Viral DNA polymerase inhibitors

Terminase inhibition is an antiviral approach that may also be of consequence for other members of the herpesvirus group. In addition, since a similar DNA maturation process does not occur in higher cells, this principle offers the potential for high selectivity, in contrast to many of the viral DNA polymerase inhibitors, which also interact with cellular enzymes and hence can have severe side effects. 

Gerona-Navarro and coworkers in 2004 have reported the synthesis and the evaluation of a series of new 2-azetidinones (Fig. 50), derived from phenylalanine [281], which were designed on the basis of the structure of the reported (3-lactam inhibitors [367] and the residues implicated in the active site of the HCMV protease [417]. These compounds have been evaluated against HCMV in human embryonic lung cells [418], and the results compared to those obtained for the reference compounds, which were the model (3-lactam la of Fig. 49, the viral DNA polymerase inhibitors DHPG (ganciclovir), and HPMPC (cidofovir). 

Andrei G, De Clereq E, Snoeck R (2009) Viral DNA polymerase inhibitors. In Raney KD, Gotte M, Cameron CE (eds) Viral genome replieation. Springer, New York, p 481-526... 

To summarize, terminase inhibitors point the way toward a switch in strategy for developing HCMV inhibitors, with the aim of achieving a quality different from that of established DNA polymerase inhibitors. Intervention with viral DNA maturation arrests the replicative cycle at the DNA cleavage and packaging step, leading to an accumulation of empty procapsids and unprocessed concatemeric DNA. 

The active metabolite of acyclovir inhibits herpesvirus DNA replication in two ways. Acyclovir triphosphate acts as a competitive inhibitor for the incorporation of deoxyguanosine triphosphate (dGTP) into the viral DNA. In addition, acyclovir that is incorporated into viral DNA acts as a chain terminator because it lacks the 3 -hydroxy group necessary for further chain elongation. Viral DNA polymerase becomes irreversibly bound to an acyclovir-terminated DNA chain and is unavailable for further replicative activity. The effect of acyclovir on host cell DNA synthesis is much smaller than its effect on the viral enzyme. Concentrations of acyclovir significantly beyond the therapeutic range are required to inhibit host cell growth. 

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. 

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. 

A. Oseltamivir inhibits neuraminidase, an enzyme that cleaves neuraminic acid from oligosaccharides. Neuraminidase activity aids the movement of viral particles through neuraminic acid-rich respiratory secretions and is required for the release of progeny virions. Inhibition of viral DNA polymerase is the mechanism of action of nucleoside analogue antiviral drugs. Interferons do stimulate the JAK-STAT signaling pathway but do not stimulate proliferation of immune cells. Ribavirin inhibits GTP synthesis, and the antiretroviral protease inhibitors (e.g., ritonavir) inhibit HIV protease. 

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... 

Acyclovir (ACV) is not a true nucleoside, because the guanine residue is attached to an open-chain structure, but it mimics deoxyribose well enough for the compound to be accepted as a substrate by a thymidine kinase specified by certain herpes-type viruses. The normal thymidine kinase in mammalian cells does not recognize ACV as a substrate, however, so only virus-infected cells convert ACV to its monophosphate. Once the first phosphate has been added, the second phosphate is added by cellular guanylate kinase several other cellular kinases can add the third phosphate. The triphosphate is a more potent inhibitor of the viral DNA polymerases than of cellular DNA polymerases and also inactivates the former but not the latter. The net result is that ACV has been an effective treatment of, and prophylaxis for, genital herpes. Also it can result in dramatic relief of pain associated with shingles caused by reactivation of latent varicella-zoster virus, and has been successful in many patients with herpes encephalitis. 

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. 

It should also be noted that the activation of a mechanism-based inhibitor by its target enzyme is, formally, an example of metabolic activation. However, there is a clear distinction between the activation of a mechanism-based inhibitor described above and the metabolic activation of a prodrug. In the latter case, an inactive precursor is metabolized in the body (either chemically or enzymatically) to metabolites that possess the desired activity. For example. Acyclovir (3a) must be metabol-ically converted to the triphosphate (3b) and released into the medium before it will inhibit viral DNA polymerase. Further discussion on prodrugs may be found in volume 2, chapter 14. 

