HSV-DNA polymerase

In particular, acyclovir 1 has become important because of its potent antiherpes activity. Interest in this compound was further enhanced by the finding that, in HS V infected cells, its mechanism of action is due to its specific phosphorylation by the viral-encoded (but not cellular) thymidine kinase to acyclo GMP followed by further phosphorylation by cellular GMP and GDP kinases to the triphosphate, acyclo GTP, which is a selective and potent inhibitor of the HSV DNA polymerase.DHPA (3) and DHPG (2) "" also show significant antiviral activity. These findings have stimulated a widespread interest in. and led to the synthesis of. a large number of purine (and pyrimidine) acyclo nucleosides. " "... 

MECHANISMS OF ACTION AND RESISTANCE Cidofovir inhibits viral DNA synthesis by slowing and eventually terminating chain elongation. Cidofovir is metabohzed to its active diphosphate form by cellular enzymes the levels of phosphorylated metabolites are similar in infected and uninfected cells. The diphosphate both acts as a competitive inhibitor with respect to dCTP and an alternative substrate for viral DNA polymerase. The diphosphate has a prolonged intracellular tj 2 and competitively inhibits CMV and HSV DNA polymerases at concentrations one-eighth to one six-hundredth of those required to inhibit human DNA polymerases. A phosphochoUne metabolite has a prolonged intracellular hours) and may serve as an intracellular drug reser-... 

BVdU differs from IdU and F TdU by being specifically phosphorylated in the 5 -position by herpes simplex vims type-1 (HSV-1) induced thymidine kinase. This restricts its action to cells infected by HSV-1. It is less active against genital herpes (HSV-2). HSV-l-induced thymidine kinase converts BVdU to the corresponding 5 -mono- and diphosphate, but HSV-2-induced thymidine kinase stops at the stage of the 5 -phosphate of BVdU. Apparendy, cellular kinases phosphorylate BVdU-5 -diphosphate to the corresponding 5 -triphosphate, which inhibits HSV-1 DNA polymerase to a greater extent than similar cellular DNA polymerases. 

It is likely that ara-HxMP similarly exerts its antiviral activity in the form of the triphosphate, ara-HxTP, since ara-HxTP inhibits HSV-1 DNA polymerase (49). Another possible explanation of the antiviral activity of ara-HxTP is that it is metaboHcaHy converted to ara-AMP. In fact, it has been shown at Wellcome Research Laboratories that ara-HxMP is a substrate for adenylosuccinate synthetase, and that the resulting arabinofuranosyladenylosuccinate is cleaved to ara-AMP by adenylosuccinate lyase (1). The selective action of ara-A against HSV appears to be a consequence of the preferential inhibition of ara-ATP against HSV-1 and HSV-2 polymerases. Ara-ATP also inhibits normal cellular DNA polymerases, which may be the reason for its cellular toxicity. Also, it has been observed that ara-A is incorporated uniformly throughout the HSV-1 genome, which may result in defective viral DNA (50). 

The antiviral mechanism of action of acyclovir has been reviewed (72). Acyclovir is converted to the monophosphate in herpes vims-infected cells (but only to a limited extent in uninfected cells) by viral-induced thymidine kinase. It is then further phosphorylated by host cell guanosine monophosphate (GMP) kinase to acyclovir diphosphate [66341 -17-1], which in turn is phosphorylated to the triphosphate by unidentified cellular en2ymes. Acyclovir triphosphate [66341 -18-2] inhibits HSV-1 viral DNA polymerase but not cellular DNA polymerase. As a result, acyclovir is 300 to 3000 times more toxic to herpes vimses in an HSV-infected cell than to the cell itself. Studies have shown that a once-daily dose of acyclovir is effective in prevention of recurrent HSV-2 genital herpes (1). HCMV, on the other hand, is relatively uninhibited by acyclovir. 

The incorporation of acyclovir triphosphate into calf thymus DNA primer template has been shown to be much more rapid and extensive with HSV-1 DNA polymerase than with vero cell DNA polymerase a. This incorporation of acyclovir ceased after 15 min since the template is chain terminated by the acyclovir incorporation, as there is no 3 -hydroxyl group on which to continue elongation. The viral DNA polymerase is also inactivated by tight binding to the terminated template. 

