FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024382021> ?p ?o ?g. }

• W2024382021 endingPage "23059" @default.
• W2024382021 startingPage "23055" @default.
• W2024382021 abstract "5′-Triphosphates of β-D and β-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 α, β, γ, δ, and ɛ. 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 γ and β 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 Ki values of their 5′-triphosphates for DNA polymerase γ were found to be in the following order: D-ddC < D-FddC ≤ L-OddC ≤ D-FOddC < L-FOddC L-FddC. The Ki values of L-OddCTP for the reactions catalyzed by DNA polymerases α, δ, ɛ, β, and γ were 6.0, 1.9, 0.4, 3.0, and 0.014 μM, respectively, and those of L-FOddCTP were 6.5, 1.9, 0.7, 19, and 0.06 μM, respectively. The Km values for incorporation of L-OddCTP into the standing points of primer extension were also evaluated and determined to be 1.3, 3.5, 1.5, 2.8, and 0.7 μM for DNA polymerases α, δ, ɛ, β, and γ, respectively. The incorporation of dioxolane analogs into DNA by replicative DNA polymerases could explain their potent cellular toxicity. 5′-Triphosphates of β-D and β-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 α, β, γ, δ, and ɛ. 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 γ and β 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 Ki values of their 5′-triphosphates for DNA polymerase γ were found to be in the following order: D-ddC < D-FddC ≤ L-OddC ≤ D-FOddC < L-FOddC L-FddC. The Ki values of L-OddCTP for the reactions catalyzed by DNA polymerases α, δ, ɛ, β, and γ were 6.0, 1.9, 0.4, 3.0, and 0.014 μM, respectively, and those of L-FOddCTP were 6.5, 1.9, 0.7, 19, and 0.06 μM, respectively. The Km values for incorporation of L-OddCTP into the standing points of primer extension were also evaluated and determined to be 1.3, 3.5, 1.5, 2.8, and 0.7 μM for DNA polymerases α, δ, ɛ, β, and γ, respectively. The incorporation of dioxolane analogs into DNA by replicative DNA polymerases could explain their potent cellular toxicity. INTRODUCTIONSeveral 2′,3′-dideoxynucleoside analogs have been approved for the treatment of patients with AIDS(1Mitsuya H. Broder S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1911-1915Google Scholar, 2Mitsuya H. Weinhold K.J. Furman P.A. Clair St., M.H. Lehrman S.N. Gallo R.C. Bolognesi D. Barry D.W. Broder S. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7096-7100Google Scholar). Among them, ddC 1The abbreviations used are: ddC, ddCMP, and ddCTP2′,3′-dideoxycytidine and its mono- and triphosphates, respectively, all other nucleosides and their 5′-triphosphates have the same abbreviations throughout the paper. pol α, pol δpol β pol γ, and pol ɛDNA polymerases α, δ, β, γ, and ɛ, respectivelydNTP2′-deoxynucleoside 5′-triphosphateHIVhuman immunodeficiency virusssDNAsingle-stranded DNABuAdATP2[p-(n-butyl)anilino]dATP. Abbreviations of nucleotide analogs used are shown in Fig. 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 (3Belleau, B., Dixit, D., Nguyen-Ba, N., Kraus, J. L., (1989) Fifth International Conference on AIDS, Montreal, Canada, June 4-9, 1989, p. 515.Google Scholar, 4Soudeyns H. Yao X.J. Gao Q. Belleau B. Kraus J.L. Nguyen-Ba N. Spira B. Wainberg M.A. Antimicrob. Agents Chemother. 1991; 35: 1386-1390Google Scholar) and hepatitis B virus (5Doong S.L. Tsai C.H. Schinazi R.F. Liotta D.C. Cheng Y.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8495-8499Google Scholar) in cell culture. It was subsequently shown that the stereoisomer L-SddC (3TC) was responsible for the antiviral effect, while cytotoxicity was associated mainly with the D-enantiomer(6Chang C.N. Skalski V. Zhou J.H. Cheng Y. J. Biol. Chem. 1992; 267: 22414-22420Google Scholar, 7Coates J.A.V. Cammack N. Junkinson H.J. Mutton I.M. Pearson B.A. Storer R. Cameron J.M. Penn C.R. Antimicrob. Agents Chemother. 