T-705-Derived Prodrugs Show High Antiviral Efficacies against a Broad Range of Influenza A Viruses with Synergistic Effects When Combined with Oseltamivir

Figure 1. Chemical structures of T-705 1a and T-1105 1b, T-1106 2a and the modified T-1106 nucleosides 2b–f, as well as the T-1106-DiPPro-prodrugs 3a and 3b and the T-1106-TriPPPro-prodrugs 4a–4f. After nucleobases 1a and 1b reach the inside of the infected cell via passive diffusion, they are metabolized into their 5′-monophosphates via the humane enzyme HGPRT. The 5′-monophosphates are then further metabolized by human kinases into the antiviral active 5′-triphosphates. The nucleosides 2a–f are transported into the infected cell by nucleoside transporters. Afterward, the nucleosides are metabolized by human kinases into the 5′-triphosphates. The T-1106-TriPPPro-prodrugs 4a–4f are able to diffuse through the cell membrane due to the lipophilic masking units. Once they are inside the cell, lipophilic masking units are cleaved, and the 5′-triphosphates are released. The T-1106-DiPPro-prodrugs 3a and 3b release the 5′-diphosphates instead, which are then metabolized into their 5′-triphosphates.

Figure 2. Antiviral activity of T-705 (1a) and its derivates against seasonal influenza A virus (IAV) replication. (a) MDCKII cells were infected with H3N2 IAV at a multiplicity of infection of 0.01 and subsequently treated with T-705 and its derivates at a final concentration of 50 µM. Cell culture supernatants were collected at 24 h post-infection, and infectious virus titers were determined by plaque assay. DMSO treatment was used as a negative control (0% inhibition). Shown are the average virus titers as log10 plaque forming units per ml (pfu/mL) ± SD of two independent biological replicates, each performed with technical triplicates. Individual data points are shown as well. The detection limit (DL) is indicated with a dashed line. Statistically significant differences were calculated using Kruskal-Wallis test in combination with Dunn’s multiple comparisons tests, and p values are displayed. (b–e, left y-axis) MDCKII cells were infected with H1N1 IAV at a multiplicity of infection of 0.01 and subsequently treated with T-705 1a (b) or its prodrug derivates 3a (c), 3b (d), and 4a (e) at eight different concentrations (range: 0.01–50 µM). Cell culture supernatants were collected at 24 h…

Figure 3. Antiviral activity of T-705 (1a) and the T-1106 prodrugs (3a, 3b, and 4a) against highly pathogenic avian influenza viruses (HPAIV). (a–h, left y-axis) MDCKII cells were infected with H5N1 (a–d) or H7N9 (e–h) HPAIV at a multiplicity of infection of 0.01 and subsequently treated with T-705 1a (a,e) or its prodrug derivates 3a (b,f), 3b (c,g), and 4a (d,h) at eight different concentrations (range: 0.001–50 µM). Cell culture supernatants were collected at 24 h post-infection, and infectious virus titers were determined by plaque assay. DMSO treatment was used as a negative control. Shown is the average inhibition of viral replication (%) ± SD by the respective compound compared to the DMSO control treatment (0% inhibition). Non-linear regression (red curve) was performed to determine the inhibitory concentration 50 (IC50) values. (a–h, right y-axis) MDCKII cells were treated with T-705 1a (a,e) or its prodrug derivates 3a (b,f), 3b (c,g), and 4a (d,h) at eight different concentrations (range: 0.001–50 µM). At 24 h post-treatment, cell viability was measured using an MTT-based assay according to the manufacturer’s instructions. DMSO treatment was used as a control. The average…

Figure 4. Antiviral activity of T-1106-derived DP prodrug 3a in combination with the neuraminidase inhibitor oseltamivir (OS) against H5N1 HPAIV replication. (a–d) MDCKII cells were infected with H5N1 HPAIV at a multiplicity of infection of 0.01 and subsequently treated with combinations of the T-1106 prodrug 3a and the neuraminidase inhibitor oseltamivir. The tested concentrations are based on the respective IC1, IC10, IC50, and IC90 values for each drug. Cell culture supernatants were collected at 24 h post infection and infectious virus titers were determined by plaque assay. DMSO treatment was used as a negative control (0% inhibition). Bliss independence (Bliss; a), Zero Interaction Potency (ZIP; b), highest single agent (HSA; c), and Loewe additivity (Loewe; d) reference models were used to calculate and visualize synergistic areas. The shown landscapes are color-coded into synergistic (red) and antagonistic (green) interactions. Data are derived from three independent biological replicates, each performed in technical triplicates.