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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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/10.1101/2020.03.24.992230 doi: bioRxiv preprint To gain insight into the mechanism underlying the improvement of R1479 efficacy with CPU, we deployed a replicon assay that bypasses viral entry by electroporation of DENV RNA (Alvarez et al., 2005; Marceau et al., 2016) . Such replicon systems include the coding region needed for viral RNA translation and replication, while lacking the structural genes necessary for the production of viral particles. Consequently, viral genome replication is examined independent of viral entry and particle assembly (Kato and Hishiki, 2016) . In this assay (Figure 7) , the EC50 of R1479 alone was ~90 µM. Inhibition of de novo pyrimidine biosynthesis alone with GSK983 did not alter this value (~96 µM), whereas addition of 250 µM CPU shifted it to ~56 µM. Targeting both the de novo and salvage pathways with a combination of 1 µM GSK and 250 µM CPU lowered the EC50 of R1479 by over 4fold to ~19 µM. These results suggest that intracellular nucleotide depletion is the major driver of CPU's antiviral activity. Together, these findings highlight the potential of combining modulators of pyrimidine metabolism with inhibitors of viral genome replication.