1992; 36: 202-205Google Scholar, 8Shinazi R.F. Chu C.K. Peck A. McMillan A. Mathis R. Cannon D. Jeong L.S. Beach J.W. Choi W.B. Yeola S. Liotta D.C. Antimicrob. Agents Chemother. 1992; 36: 672-676Google Scholar, 9Beach J.W. Jeong L.S. Alves A.J. Pohl D. Kim H.O. Chang C.N. Doong S.L. Schinazi R.F. Cheng Y.C. Chu C.K. J. Org. Chem. 1992; 57: 2217-2219Google Scholar). L-ddC and L-FddC were also found to be active against hepatitis B virus and HIV in culture without much toxicity(10Lin T.S. Luo M.Z. Liu M.C. Pai B. Dutschman G.E. Cheng Y.C. Biochem. Pharmacol. 1994; 47: 171-174Google Scholar, 11Lin T.S. Luo M.Z. Liu M.C. Pai B. Dutschman G.E. Cheng Y.C. J. Med. Chem. 1994; 37: 798-803Google Scholar, 12Schinazi R.F. Gosselin G. Faraj A. Korba B.E. Liotta D.C. Chu C.K. Mathe C. Imbach J.L. Sommadossi J.P. Antimicrob. Agents Chemother. 1994; 38: 2172-2174Google Scholar). 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 (13Kim H.O. Shanmuganathan K. Alves A.J. Jeong L.S. Beach J.W. Shinazi R.F. Chang C.N. Cheng Y.C. Chu C.K. Tetrahedron Lett. 1992; 33: 6899-6902Google Scholar). 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(14Grove K.L. Guo X. Liu S.H. Gao Z. Chu C.K. Cheng Y.C. Cancer Res. 1995; 55: 3008-3011Google Scholar). 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 5′-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 5′-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 5′-triphosphates of D- and L-enantiomers of 2,3-dideoxycytidine and 1,3-dioxolane-cytidine as well as their 5-fluoro-derivatives with human pol α, pol β, pol ɛ, pol δ, and pol γ is reported.EXPERIMENTAL PROCEDURESMaterials and CompoundsL- and D-stereoisomers of ddC and OddC and their 5-fluoro-derivatives were synthesized as described previously(11Lin T.S. Luo M.Z. Liu M.C. Pai B. Dutschman G.E. Cheng Y.C. J. Med. Chem. 1994; 37: 798-803Google Scholar, 13Kim H.O. Shanmuganathan K. Alves A.J. Jeong L.S. Beach J.W. Shinazi R.F. Chang C.N. Cheng Y.C. Chu C.K. Tetrahedron Lett. 1992; 33: 6899-6902Google Scholar). The triphosphate forms of these compounds were synthesized in our laboratory(15Chang C.N. Doong S.L. Zhou J.H. Beach J.W. Jeong L.S. Chu C.K. Tsai C. Cheng Y. J. Biol. Chem. 1992; 267: 13938-13942Google Scholar). Unlabeled dNTP and ddNTP were purchased from Boehringer Mannheim. Poly(rA)•oligo(dT)10 and poly(dA)•oligo(dT)12-18 were obtained from Pharmacia Biotech Inc.ssDNA cellulose was obtained from Sigma. DEAE cellulose (DE-52), phosphocellulose (P11), and S-Sepharose fast flow were purchased from Whatman. [α-32P]dCTP (3000 Ci/mmol), [γ-32P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase were obtained from Amersham Corp. M13 mp10 and M13 mp18 phage ssDNA were isolated as described previously(16Sambrook J. Fritch E.F. Manniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1059-1061Google Scholar). Oligonucleotides were synthesized on an Applied Biosystems 380A DNA synthesizer at the Yale Oligonucleotide Synthesis Facility. The primer oligonucleotides were labeled at the 5′ position with T4 polynucleotide kinase using [γ-32P]ATP, annealed to M13 phage ssDNA as described in (16Sambrook J. Fritch E.F. Manniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1059-1061Google Scholar), purified on a Sephadex G-25 column, and used as substrates for elongation reactions. BuAdATP was a kind gift from Professor George Wright (University of Massachussetts).EnzymesHuman pol α, pol δ, and pol γ from K562 chronic myelogenous leukemia cells and pol β from KB cells were partially purified using P11, DE-52, S-Sepharose, and ssDNA cellulose chromatography(6Chang C.N. Skalski V. Zhou J.H. Cheng Y. J. Biol. Chem. 1992; 267: 22414-22420Google Scholar). All DNA polymerases were distinguished from each other using several biochemical criteria, including chromatographic behavior, template preference, sensitivity to specific inhibitors and associated exonuclease activities. The ability to use poly(rA)•oligo(dT)10 as template and primer in the presence of 80 mM KCl at pH 7.5-8.0 and high sensitivity to ddNTP indicated that the DNA polymerase activity was that of pol γ(17Knopf K.W. Yamada M. Weissbach A. Biochemistry. 