Cidofovir (Figure 24.4) is an antiviral cytidine nucleotide analog with inhibitory activity against HCMV and other herpes viruses. Cidofovir is first converted to an active diphosphate form by cellular enzymes. Antiviral effects of cidofovir are due to inhibition of viral DNA polymerase by the diphosphate metabolite (Neyts and De Clercq, 1994 Plosker and Noble, 1999 Scholar and Pratt, 2000). The diphosphate probably interacts with DNA polymerase either as an alternate substrate (incorporation at the 3 end or within the interior of the DNA chain) or as a competitive inhibitor (with respect to the normal substrate dCTP). Cidofovir inhibits HCMV DNA synthesis at intracellular concentrations 1000-fold lower than are required to inhibit cellular DNA synthesis (Neyts and De Clercq, 1994). For HSV-1 and HSV-2 corresponding concentrations are at least 50-fold lower. 

For HSV at least three mechanisms have been described that generate resistance to AC V deficiency or loss of viral TK activity, alteration in substrate specificity of the virus-encoded TK, and alteration in the substrate specificity of the viral DNA polymerase (1,8). Most of the ACVr mutants that have been isolated in vitro and recovered from clinical specimens are TK-deficient (TK). However, resistant clinical mutants that have an altered TK or altered DNA polymerase activity have occasionally been described too. Although TK mutants are crossresistant with drugs that also depend on viral TK for their activation (i.e., GCV, penciclovir and brivudin (BVDU), they remain sensitive to agents, such as PFA, vidarabine (Ara-A), and the acyclic nucleoside phosphonate (ANP) analogs. PFA, a pyrophosphate analog, is a direct inhibitor of the viral DNA poly-merase in which it binds to the site involved in releasing the pyrophosphate product of DNA synthesis. Phosphorylation of Ara-A to Ara-A triphosphate is carried out by cellular enzymes phosphorylation of ANP derivatives to their mono- and diphosphoryl derivatives is also carried out by cellular enzymes. 

Monophosphorylated by viral thymidine kinase (TK), then further bioactivated by host-cell kinases to the triphosphate. Acyclovir-triphosphate is both a substrate for and inhibitor of viral DNA polymerase when incorporated into the DNA molecule, it acts as a chain terminator because it lacks the ribosyl 3 hydroxyl group. 

The antiherpes drugs include acyclovir, ganciclovir, and foscarnet. Famciclovir and valacydovir are j newer drugs very similar to acyclovir. All inhibit viral DNA polymerase. Acyclovir and ganciclovir do so j by first being phosphorylated by viral enzymes. As well as acting as a polymerase inhibitor, acyclovir j j triphosphate is incorporated into the viral DNA where it acts as a chain terminator. The mechanisms of j j action, activities, clinical uses, and adverse effects are discussed, j... 

Adefovir dipivoxil enters cells and is deesterified to adefovir. Adefovir is converted by cellular enzymes to the diphosphate, which acts as a competitive inhibitor of viral DNA polymerases and reverse transcriptases with respect... 

Tenofovir disoproxil fumarate is hydrolyzed rapidly to tenofovir and then is phosphorylated by cellular kinases to its active metabohte, tenofovir diphosphate the active moiety is, in fact, a triphosphate compound because the parent drug starts out as the monophosphate. The intracellular diphosphate is a competitive inhibitor of viral reverse transcriptases and is incorporated into HIV DNA to cause chain termination because it has an incomplete ribose ring. Although tenofovir diphosphate has broad-spectrum activity against viral DNA polymerases, it has low affinity for human DNA polymerases-a, -P, and -y, which is the basis for its selective toxicity. 

Vidarabine is an inhibitor of viral DNA synthesis. Cellular enzymes phosphorylate vidarabine to the triphosphate, which inhibits viral DNA polymerase activity in a manner that is competitive with deoxyadenosine triphosphate. Vidarabine triphosphate is incorporated into both cellular and viral DNA, where it may act as a chain terminator. Vidarabine triphosphate also inhibits ribonucleoside reductase, RNA polyadenylation, and 5 -adenosylhomocysteine hydrolase, an enzyme involved in transmethylation reactions. Resistant variants due to mutations in viral DNA polymerase can be selected in vitro. 

Inhibitors of viral DNA polymerase, RNA polymerase, reverse transcriptase, helicase, primase, or integrase... 

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-... 

Acyclovir-triphosphate is both a substrate for and inhibitor of viral DNA polymerase... 

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