Biosynthesis and half-life of MBX-2168-triphosphate in herpes virus-infected cells

Abstract

The third generation of methylenecyclopropane nucleoside analogs (MCPNAs) elicit an anti-viral effect against all three sub-classes of herpes viruses without inducing cytotoxicity in vitro. It has been previously established that the mechanism of action of MCPNAs is similar to that of ganciclovir (GCV) or acyclovir (ACV). However, the activation of MBX-2168, a third generation MCPNA, involves additional and unique enzymatic steps and this process has not been examined in virus-infected cells. To that end, herpes virus-infected cells were incubated with MBX-2168, synguanol, GCV, or ACV. Incubation of HCMV-infected cells with five times the EC₅₀ of MBX-2168 (4.0 μM), synguanol (10.5 μM), or GCV (25 μM) resulted in a time-dependent increase in triphosphate accumulation reaching a maximum of 48.1 ± 5.5, 45.5 ± 2.5, and 42.6 ± 3.7 pmol/10⁶ cells at 120 h, respectively. Additionally, half-lives of these compounds were similar in HCMV-infected cells (GCV-TP = 25.5 ± 2.7 h; MBX-2168-TP/synguanol-TP = 23.0 ± 1.4 h). HSV-1-infected cells incubated with five times the EC₅₀ of MBX-2168 (33.5 μM) or ACV (5.0 μM) demonstrated a time-dependent increase in triphosphate levels reaching a maximum of 12.3 ± 1.5 and 11.6 ± 0.7 pmol/10⁶ cells at 24 h, respectively. ACV-TP and MBX-2168-TP also had similar half-lives under these conditions (27.3 ± 4.8 h and 22.2 ± 2.2 h, respectively). We therefore conclude that although MBX-2168 does not follow the classical route of nucleoside analog activation, the metabolic profile of MBX-2168 is similar to other nucleoside analogs such as GCV and ACV that do.

Keywords: Cytomegalovirus; Drug metabolism; Herpes simplex virus; Methylenecyclopropane nucleoside analogs.

Figure 1.

Structures of acyclovir (ACV), ganciclovir (GCV), synguanol, and MBX-2168.

Figure 2.

a. Biosynthesis of GCV-TP, MBX-2168-TP, and Synguanol-TP in HCMV-infected HFF cells. HCMV-infected cells were incubated with five times the EC₅₀ of either GCV (25 μM), MBX-2168 (4.1 μM), or synguanol (10.5 μM). Samples were taken and processed at designated times following the addition of drugs. Maximum accumulations of GCV-TP (▲; 42.6 ± 3.7 pmol/10⁶ cells), MBX-2168-TP (■; 48.1 ± 5.5 pmol/10⁶ cells), and synguanol-TP (◆; 45.5 ± 2.5 pmol/10⁶ cells) were equivalent. b. Depletion of GCV-TP and MBX-2168-TP in HCMV-infected HFF cells. HCMV-infected cells were incubated as above with either GCV (25 μM) or MBX-2168 (4.1 μM). After five days drug-containing media was replaced with fresh media without drug. Samples were taken and processed at designated time points following drug removal. Both compounds demonstrated first order kinetics and half-lives were calculated accordingly. The half-lives of GCV-TP (▲) and MBX-2168-TP (■) were not statistically different, 25.5 ± 2.7 hours and 23.0 ± 1.4 hours, respectively.

Figure 3.

a. Biosynthesis of ACV-TP and MBX-2168-TP in HSV-infected Vero cells. HSV-infected cells were incubated with five times the EC₅₀ of either ACV (5.0 μM) or MBX-2168 (33.5 μM). Samples were taken and processed at designated times following the addition of drug. Time-dependent increases were observed in ACV-TP (●) and MBX-2168-TP (■); maximum concentrations were 11.6 ± 0.7 and 12.3 ± 1.5 pmol/10⁶ cells, respectively. b. Depletion of ACV-TP and MBX-2168-TP in HSV-infected Vero cells. HSV-infected cells were incubated as above with either ACV (5.0 μM) or MBX-2168 (33.5 μM). After 24 hours drug-containing media was replaced with fresh media without drug. Samples were taken and processed at designated time points following drug removal. Both compounds demonstrated first order kinetics and half-lives were calculated accordingly. The half-lives of ACV-TP (●) and MBX-2168-TP (■) were not statistically different, 22.2 ± 2.2 hours and 27.3 ± 4.8 hours, respectively.