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

The effect of 250 µM of each CPU analog on UCK2 activity was evaluated in the presence of 50 µM uridine. CPU and 5-F-CPU showed much higher ATP consumption compared to other analogs ( Figure 3A ), suggesting these two compounds were substrates of UCK2. We then individually measured the steady state kinetic parameters of UCK2 using uridine, CPU and 5-F-CPU as substrates. Their KM values were 86, 25, and 41 µM, and their kcat / KM values were 2.6´10 5 , 1.5´10 5 , and 7.3´10 4 s -1 ×M -1 ( Figure 3B ). As predicted by the relative magnitude of these kinetic parameters, addition of CPU or 5-F-CPU to an assay mixture containing UCK2, uridine and ATP resulted in a dose-dependent decrease in the rate of UMP synthesis ( Figure 3C ).