GSK983, a potent broad-spectrum antiviral compound in vitro, has been identified as an inhibitor of the de novo pyrimidine nucleotide biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) in our previous genome-wide screening. However, this compound was not active in vivo due to the compensation of pyrimidine nucleotides by the salvage pathway. Guided by the screening result that the knockdown of pyrimidine metabolism genes uridine-cytidine kinase 2 (UCK2) and cytidine monophosphate kinase 1 (CMPK1) sensitized cells to GSK983, we designed and synthesized a series of cyclopentenyl uracil (CPU) analogues as the UCK2 inhibitors for the combination antiviral therapy with GSK983. Enzymatic and cellular assays were then developed to evaluate these analogues. In conjugation with a LC-MS/MS assay, we found that CPU was not only an inhibitor of UCK2, but also a substrate. Notable, the resulting CPU monophosphate then served as a substrate and inhibitor of CMPK1, and ultimately it was converted into the triphosphate.<br/>On one hand, the afflux of CPU toward its triphosphate in cells depleted intracellular pyrimidine nucleotide pool to inhibit the replication of viral RNA. On the other hand, its triphosphate metabolite was a putative RNA-dependent RNA polymerase (RdRp) inhibitor, which in turn enhanced its antiviral activity. Taken together, our biochemical characterization of CPU analogues confirmed that inhibition of UCK2 and CMPK1 enzymes synergized with DHODH inhibitor GSK983 to effectively inhibit viral replication. Particularly, targeting UCK2, CMPK1 and potentially RdRp, CPU exhibited poly-pharmacologic effects in cells.

Biochemical characterization of cyclopentenyl uracil analogues: Application in the combination antiviral therapy

2019 Fall National Meeting

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