1976; 15: 4540-4548Google Scholar, 18Wernette C.M. Conway M.C. Kaguni L.S. Biochemistry. 1988; 27: 6046-6054Google Scholar, 19Insdorf N.F. Bogenhagen D.F. J. Biol. Chem. 1989; 264: 21491-21497Google Scholar). pol δ and pol α were eluted from a ssDNA-cellulose column at 0.32 M and 0.4 M KCl, respectively. Additional purifications were performed on DE-52 and S-Sepharose columns. pol δ and pol α activities were monitored on poly(dA)•oligo(dT)12-18 in the presence of proliferating cell nuclear antigen and activated DNA, respectively. pol α was very sensitive to the inhibitor BuAdATP, displaying 25% residual activity in the presence of 5 μM of this analog. In contrast, pol δ was much less sensitive, showing 80-90% residual activity with the same concentration of drug. pol δ displayed significant 3′ ⅉ 5′ exonuclease activity in contrast with the pol α preparation, which showed only traces of the 3′ⅉ 5′ exonuclease activity, which were probably due to cross-contamination. As expected, the activity of pol δ was stimulated by proliferating cell nuclear antigen using poly(dA)•oligo(dT)12-18 as template-primer, but proliferating cell nuclear antigen had no effect on pol α(20Hart G.J. Orr D.C. Penn C.R. Figueiredo H.T. Gray N.M. Boehme R.E. Cameron J.M. Antimicrob. Agents Chemother. 1992; 36: 1688-1694Google Scholar, 21Weissbach A. Enzyme. 1981; 14: 67-86Google Scholar, 22Nickel W. Austermann S. Bialek G. Grosse F. J. Biol. Chem. 1992; 267: 848-854Google Scholar). pol β that was separated from other polymerases using ssDNA cellulose chromatography was resistant to 3 mMN-ethylmaleimide(23Copeland W.C. Chen M.S. Wang T.S.F. J. Biol. Chem. 1992; 267: 21459-21464Google Scholar). pol ɛ was purified to near homogeneity from human placenta(24Mozzherin D.Yu. Atrazhev A.M. Kukhanova M.K. Mol. Biol. (Mosc.). 1992; 26: 665-671Google Scholar).pol α and pol δ activities were routinely assayed in 20 μl of 20 mM Tris-HCl buffer (pH 7.4) containing 6 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EDTA, 200 μg/ml heat-inactivated bovine serum albumin, 150 μg/ml activated calf thymus DNA, 20 μM each of dATP, dTTP, and dGTP, and 1 μCi of [3H]dCTP or [α-32P]dCTP at concentrations no less than the Km values for the appropriate enzymes. The same assay conditions were used for other polymerases with the following modifications: pol β, pH 8.5; pol γ, pH 8.0 and 80 mM KCl; and pol ɛ, 8 mM MgCl2. Assays for DNA polymerases contained 2 μl of enzyme (1 unit of pol α, pol ɛ, or pol γ, 2 units of pol δ, 0.75 unit of pol β). One unit of enzyme activity is defined as the amount of enzyme needed to incorporate 1 nmol of [3H]dTMP/h into the acid-insoluble fraction at 37°C. The reactions were allowed to proceed at 37°C for 10-60 min, after which 15-μl aliquots were removed and spotted onto Whatman DE-81 discs. The filters were washed with 0.3 M KCl containing 0.5 mM EDTA and then fixed with ethanol. The incorporation of the radiolabeled substrate into the DNA chain was measured by liquid scintillation counting.The Ki values of each compound for all DNA polymerases studied were determined using a competitive inhibition equation as described in (25Fersht A. Enzyme Structure and Mechanism. W. H. Freeman and Company, Reading1977: 84-100Google Scholar).Chain Termination AssaysTriphosphates of ddCTP analogs were analyzed for their chain terminating activity using M13 phage DNA hybridized with a 5′-32P-primer. Incorporation of one template-complementary nucleotide residue into