Author:

Abstract:

Several base- and/or sugar-modified UTP and GTP derivatives were observed to exert an inhibitory effect on the influenza polymerase, using an RNA elongation assay with virion-derived vRNPs. The most potent inhibitors were 5-bromo-UTP (IC50: 7.1 µM), 2’-fluoro-5-methyl-UTP (IC50: 9.5 µM), 2’-fluoro-2’-deoxy-UTP (IC50: 14 µM), 7-deaza-GTP (IC50: 4.1 µM) and 2’-fluoro-2’-deoxy-GTP (IC50: 3.7 µM). These data provide a rationale to design nucleoside analogues for selective inhibition of influenza virus in cell culture. To improve the intracellular bioavailability and activation, the phosphoramidate ProTide technology, delivering the activated (monophosphorylated) nucleoside analogue, was applied to the modified uridines and guanosines. We identified two 2’-fluoro-2’-deoxyuridine ProTides with moderate antiviral activity in influenza virus-infected cells, while the parent nucleoside analogue was inactive. To the 2’-fluoro-2’-deoxy-guanosine, we applied the double prodrug approach, combining a ProTide motif and a 6-O-substituted guanine. The lipophilicity of the latter substituent further enhances the cellular uptake of the ProTide. In a PCR-based virus yield assay, the ProTides derived from 6-O-methoxy-2’-fluoro-2’-deoxy-GMP, 6-O-ethoxy-2’-fluoro-2’-deoxy-GMP and 6-Cl-2’-fluoro-2’-deoxy-GMP displayed EC99 values of 15, 14 and 12 µM, respectively, while the corresponding nucleoside analogues showed no activity at 100 µM. The use of 5-methyl-UTP, 5-bromo-UTP and 7-deaza-GTP as alternative substrates for the natural UTP or GTP substrate, was demonstrated in the influenza polymerase assay. The RNA elongation reaction was shown to proceed equally efficient when UTP was replaced by 5-methyl-UTP or 5-bromo-UTP. A similar observation was made with 7-deaza-GTP. The implications of their incorporation on viral RNA transcription and translation are the subject of current investigation.