Laboratory confirmation is vital to effective treatment of HSV, especially in individuals in whom a clinical diagnosis cannot be obtained. There are several methods by which a definitive diagnosis may be acquired, and these include virologic typing, serologic diagnosis, rapid point-of-care antigen detection, enzyme-linked immunosorbent assay (ELISA), immunoblot, and DNA polymerase chain reaction.27... 

The Li+-induced inhibition of the production of the HSV virus may be related to its actions upon viral DNA polymerase production and activity. Li+ reduces both the synthesis of DNA polymerase in tissue culture and the activity of DNA polymerase in vitro, each by about 50%. It has been proposed that Li+ reduces the biosynthesis of viral polypeptides and nucleic acids, and hence inhibits viral DNA replication by competition with Mg2+, a cofactor of many enzymes [243]. However, the inhibitory effect of Li+ on HSV replication in tissue culture is not affected by Mg2+ levels. A more likely hypothesis is the alteration of the intracellular K+ levels, possibly modifying levels of the high-energy phosphate compounds by replacement of either Na+ or K+ in Na+/K+-ATPase [244]. In tissue culture, HSV replication has been shown to be affected by the... 

Li+ is currently administered topically for the relief of HSV and, in addition, it has been demonstrated that the recurrence of HSV infection is inhibited in Li+-treated patients, indicating another potential prophylactic effect of Li+ [245]. Ointment containing 8% lithium succinate has been shown to reduce the severity and the incidence of recurrent genital HSV infection in man [246]. It has also been proposed that Li+ might be efficacious in treating HIV-infected patients, although any benefits have not yet been demonstrated [247]. While HTV is a RNA virus and as such might not be predicted to be affected by Li+, it is a retrovirus and utilizes a DNA intermediate for its replication and it uses a DNA polymerase. 

Famciclovir (Famvir) is the diacetyl ester prodrug of the acyclic guanosine analogue 6-deoxypenciclovir Dena-vir). Penciciovir has activity against HSV-1, HSV-2, VZV, and HB V. After oral administration, famciclovir is converted to penciciovir by first-pass metabolism. Penciciovir has a mechanism of action similar to that of acyclovir. It is first monophosphorylated by viral thymidine kinase then it is converted to a triphosphate by cellular kinases. Penciciovir triphosphate acts as a competitive inhibitor of viral DNA polymerase, but unlike acyclovir, it does not cause chain termination. 

Mutations in DNA polymerase or thymidine kinase may result in resistance. Acyclovir-resistant HSV strains that exhibit thymidine kinase deficiency are also resistant to famciclovir and penciciovir. 

Foscamet (Foscavir) is an inorganic pyrophosphate analogue that acts in vitro against HSV-1, HSV-2, VZV, CMV, EB V HBV, and HIV. It acts as a noncompetitive inhibitor of viral DNA polymerase and reverse transcriptase by reversibly binding to the pyrophosphate-binding site of the viral enzyme and preventing the cleavage of pyrophosphate from deoxynucleoside triphosphates. 

Trifluridine (Viroptic) is a fluorinated pyrimidine nucleoside that has in vitro activity against HSV-1 and HSV-2, vaccinia, and to a lesser extent, some adenoviruses. Activation of trifluridine requires its conversion to the 5 monophosphate form by cellular enzymes. Trifluridine monophosphate inhibits the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) by thymidylate synthetase. In addition, it competes with deoxythymidine triphosphate (dTTP) for incorporation by both viral and cellular DNA polymerases. Trifluridine-resistant mutants have been found to have alterations in thymidylate synthetase specificity. 

Viral DNA polymerase is an important catalyst for the synthesis of viral nucleic acids. DNA polymerase inhibitors have already been encountered as antitumor agents. Ara-A (9.5, vidarabine) is a DNA polymerase inhibitor that has demonstrated activity against herpes simplex virus type I (HSV-1) infections, responsible for cold sores on... 

