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

. CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 //doi.org/10. /2020 To continuously monitor reaction progress spectrophotometrically, ATP hydrolysis was coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH) (Tomoike et al., 2017) . Reactions were conducted at room temperature in 100 µL in 96-well plates (Greiner Bio-One, UV-Star, Half Area). Mixtures contained 20 mM HEPES (pH 7.2), 100 mM KCl, 2 mM MgCl2, 300 µM ATP, 0-500 µM uridine, 0-500 µM CPU analogs, 10 nM UCK2 (unless otherwise noted), 1 mM phosphoenolpyruvate, 500 µM NADH and 20 units/mL of PK and LDH. Progress was monitored in the linear region using a Biotek Synergy HT and kinetic and inhibition constants were determined using GraphPad Prism 7 (GraphPad Software).