Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Abstract

The antiviral activity of a new series of thymidine analogs was determined against vaccinia virus (VV), cowpox virus (CV), herpes simplex virus, and varicella-zoster virus. Several compounds were identified that had good activity against each of the viruses, including a set of novel 5-substituted deoxyuridine analogs. To investigate the possibility that these drugs might be phosphorylated preferentially by the viral thymidine kinase (TK) homologs, the antiviral activities of these compounds were also assessed using TK-deficient strains of some of these viruses. Some of these compounds were shown to be much less effective in the absence of a functional TK gene in CV, which was unexpected given the high degree of amino acid identity between this enzyme and its cellular homolog. This unanticipated result suggested that the CV TK was important in the mechanism of action of these compounds and also that it might phosphorylate a wider variety of substrates than other type II enzymes. To confirm these data, we expressed the VV TK and human TK1 in bacteria and isolated the purified enzymes. Enzymatic assays demonstrated that the viral TK could efficiently phosphorylate many of these compounds, whereas most of the compounds were very poor substrates for the cellular kinase, TK1. Thus, the specific phosphorylation of these compounds by the viral kinase may be sufficient to explain the TK dependence. This unexpected result suggests that selective phosphorylation by the viral kinase may be a promising new approach in the discovery of highly selective inhibitors of orthopoxvirus replication.

Antiviral Activities and Phosphorylation of 5-halo-2'-Deoxyuridines and N-Methanocarbathymidine in Cells Infected with Vaccinia Virus

“…CldU [8]. The phosphorylation studies we conducted using vaccinia-virus-infected cells treated with thymidine, BrdU and IdU confirmed that higher levels of nucleoside triphosphate were produced in TK + virus-infected LLC-MK 2 cells.…”

“…In the cell lines used in the research, viral TK made an additional contribution toward enhancing phosphorylation and increasing antiviral potency of the 2′-deoxyuridines. Other investigators have indicated that poxvirus TK is responsible for activation (monophosphorylation) of certain nucleoside analogues related to thymidine [7,8]. Some of those compounds were highly dependent upon viral TK for antiviral activity, suggesting that phosphorylation by cell TK was minimally contributory.…”

“…This feature of the virus may be exploited to identify selective new poxvirus inhibitors. Indeed, Prichard and colleagues [8] later reported a series of 2′-deoxyuridine derivatives whose antiviral activity was highly dependent upon poxvirus TK.The antipoxvirus activities of certain compounds may be dependent upon the cell line in which the assay was conducted. For example, we found that BrdU was more potent against TK + than TK -vaccinia virus, but this effect was only seen in Vero monkey cells and A549…”

“…This feature of the virus may be exploited to identify selective new poxvirus inhibitors. Indeed, Prichard and colleagues [8] later reported a series of 2′-deoxyuridine derivatives whose antiviral activity was highly dependent upon poxvirus TK.…”

Single-day famciclovir for the treatment of genital herpes: follow-up results of time to next recurrence and assessment of antiviral resistance

“…HSV isolates were evaluated in a blinded fashion (University of Alabama, Birmingham, AL, USA) for potential resistance to penciclovir, the active metabolite of famciclovir, by plaque reduction assays 13 . An isolate was considered sensitive to penciclovir if the 50% effective concentration (EC50) value was <5 mg/mL.…”