Resistance to acyclovir can develop in HSV or VZV through alteration in either the viral thymidine kinase or the DNA polymerase, and clinically resistant infections have been reported in immunocompromised hosts. Most clinical isolates are resistant on the basis of deficient thymidine kinase activity and thus are cross-resistant to valacyclovir, famciclovir, and ganciclovir. Agents such as foscarnet, cidofovir, and trifluridine do not require activation by viral thymidine kinase and thus have preserved activity against... 

Famciclovir is the diacetyl ester prodrug of 6-deoxypencidovir, an acyclic guanosine analog (Figure 49-2). After oral administration, famciclovir is rapidly deacetylated and oxidized by first-pass metabolism to penciclovir. It is active in vitro against HSV-1, HSV-2, VZV, EBV, and HBV. As with acyclovir, activation by phosphorylation is catalyzed by the virus-specified thymidine kinase in infected cells, followed by competitive inhibition of the viral DNA polymerase to block DNA synthesis. Unlike acyclovir, however, penciclovir does not cause chain termination. Penciclovir triphosphate has lower affinity for the viral DNA polymerase than acyclovir triphosphate, but it achieves higher intracellular concentrations. The most commonly encountered clinical mutants of HSV are thymidine kinase-deficient these are cross-resistant to acyclovir and famciclovir. 

Trifluridine (trifluorothymidine) is a fluorinated pyrimidine nucleoside that inhibits viral DNA synthesis in HSV-1, HSV-2, CMV, vaccinia, and some adenoviruses. It is phosphorylated intracellularly by host cell enzymes, and then competes with thymidine triphosphate for incorporation by the viral DNA polymerase (Figure 49-3). Incorporation of trifluridine triphosphate into both viral and host DNA prevents its systemic use. Application of a 1% solution is effective in treating keratoconjunctivitis and recurrent epithelial keratitis due to HSV-1 or HSV-2. Cutaneous application of trifluridine solution, alone or in combination with interferon alfo, has been used successfully in the treatment of acyclovir-resistant HSV infections. 

Foscarnet (phosphonoformic acid) is an inorganic pyrophosphate analog (Figure 49-2) that inhibits viral DNA polymerase, RNA polymerase, and HIVreverse transcriptase directly without requiring activation by phosphorylation. Foscarnet blocks the pyrophosphate binding site of these enzymes and inhibits cleavage of pyrophosphate from deoxynucleotide triphosphates. It has in vitro activity against HSV, VZV, CMV, EBV, HHV-6, HHV-8, and HIV-1. 

Inhibits HSV-2 polymerase competitively with deoxyguanosine triphosphate, selectively inhibiting herpes viral DNA synthesis and replication... 

Penciclovir triphosphate has lower affinity for the viral DNA polymerase than acyclovir triphosphate, but it achieves higher intracellular concentrations and has a more prolonged intracellular effect in experimental systems. The most commonly encountered clinical mutants of HSV are thymidine kinase-deficient and are cross-resistant to acyclovir and famciclovir. 

Cidofovir is a cytosine nucleotide analog with in vitro activity against CMV, HSV-1, HSV-2, VZV, EBV, HHV-6, HHV-8, adenovirus, poxviruses, polyomaviruses, and human papillomavirus. In contrast to ganciclovir, phosphorylation of cidofovir to the active diphosphate is independent of viral enzymes. After phosphorylation, cidofovir acts both as a potent inhibitor of and as an alternative substrate for viral DNA polymerase, competitively inhibiting DNA synthesis and becoming incorporated into the viral DNA chain. Isolates with resistance to cidofovir have been selected in vitro these isolates tend to be cross-resistant with ganciclovir but retain susceptibility to foscamet. Clinically significant resistance to cidofovir has not been reported to date. 

As enzymic reactions are in theory reversible, an analogue of a nucleoside triphosphate (e.g. I or II) may be formed in which the S,Y phosphoryl residues of the triphosphate moiety are replaced by PAA or PFA residues. We have prepared the ATP analogue of PAA. This compound has, not unexpectedly, no effect on enzymes such as hexokinase which transfer the Y phosphoryl residue of ATP to a substrate. This ATP analogue is also not a substrate RNA polymerase from E. aoli or from influenza virus. Furthermore, the dTTP analogue is not a substrate for the DNA polymerase of HSV (7). 

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