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

From above enzymological and metabolic data, we hypothesized that the combination of CPU and GSK983 would be more effective at inhibiting dengue virus replication than any other combination. Using an infectious clone of dengue serotype 2 (DENV-2) strain 16681 engineered to express a luciferase reporter (Deans et al., 2016; Hierholzer and Killington, 1996; Marceau et al., 2016) , the efficacy and cytotoxicity of each combination treatment was tested in infected or uninfected cultures of the A549 lung carcinoma cell line ( Figure 5A ). In all assays, culture medium was supplemented with 20 µM uridine to mimic physiological plasma concentrations. In the presence of 20 µM exogenous uridine, 1 µM of the de novo pyrimidine synthesis inhibitor GSK983 alone did not significantly inhibit viral infection or cellular viability ( Figure 5A ). In line with our original hypothesis, combination of GSK983 with either 500 µM CPU and 5-F-CPU caused significant reductions in viral infection unlike the other cyclopentenyl compounds tested ( Figure 5A ). However, only CPU appeared to have a workable therapeutic index at this concentration and time point. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10. 1101 To further examine the efficacy and cytotoxicity of CPU, we conducted a more comprehensive checkerboard analysis of the CPU-GSK983 combination. While neither GSK983 nor CPU were effective as single agents in physiological uridine levels, the combination of both molecules successfully suppressed dengue virus infection ( Figure 5B ). For example, combining 0.2 µM GSK983 and 250 µM CPU inhibited ~ 50% of virus replication ( Figure 5B ). At a CPU dose of 1 mM, virus replication was suppressed almost completely. Notably, the combination treatment had much less effect on A549 cell growth, suggesting that combinations of GSK983 and CPU could be selective for inhibition of virus but not host replication.