acyclovir-monophosphate and acyclovir-triphosphate

Acyclovir, 9-(2-hydroxyethoxymethyl)guanine, is an acyclic nucleoside analogue which has a high activity and selectivity for herpes viruses, particularly herpes simplex viruses types 1 and 2 and varicella zoster virus. This selectivity is due to the initial activation of the drug by phosphorylation by a herpes virus-specified thymidine kinase. Normal cellular enzymes do not phosphorylate acyclovir to any significant degree. Acyclovir monophosphate is subsequently converted to a triphosphate which is a more potent inhibitor of herpes virus DNA polymerases than of cellular DNA polymerases. The relationship between the amount of acyclovir triphosphate formed and its inhibition constant (Ki) for the particular viral or cellular DNA polymerase is predictive of the inhibitory activity of acyclovir on DNA replication.

Topics: Acyclovir; DNA Polymerase II; DNA Replication; DNA, Viral; Herpesvirus 3, Human; Peptide Chain Termination, Translational; Phosphorylation; Simplexvirus; Thymidine Kinase

Acyclovir, an acrylic purine nucleoside analog, is a highly potent inhibitor of herpes simplex virus (HSV), types 1 and 2, and varicella zoster virus, and has extremely low toxicity for the normal host cells. This selectivity is due to the ability of these viruses to code for a viral thymidine kinase capable of phosphorylating acyclovir to a monophosphate; this capability is essentially absent in uninfected cells. The acyclovir monophosphate (acyclo-GMP) is subsequently converted to acyclovir triphosphate (acyclo-GTP) by cellular enzymes. Acyclo-GTP persists in HSV-infected cells for many hours after acyclovir is removed from the medium. The amounts of acyclo-GTP formed in HSV-infected cells are 40 to 100 times greater than in uninfected Vero cells. Acyclo-GTP acts as a more potent inhibitor of the viral DNA polymerases than of the cellular polymerases. The DNA polymerases of HSV-1 and HSV-2 also use acyclo-GTP as a substrate and incorporate acyclo-GMP into the DNA primer-template to a much greater extent than do the cellular enzymes. The viral DNA polymerase binds strongly to the acyclo-GMP-terminated template, and in thereby inactivated.

Topics: Acyclovir; Animals; Antiviral Agents; Cell Line; Guanine; Humans; Nucleic Acid Synthesis Inhibitors; Simplexvirus; Thymidine Kinase

The potent inhibition of herpes simplex type 1 (HSV-1) DNA polymerase by acyclovir triphosphate has previously been shown to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'-end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism of inhibition of HSV-1 DNA polymerase has been used here to design an affinity column for the enzyme. A DNA hook template-primer containing an acyclovir monophosphate residue on the 3'-primer terminus has been synthesized and attached to a resin support. In the absence of added nucleotides, the column behaves as a simple DNA-agarose column, and HSV-1 DNA polymerase can be chromatographed using a salt gradient. The presence of the next required nucleotide encoded by the template (dGTP) increases the affinity of HSV-1 DNA polymerase for the acyclovir monophosphate terminal primer-template attached to the resin, and the enzyme is retained even in the presence of 1 M salt. The enzyme can be eluted from the column with a salt gradient after removal of the nucleotide from the buffer. Traditionally, the affinity purification of an enzyme relies on elution by a salt gradient, pH gradient, or more selectively by addition of a competing ligand (substrate/inhibitor) to the elution buffer. In the present example, elution of HSV-1 polymerase is facilitated by removal of the substrate from the buffer. This represents an example of mechanism-based affinity chromatography.

Topics: Acyclovir; Antiviral Agents; Base Sequence; Chromatography, Affinity; DNA-Directed DNA Polymerase; HeLa Cells; Humans; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Synthesis Inhibitors; Oligonucleotides; Simplexvirus; Templates, Genetic

The metabolism of acyclovir to its mono-, di-, and triphosphate derivatives was examined in uninfected and virus-infected cells. The level of phosphorylation of acyclovir was dependent upon virus type, cell line, exogenous drug concentration, and exposure time. Acyclovir phosphorylation was inhibited by exogenously added nucleosides. The order of inhibition was deoxythymidine greater than deoxycytidine greater than guanosine greater than or equal to deoxyguanosine. Acyclovir triphosphate persisted in infected cells after removal of the drug from the medium. The initial half-life of the triphosphate was 1.2 h in the absence of the drug in the medium, but triphosphate levels reached a plateau after 6 h. The presence of low concentrations of the drug in the medium resulted in a longer persistence of the intracellular triphosphate and a higher plateau level.

Topics: Acyclovir; Antiviral Agents; Cell Line; Chromatography, High Pressure Liquid; Culture Media; Deoxyribonucleosides; Guanine; Phosphorylation; Simplexvirus; Time Factors