Biological activities and modes of action of 9-α-d-arabinofuranosyladenine and 9-α-d-arabinofuranosyl-8-azaadenine

Abstract

From earlier studies it is known that 9-cy-D-arabinofuranosyladenine ((Y-araA) and 9-cY-D-arabinofuranosyl-8-azaadenine (cu-ara-8-azaA) have in vitro antiviral activity, are cytotoxic, and are metabolized in mammalian cells to the triphosphates. This study was designed to compare the in uiuo antiviral activities of these compounds and their loci of action with those of 9-j?-D-arabinofuranosyladenine (/3-araA). The latter compound selectively inhibits DNA synthesis in intact cells, and its triphosphate is a known inhibitor of DNA polymerases and ribonucleotide reductase. Whereas /?-araA was significantly effective in the treatment of systemic herpes simplex virus type 1 (HSV-1) infections in mice, cu-araA and a-ara-8-azaA were therapeutically ineffective. cu-AraATP at a concentration of-1 mM did not inhibit (1) DNA polymerases present in crude extracts of cultured H.Ep.-2 cells; (2) DNA polymerases present in extracts of KB cells; (3) partially purified DNA polymerase-cufrom mouse embryo cells; or (4) DNA polymerases induced by HSV-1 and HSV-2. DNA polymerasej3 from mouse embryo cells was inhibited to a small extent by 10e4M cY-araATP. In contrast, all of these enzymes were inhibited by /3-araATP at a concentration of 10m5M (as shown in these or in earlier studies). The reductions of CDP and UDP by ribonucleotide reductase from L1210 cells were not inhibited by LY-araATP (-10e3M), whereas /3-araATP produced 70-80 per cent inhibition at this concentration. In cultured H.Ep.-2 cells, a+ara-8-azaA inhibited the incorporation of thymidine, uridine, and formate into macromolecules, but it was without effect on the incorporation of adenine and hypoxanthine, and produced marginal inhibition of the incorporation of leucine. a-Ara-8-azaA produced a dose-dependent inhibition of the accumulation of ['"Cl formyl-glycinamide ribonucleotide in H.Ep.-2 cells treated with azaserine and ['"Cl formate. These results indicate that the cY-nucleosides inhibit nucleic acid synthesis by mechanisms different from those of /3-araA.

DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs

“…The sheet was developed stepwise in increasing concentrations of LiC1 in 1 M acetic acid [16], sectioned, eluted and counted as described previously [19]. Degradation in whole cell extracts was measured in the same manner near the K m concentration of 1.5 #M using extraction and DNA polymerase assay conditions described previously [20]. [21] and disrupted by the addition of 5% sarkosyl in TES (30 mM Tris-HC1 (pH 8.0)/5 mM EDTA/50 mM NaCI).…”

“…[3H]dATP (1500-4500 cpm/pmol) was used as labeled substrate; all other conditions have been published [20].…”

Inhibition of herpes simplex virus DNA replication by ara-tubercidin

“…The mechanistic basis for the differential inhibition of viral and cellular DNA synthesis was investigated by measuring the effect of the S-triphosphate of aratubercidin (ara-tubercidin-TP) on partially purified DNA polymerases. The viral DNA polymerases specified by HSV-1 and HSV-2 and cellular DNA polymerases (Y and p were isolated and assayed as described previously (Bennett et al, 1981;Reinke et al, 1978). Table 1 lists the inhibitory constants determined for ara-tubercidin-TP and illustrates that this compound was an effective inhibitor of the viral polymerases., To our knowledge, this is the first report of a herpesvirus DNA polymerase being inhibited by a pyrrolopyrimidine nucleotide.…”