Metabolic pathways of N-methanocarbathymidine, a novel antiviral agent, in native and herpes simplex virus type 1 infected Vero cells.
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 IC50s were 0.02 and 0.25 microM for (N)-MCT and GCV, respectively. (N)-MCT showed no cytotoxic effect on uninfected Vero cells (CC50>100 microM). 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.
Zalah
Livnat
L
Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel.
Huleihel
Mahmoud
M
Manor
Esther
E
Konson
Alexander
A
Ford
Harry
H
Marquez
Victor E
VE
Johns
David G
DG
Agbaria
Riad
R
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D015774:Ganciclovir / Q000378:metabolism / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000187:drug effects* / Q000502:physiology; D010766:Phosphorylation; D011122:Polyphosphates / Q000378:metabolism; D013936:Thymidine / Q000031:analogs & derivatives / Q000494:pharmacology*; D014709:Vero Cells
D000998:Antiviral Agents; D011122:Polyphosphates; C469995:(north)-methanocarbathymidine; C005692:triphosphoric acid; D015774:Ganciclovir; D013936:Thymidine