BDBM50366389 GENTAMICIN
BDBM50118510 FMS2-gentamicin C1 CHEMBL384757
FMS3-gentamicin C1 CHEMBL264831 BDBM50118508
SCH-9724 Uromycine BDBM50476074 Gentamicin
- Viral Plaque Reduction Assay African green monkey kidney BSC-1 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL Life Technologies, Gaithersburg, Md.) and 0.1% gentamicin antibiotic at 37° C. in a humidified 5% CO2 environment. Confluent BSC-1 cells were infected with vaccinia virus at an MOI of 0.005 in 48-well plate. The test compounds and control cidofovir were dissolved in DMSO and diluted with the medium. One hour post infection, 400 uL of the test compounds and control were added per well at concentrations ranging from 200 nM to 200 uM and incubated at 37° C. for 16 hours. A 5% solution of formaldehyde in PBS was used to fix the cells. After washing twice with PBS, the plate was stained with 0.2% crystal violet in 50% ethanol.
- Cytopathic Effect Assay with RSV A2 in HEp-2 Cells Test compounds were dissolved in DMSO and then prepared in eight half-log dilutions in MEM medium with 50 μg/mL gentamicin and 2% FBS (MA-104 cells only). Each dilution was added to 5 wells of a 96-well plate with 80-100% HEp-2 cells. Three wells of each dilution were infected with respiratory syncytial virus A2 (ATCC VR-1540) and two wells remained uninfected as toxicity controls. After maximum cytopathic effect (CPE) was observed microscopically in untreated virus control wells, plates were stained with neutral red dye for approximately 2 hours, then supernatant dye was removed and the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol and read on a spectrophotometer. Optical density values were normalized based on cell and virus controls, then the concentration of test compound required to inhibit CPE by 50% (EC50) was calculated by regression analysis. The reported EC50 value is the mean of at least two experiments.
- Cytopathic Effect Assay with RSV A2 in MA-104 Test compounds were dissolved in DMSO and then prepared in eight half-log dilutions in MEM medium with 50 μg/mL gentamicin and 2% FBS (MA-104 cells only). Each dilution was added to 5 wells of a 96-well plate with 80-100% MA-104 cells. Three wells of each dilution were infected with respiratory syncytial virus A2 (ATCC VR-1540) and two wells remained uninfected as toxicity controls. After maximum cytopathic effect (CPE) was observed microscopically in untreated virus control wells, plates were stained with neutral red dye for approximately 2 hours, then supernatant dye was removed and the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol and read on a spectrophotometer. Optical density values were normalized based on cell and virus controls, then the concentration of test compound required to inhibit CPE by 50% (EC50) was calculated by regression analysis. The reported EC50 value is the mean of at least two experiments.
- D3 Radioligand Binding Assay Compounds were tested for their ability to compete with the orthosteric radioligand [3H]methylspiperone for binding to the D3 DAR using stable HEK cell lines expressing the D3 DAR (Codex Biosciences, Gaithersburg, Md., Catalogue no. CB-80300-206) as described in the detailed protocol below presented in Table 5. Cells were cultured in Dulbecco's modified Eagle's Medium (Corning, catalogue no. 10-013) containing 10% FBS, 1,000 units/mL Penicillin, 1,000 mg/mL Streptomycin, 100 mM sodium pyruvate, 1 μg/mL Gentamicin, and 250 mg/mL G418. All cells were maintained at 37° C. in 500 CO2 and 90% humidity. For radioligand binding assays cells were removed mechanically using calcium-free Earle's balanced salt solution (EBSS). Intact cells were collected by centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl2 at pH 7.4. Homogenates were centrifuged at 30,000×g for 30 minutes. The membranes were re-suspended in EBSS (US Biological, catalogue no. E0249-05) pH 7.4 to a concentration of 16 μg/mL. For competition binding studies, membrane preparations were incubated for 90 minutes at room temperature with various concentrations of compound and a single concentration of [3H]methylspiperone (Perkin Elmer, NET856) in a reaction volume of 250 μL. Non-specific binding was determined in the presence of 4 μM (+)-butaclamol (Sigma-Aldrich, catalogue no. D033). Bound ligand was separated from unbound by filtration through GF/C filters using a PerkinElmer cell harvester and quantified on a Top-count (PerkinElmer). Ki values were determined using Cheng-Prusoff equation from observed IC50 values and ligand Kd values from separate saturation experiments.