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

In a similar manner as UCK2 assay, ATP hydrolysis was coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH) to continuously monitor reaction progress spectrophotometrically. Reactions were conducted at room temperature in 50 µL in 96-well plates (Greiner Bio-One, UV-Star, Half Area). Mixtures contained 50 mM TrisHCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 2 mM DTT, 500 µM NADH, 1 mM PEP, 20 units/mL of PK and LDH. To 39.5 µL above reaction mixtures were add 5 µL 0-1 mM stock solutions of substrates (CMP from the commercial vendor and denatured UCK2 reaction mixtures containing newly synthesized CMP, CPU-MP and 5-F-CPUMP) in UCK2 assay buffer [20 mM HEPES (pH 7.2), 100 mM KCl, 2 mM MgCl2]. After the UV-readout at 340 nm reached stable in ca. 5 minutes, a mixture of ATP and CMPK1 in assay buffer [50 mM TrisHCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 2 mM DTT] was added to give a final reaction volume of 50 µL and a final ATP concentration of 500 µM and CMPK1 of 20-100 nM. Progress was monitored in the linear region using a Biotek Synergy HT, and kinetic constants were determined using GraphPad Prism 7 (GraphPad Software). Generation of CPU-DP and 5-F-CPU-DP was confirmed by LC-MS/MS ( Figure S2 ).