyclonal anti-dengue virus, Abcam, Cambridge, MA) with a dilution of 1:1,000 was added to the blot. The blot was incubated for 2 h at 4°C on an orbital shaker. The membrane was then thoroughly washed with TBST (25 mM Tris•HCl, pH 8.0, 125 mm NaCl, 0.1% Tween 20) thrice for 10 min each. After-which, the blot was incubated with secondary antibody goat anti-rabbit conjugated with HRP (Zymed, San Francisco, CA) at dilution of 1:1,000. The incubation was carried at room temperature for 1 hr on an orbital shaker. After the incubation period, the blot was washed extensively with TBST (25 mM Tris•HCl, pH 8.0, 125 mm NaCl, 0.1% Tween 20) for 3 times with 10 min each time. The blot was developed by addition of Immun-Star HRP luminal substrate for 10 mins and the chemiluminescence signal was detected.