Mechanism of action of T-705 against influenza virus

Abstract

T-705, a substituted pyrazine compound, has been found to exhibit potent anti-influenza virus activity in vitro and in vivo. In a time-of-addition study, it was indicated that T-705 targeted an early to middle stage of the viral replication cycle but had no effect on the adsorption or release stage. The anti-influenza virus activity of T-705 was attenuated by addition of purines and purine nucleosides, including adenosine, guanosine, inosine, and hypoxanthine, whereas pyrimidines did not affect its activity. T-705-4-ribofuranosyl-5'-triphosphate (T-705RTP) and T-705-4-ribofuranosyl-5'-monophosphate (T-705RMP) were detected in MDCK cells treated with T-705. T-705RTP inhibited influenza virus RNA polymerase activity in a dose-dependent and a GTP-competitive manner. Unlike ribavirin, T-705 did not have an influence on cellular DNA or RNA synthesis. Inhibition of cellular IMP dehydrogenase by T-705RMP was about 150-fold weaker than that by ribavirin monophosphate, indicating the specificity of the anti-influenza virus activity and lower level of cytotoxicity of T-705. These results suggest that T-705RTP, which is generated in infected cells, may function as a specific inhibitor of influenza virus RNA polymerase and contributes to the selective anti-influenza virus activity of T-705.

FIG. 1.

Time-of-addition experiment. MDCK cells were inoculated with influenza A/PR/8/34 virus at a multiplicity of infection of 0.001. T-705 was added at the indicated times. Viral yields were determined at 10 h postinfection by plaque assays. Open columns, the mean viral yield for control cells; closed columns, the mean viral yield for cells treated with T-705 (10 μg/ml); vertical lines, standard deviations (n = 3). Two independent experiments were done, and representative data are shown. *, results significantly different from that for each control by Student's t test (P < 0.05), **, results significantly different from that for each control by Student's t test (P < 0.01).

FIG. 2.

HPLC radiochromatogram of intracellular extracts from MDCK cells incubated with [¹⁴C]T-705 for 24 h. Extracts were analyzed by HPLC on a SAX column, as described in Materials and Methods.

FIG. 3.

Inhibitory effects of T-705RTP, rivabirin TP, T-705, and T-705RMP on influenza virus RNA polymerase activity. Results are means ± standard deviations (n = 3). *, results significantly different from those for the controls by the Tukey test (P < 0.01).

FIG. 4.

Inhibitory effects of T-705RMP, ribavirin MP, and mycophenolic acid on cellular IMPDH activity. The formation of [¹⁴C]XMP from [¹⁴C]IMP was determined with a radiometric 525TR HPLC system and is given as the percentage of that for the vehicle-treated control. The data are means ± standard deviations (n = 3). Two independent experiments were done, and representative data are shown.

FIG. 5.

Hypothetic mechanism of action of T-705. T-705 may be converted to T-705 ribofuranosyl phosphates by host cell enzymes. The triphosphate form, T-705RTP, strongly inhibited by influenza virus RNA polymerase activity. Meanwhile, T-705 and its phosphates showed little inhibitory effect on the replication of the host cell.