L- and D-Enantiomers of 2′,3′-Dideoxycytidine 5′-Triphosphate Analogs as Substrates for Human DNA Polymerases

Abstract

5-Triphosphates of ␤-D and ␤-L-enantiomers of 2,3-dideoxycytidine (ddC), 2,3-dideoxy-5-fluorocytidine (FddC), 1,3-dioxolane-cytidine (OddC), and 1,3-dioxolane-5-fluorocytidine (FOddC) were evaluated as inhibitors and substrates for human DNA polymerases ␣, ␤, ␥, ␦, and ⑀. L-ddCTP was not a substrate or inhibitor for any DNA polymerase studied; L-FddCTP was not an inhibitor or substrate for replicative DNA polymerases and was a less potent inhibitor of DNA polymerases ␥ and ␤ than its D-enantiomer by 2 orders of magnitude. In contrast, all L-dioxolane analogs were potent inhibitors and chain terminators for all cellular DNA polymerases studied. The K i values of their 5-triphosphates for DNA polymerase ␥ were found to be in the following order: Several 2Ј,3Ј-dideoxynucleoside analogs have been approved for the treatment of patients with AIDS (1, 2). Among them, ddC 1 has been shown to be one of the most potent inhibitors of HIV replication. Recently, SddC was found to have potent activity against HIV (3, 4) and hepatitis B virus (5) in cell culture. It was subsequently shown that the stereoisomer LSddC (3TC) was responsible for the antiviral effect, while cytotoxicity was associated mainly with the D-enantiomer (6 -9). L-ddC and L-FddC were also found to be active against hepatitis B virus and HIV in culture without much toxicity (10 -12). In contrast to L-SddC or L-FddC, L-OddC and L-FOddC were markedly more toxic than their D-enantiomers against human leukemic CEM cells (13). The spectrum of L-OddC toxicity against human tumor cells differed from that of cytosine arabinoside. L-OddC was also shown to have activity against solid human tumors in nude mice (14). Currently, L-OddC is being evaluated further as an anticancer agent. The reason why L-OddC but not L-ddC or L-SddC has potent antitumor activity is not clear, but it is probably associated with its interference in cellular DNA synthesis. L-SddC and L-OddC were shown to be phosphorylated by cellular kinases to the corresponding 5Ј-triphosphate metabolites inside cells. The antiviral effects of nucleoside analog are due to preferential interference with viral replication caused by incorporation into DNA by viral DNA polymerase. The interaction of 5Ј-triphosphate metabolites of L-OddC or L-FddC with cellular DNA polymerases may be one of the key factors in determining L-OddC cytotoxicity.In the present paper, the interaction of 5Ј-triphosphates of Dand L-enantiomers of 2,3-dideoxycytidine and 1,3-dioxolanecytidine as well as their 5-fluoro-derivatives with human pol ␣, pol ␤, pol ⑀, pol ␦, and pol ␥ is reported. EXPERIMENTAL PROCEDURES Materials and Compounds-L-and D-stereoisomers of ddC and OddCand their 5-fluoro-derivatives were synthesized as described previously (11,13). The triphosphate forms of these compounds were synthesized in our laboratory (15). Unlabeled dNTP and ddNTP were purchased from Boehringer Mannheim. Poly(rA)⅐oligo(dT) 10 and poly(dA)⅐oli-go(dT) 12-18 were obtained from Pharmacia Biotech Inc.ssDNA cellulose was obtained from Sigma. DEAE c...

Nucleoside analogs: molecular mechanisms signaling cell death

“…Like other Responses to nucleoside analogs B Ewald et al nucleoside analogs, the main mechanism causing apoptosis is believed to be incorporation of the triphosphate into DNA. However, as troxacitabine lacks a 3 0 -hydroxyl group, incorporation of a single troxacitabine molecule does not permit further extension (Kukhanova et al, 1995). Thus, once incorporated into DNA, this nucleotide acts as a de facto chain terminator.…”

Substrate specificity of the p53-associated 3′-5′ exonuclease

“…Utilizing less than one unit of SmaI reduces the star activity, however it also significantly lowers the efficiency of the reaction (less than 50 per cent of the total DNA is digested to produce double-strand breaks). The annealing of restricted sequences was verified as previously described (Kukhanova et al, 1995). Briefly, 5'-3' incorporation of cold nucleotides into the BamHI-generated, 5'-end radiolabeled 15-mer along the M13mp19 (+) template was examined in reactions catalyzed by the Klenow fragment of DNA polymerase I after 45 and 90 min at 378C.…”

“…Act. 6000 Ci/mmol) and puri®ed on G25 Sephadex columns as previously described (Kukhanova et al, 1995). Mismatched DNA substrates were prepared by annealing 5'-radiolabeled 17-mers (synthesized with A, T or G instead of C at their 3'-terminals) with a threefold molar excess of M13mp19 (+) DNA to create G : A, G : T and G : G mispairs.…”

The Enantioselectivity of Enzymes Involved in Current Antiviral Therapy Using Nucleoside Analogues: A New Strategy?

“…The lack of enantioselectivity of the replicative human DNA polymerases α, β, ε is particularly apparent in the case of the dioxolane-cytidine derivatives OddCTP and 5-FOddCTP, and concerns both their inhibitor and substrate properties (Kukhanova et al, 1995). However, there are exceptions, and the enantioselectivity in the substrate or inhibition properties of these enzymes appears to be fairly high with respect to the enantiomers of β-FMAUTP, β-dTTP, β-dCTP (Table 1) and BHCGTP (Terry et al, 1991).…”