Cells were infected with the specified myxovirus at an MOI = 0.1 pfu/cell (all paramyxoviruses assessed), 0.05 pfu/cells (influenza viruses), 1.0 (vaccinia virus), or 10 sindbis virus) in the presence of a range of compound concentrations or equivalent volumes of solvent (DMSO) only, and incubated in the presence of compound at 37uC. When vehicle treated controls approached the end of the logarithmical growth phase, progeny viral particles were harvested and titered by TCID 50 titration, plaque assay or TaqMan real-time PCR, respectively, as described above. Plotting virus titers as a function of compound concentration allowed quantitative assessment of resistance. Where applicable, 50% inhibitory concentrations were calculated using the variable slope (four parameters) non-linear regression-fitting algorithm embedded in the Prism 5 software package (GraphPad Software). In vitro assessment of metabolic and plasma stability JMN3-003 was mixed with liver S9 fractions (protein concentration 2.5 mg/ml) from pooled mixed gender humans (Xeno-Tech) at a final concentration of 1 mM and reactions initiated by the addition of cofactors (1.14 mM NADPH, 1.43 mM glucose-6phosphate, 1.43 mM uridine 59-diphosphoglucuronic acid, 9.42 mM potassium chloride, 2.28 mM magnesium chloride) in 100 mM potassium phosphate buffer, pH 7.4. Samples were incubated at 37uC with mixing, aliquots removed after 0, 15, 30 and 60 minutes and subjected to reversed-phase LC-MS/MS (Applied Biosystems API 4000 QTRAP with heated nebulizer; Turbo IonSpray for JMN5-166) analysis. Peak areas were measured to calculate half life and percent of input compound remaining according to the formulas t 1/2 = (20.693/slope of linear regression analysis of log transformed peak area versus) and % input remaining = (peak area of test compound at t x /peak area of test compound at t 0 )*100. Positive controls to assess the metabolic competency of the liver S9 fractions were 7-Ethoxycoumarin, Propranolol, and Verapamil (Sigma), which were analyzed in parallel to the article. To determine compound plasma stability, articles were mixed with freshly prepared human plasma at a final concentration of 0.5 mM and incubated at 37uC for up to 120 minutes. Aliquots were removed at distinct time points as indicated and analyzed by LC-MS/MS with detection of the compound at 254 nm. Values are expressed as percent of compound remaining at each time relative to the amount of that compound present at the starting time point.