Cellular DNA polymerase

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 mode of action of PMEA may be quite similar to the mechanism by which (3)-HPMPA accomplishes its selective inhibitory activity against herpes vimses. Eor PMEA to reach its active triphosphate form, it needs only two phosphorylation steps. The triphosphate derivative of PMEA has a much stronger affinity for HIV-1 reverse transcriptase than for cellular DNA polymerases (175). Whether it is actually incorporated into DNA and terminates the growing DNA chain is currentiy under investigation. 

The isolation from a marine ascidian and subsequent structure determination of polycitone A (105) (Fig. 6) was first reported [52] by Kashman and coworkers in 1994. In this paper, the penta-O-methyl derivative was reported to inhibit the growth of SV40 transformed fibroblast cells at a concentration of 10 jtg/mL. Loya, Hizi and Kashman published [53] an extensive account of the biological activity of polycitone A in 1999 in which case inhibition of retroviral reverse transcriptases and cellular DNA polymerases was described. The isolation from an ascidian and structure determination of polycitone B (106) (Fig. 4) was subsequently reported [54] by Kashman and coworkers in 2000. Obviously, the presence of extensive bromination in both polycitone A and B make this family of compounds unique among the 3,4-diarylpyrrole natural products. 

In addition to drugs such as AZT, other antivirals targeted at reverse transcriptase are also being developed. AZT (and its relatives DDI and DDC) inhibits HIV replication by mimicking normal building blocks of DNA and being selectively incorporated by reverse transcriptase into viral DNA as opposed to cellular DNA. Viral DNA that has incorporated these compounds cannot be completed, and virus replication is aborted. Other compounds have been developed that directly inhibit the activity of HIV reverse transcriptase, with relatively little effect on cellular DNA polymerases. The net effect of these compounds also is to selectively inhibit HIV replication. One class of reverse transcriptase inhibitors currently being tested is referred to as TIBO inhibitors. 

Pharmacology Foscarnet exerts its antiviral activity by a selective inhibition at the pyrophosphate binding site on virus-specific DNA polymerases and reverse transcriptases at concentrations that do not affect cellular DNA polymerases. CMV strains resistant to ganciclovir may be sensitive to foscarnet. Acyclovir- or ganciclovir-resistant mutants may be resistant to foscarnet. 

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. 

The ends of a linear chromosome are not readily replicated by cellular DNA polymerases. DNA replication requires a template and primer, and beyond the end of a linear DNA molecule no template is available for the pairing of an RNA primer. Without a special mechanism for replicating the ends, chromosomes would be shortened somewhat in each cell generation. The enzyme telomerase solves this problem by adding telomeres to chromosome ends. 

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. 

Decatromicins A (1218) and B (1219) are produced by an Actinomadura sp. and are active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (1229,1230). These compounds are closely related to pyrrolosporin A (1220) from Micromonospora sp. (1231,1232). The ascidian Polycitor africanus from Madagascar has afforded the new polycitone B (1221) (1233), which is related to the known polycitone A (1), a potent inhibitor of retroviral reverse transcriptases and cellular DNA polymerases (1234). The known polycitrin B was synthesized for the first time (1235). 

Loya S, Rudi A, Kashman Y, Hizi A (1999) Polycitone A, a Novel and Potent General Inhibitor of Retroviral Reverse Transcriptases and Cellular DNA Polymerases. Biochem J 344 85... 

Mode of action AZT must be converted to the corresponding nucleoside triphosphate by mammalian thymidine kinase in order for it to exert its antiviral activity. AZT-triphosphate is then incorporated into the growing chain of viral (but not mammalian nuclear) DNA by reverse transcriptase1. Because AZT lacks a hydroxyl at the 3 position, another 5 -3 phosphodiester linkage cannot be formed. Thus, synthesis of the DNA chain is terminated, and replication of the virus cannot take place. The relative lack of discrimination of the viral reverse transcriptase is believed to favor the introduction of the AZT into the viral-catalyzed process the cellular DNA polymerase is more selective. In addition, the phosphory-... 

No sample preparation or processing is needed for testing culture supernatants. Typically, 10 pL is applied to the RT buffer A, which can be prepared without the addition of NP-40 and therefore has no NP-40 (the final concentration of NP-40 in the RT reaction will be 0.06%). Culture supernatants should be cell-tree because contamination with cellular DNA polymerases may produce false positive results. 

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