Metabolic pathways of N-methanocarbathymidine, a novel antiviral agent, in native and herpes simplex virus type 1 infected Vero cells

Abstract

N-Methanocarbathymidine (( N)-MCT), a thymidine analog incorporating a pseudosugar with a fixed Northern conformation, exhibits potent antiherpetic activity against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). This study contrasts the metabolic pathway of ( N)-MCT and the well-known antiherpetic agent ganciclovir (GCV) in HSV-1-infected and uninfected Vero cells. Treatment of HSV-1 infected Vero cells immediately after viral infection with ( N)-MCT profoundly inhibited the development of HSV-1 infection. Using standard plaque reduction assay to measure viral infection, ( N)-MCT showed a potency greater than that of ganciclovir (GCV), the IC 50s were 0.02 and 0.25 μM for ( N)-MCT and GCV, respectively. ( N)-MCT showed no cytotoxic effect on uninfected Vero cells (CC 50>100 μM). Dose and time dependence studies showed high levels of ( N)-MCT-triphosphate (( N)-MCT-TP), and GCV-triphosphate (GCV-TP) in HSV-1-infected cells incubated with ( N)-MCT or GCV, respectively. In contrast, uninfected cells incubated with ( N)-MCT showed elevated levels of ( N)-MCT-monophosphate only, while low levels of mono, di- and triphosphates of GCV were found following incubation with GCV. Although the accumulation rate of ( N)-MCT and GCV phosphates in HSV-1-infected cells were similar, the decay rate of ( N)-MCT-TP was slower than that of GCV-TP. These results suggest that: (1) the antiviral activity of ( N)-MCT against herpes viruses is mediated through its triphosphate metabolite; (2) in contrast to GCV, the diphosphorylation of ( N)-MCT in HSV-1- infected cells is the rate limiting step; (3) ( N)-MCT-TP accumulates rapidly and has a long half-life in HSV-1-infected cells; and (4) HSV-tk catalyzed the mono, and diphosphorylation of ( N)-MCT while monophosphorylating GCV only. These results provide a biochemical rational for the highly selective and effective inhibition of HSV-1 by ( N)-MCT.