Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

Qi Liu; Amita Gupta; Ayse Okesli-Armlovich; Wenjie Qiao; Curt R. Fischer; Mark Smith; Jan E. Carette; Michael C. Bassik; Chaitan Khosla

A549 cells were plated overnight at 80,000 cells/well in 24-well plates in complete DMEM. The next day, cells were treated with dipyridamole or 250 µM or 500 µM CPU, 5-F-CPU or 5'-F-CPU in DMEM additionally supplemented with 1 µM GSK983 and 5 µM uridine. Six or twelve hours after drug addition, the cell culture medium was removed and the whole plate was rapidly rinsed twice by dipping vertically into a beaker containing 37 ºC Milli-Q water. The plate was then placed on dry ice, followed by the addition of 0.5 mL -20 ºC lysis buffer (MeCN:MeOH:H2O = 2:2:1) containing 0.5 M formic acid and 450 nM uridine 15 N2 monophosphate sodium salt (Sigma Aldrich). The solution was then sonicated for 3 ´ 5 minutes on ice for the metabolite extraction. Subsequently, the sample was frozen with liquid nitrogen, freeze-dried, re-suspended with 150 µL Milli-Q water, and finally filtered through a MultiScreen 96-well filter plate prior to LC-MS analysis.