the 3′-terminus of the primer was carried out in 8 μl of a mixture containing the appropriate buffer solution as described above, enzyme, 0.01 μM primer-template complex, and the analog under investigation. After incubation, the reaction was stopped by adding 4.5 μl of formamide containing dyes and EDTA, and the reaction products were separated by denaturating 15% polyacrylamide gel electrophoresis. Chain termination assays in the presence of four natural dNTPs were based on previously published methods (26Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar) and described in Fig. 6.Figure 6:Autoradiograph of chain terminating sequencing reaction with L-OddCTP using pol α (lanes 2-6) and M13 mp10 phage DNA annealed with 5′-32P-15 mer primer. The letters on the left side of the figure indicate the sequence of the growing DNA chain after the primer. Lane 1 (control) shows the DNA sequence by Klenow fragment with 20 μM ddCTP. Lanes 2-5, DNA sequence by pol α with L-OddCTP at concentrations 10, 20, 20, and 40 μM, respectively. Lane 4, after the reaction, 100 μM mixture containing all four dNTP was added, and the reaction continued 20 min more. Track 6, reaction without L-OddCTP. The mixture for pol α contained 2.5 μM dCTP and 20 μM each of three other dNTPs.View Large Image Figure ViewerDownload (PPT)Quantitation of the Primer Extension AssayThe bands on the x-ray film, representing the incorporation of dNTP analogs into the standing position of 3′-ends of 5′-32P-primers annealed with template, were quantitated with the aid of a densitometer (Molecular Dynamics) as described previously(27Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Google Scholar). The bands were scanned, and the efficiency of the primer extension and its dependence on substrate concentration were expressed as a Lineweaver-Burk plot (28Lineweaver H. Burk D. J. Am. Chem. Soc. 1934; 56: 658-666Google Scholar). Experimental measurements were carried out within the linear range of the counts versus integration curve. The incubation time for all experiments was chosen to have a linear dependence between yield of product and time. The utilization of primer was usually less than 30%. Reaction times were 10 min for pol α and pol δ and 15 min for pol β, pol ɛ, and pol γ. Km values were estimated from these data.Complexes of M13 phage DNA and primer used in this study were as follows.M13mp18 phage DNA 3′-CATTTTGCTGCCGGTCACGG-5′ 5′-GTAAAACGACGGCCAGT-3′←17-mer primer→ M13mp10 phage DNA 3′-GGTCAAGTGCTGCAACATTTTGCTGCCGG-5′ 5′-CCAGTTCACGACGTTGTAAAACGA-3′←14-mer primer→←15-mer primer→RESULTSInhibition of Human DNA Polymerases by L- and D-Enantiomers of ddCTP AnalogsThe structures of ddCTP analogs evaluated for their ability to interact with human DNA polymerases are shown in Fig. 1. The Km values of dCTP for pol α, pol δ, pol ɛ, pol β, and pol γ were 0.6 μM, 0.4 μM, 2.0 μM, 1.9 μM, and 0.16 μM, respectively(6Chang C.N. Skalski V. Zhou J.H. Cheng Y. J. Biol. Chem. 1992; 267: 22414-22420Google Scholar, 20Hart G.J. Orr D.C. Penn C.R. Figueiredo H.T. Gray N.M. Boehme R.E. Cameron J.M. Antimicrob. Agents Chemother. 1992; 36: 1688-1694Google Scholar). Table 1 lists the Ki values of L- and D-ddCTP analogs for these enzymes. As shown in Table 1, L-OddCTP and L-FOddCTP were potent inhibitors of replicative DNA polymerases. The Ki values of L-FOddCTP were about the same as those of its D-enantiomer for pol δ, pol β, and pol γ, but were less for pol α and pol ɛ. The 5′-triphosphates of L-FddC and L-ddC at concentrations up to 20 μM had no inhibitory effect on pol α, pol β, pol δ, and pol ɛ. The results obtained for D-ddCTP and D-FddCTP were similar to those published previously(29Starnes M.C. Cheng Y. J. Biol. Chem. 1987; 262: 988-991Google Scholar, 30Wright G.E. Brown N.C. Pharmacol. & Ther. 1990; 47: 447-497Google Scholar). pol γ was much more sensitive than any other polymerase to all compounds tested with the exception of L-ddCTP. The triphosphate of L-OddC, which was shown to have anticancer activity(14Grove K.L. Guo X. Liu S.H. Gao Z. Chu C.K. Cheng Y.C. Cancer Res. 1995; 55: 3008-3011Google Scholar), was a potent inhibitor of all DNA polymerase studied.Tabled 1 Open table in a new tab Chain Termination of DNA Synthesis by L- and D-Enantiomers of ddCTP AnalogsAll analogs were evaluated for their chain-terminating activity in a system containing M13 phage DNA annealed with primer. Reaction products were monitored as described under “Experimental Procedures.” The results for pol γ, pol δ, pol ɛ, and pol β are illustrated in Figure 2:, Figure 3:. pol γ could incorporate all analogs with the exception of L-ddCTP. pol δ (Fig. 2) and pol ɛ (Fig. 3A) proved to be more selective enzymes with respect to their utilization of these analogs. Only L-OddCTP and L-FOddCTP were effective substrates for these enzymes. D-FOddCTP was 25 times less potent as a substrate for pol ɛ than its L-enantiomer. Similar results were obtained for pol α. pol β (Fig. 3B) incorporated D-ddCTP (lane 1), D-FddCTP (lanes 3 and 4), L-OddCTP (lanes 7 and 8), L-FOddCTP (lanes 9 and 10) and D-FOddCTP (lanes 11 and 12). L-ddCTP (lane 2) and L-FddCTP (lanes 5 and 6) were not substrates for pol β. The results obtained for D-ddCTP (lane 1) were similar to those previously described(23Copeland W.C. Chen M.S. Wang T.S.F. J. Biol. Chem. 1992; 267: 21459-21464Google Scholar).Figure 2:Incorporation of L- and D-enantiomers of ddCTP analogs into the 3′-end of a 17-mer primer annealed with M13 mp18 phage DNA by pol γ (lanes 1-9) and pol δ (lanes 10-17). The reactions were performed as described under “Experimental Procedures.” Lane 1, incorporation of dGMP residues into the first position after primer; lane 2, 1 μMD-FddCTP; lane 3, 10 μML-ddCTP; lanes 4 and 5, 5 and 10 μML-FddCTP, respectively; lanes 6 and 7, 1 and 6 μML-FOddCTP; lanes 8 and 9, 0.2 and 2 μML-OddCTP, respectively. Lane 10, 5 μMD-ddCTP; lane 11, 10 μMD-FddCTP; lane 12, 10 μML-ddCTP; lane 13, 10 μML-FddCTP; lanes 14 and 15, 1 and 6 μML-FOddCTP, respectively; lanes 16 and 17, 2 and 5 μML-OddCTP, respectively.View Large Image Figure ViewerDownload (PPT)Figure 3:Incorporation of L- and D-enantiomers of ddCTP analogs into the 3′-end of 5′-32P-15-mer primer annealed with M18 mp10 phage DNA by pol ɛ (A) and pol β (B). A, 10 μMD-ddCTP (lane 1); 10 μMD-FddCTP (lane 2); 10 μML-ddCTP (lane 3); 10 μML-FddCTP (lane 4); 5 and 20 μMD-FOddCTP (lanes 5 and 6); 0.5 and 2 μML-FOddCTP (lanes 7 and 8); 0.5, 1, and 2 μML-OddCTP (lanes 9, 10, and 11, respectively); 10 μML-SddCTP (lane 12). B, 5 μMD-ddCTP (lane 1); 10 μML-ddCTP (lane 2); 1 and 5 μMD-FddCTP (lanes 3 and 4, respectively); 2 and 10 μML-FddCTP (lanes 5 and 6, respectively); 2 and 10 μML-OddCTP (lanes 7 and 8); 2 and 5 μML-FOddCTP (lanes 9 and 10, respectively); 1 and 5 μMD-FOddCTP (lanes 11 and 12, respectively).View Large Image Figure ViewerDownload (PPT)Kinetic Parameters for the Incorporation of L-OddCTP into a DNA ChainThe Km values for the incorporation of L-OddCTP into a growing DNA chain were determined (Fig. 4). Panel A indicates the dose dependence of the incorporation of L-OddCTP into the 3′-end of 5′-32P-15-mer primer annealed with M13 mp10 phage DNA by pol α and pol δ. As the concentration of L-OddCTP in the reaction was increased from 0.2 μM to 10 μM, more product was observed. The bands corresponding to the 16-mer primer were scanned with a densitometer, and panel B shows a double reciprocal plot of band intensity versus substrate concentration: 1/V versus 1/[L-OddCTP]. The Km values were estimated in the concentration range from 0.2 to 10 μM (panel B). Above this concentration, the double reciprocal plot of the initial rate against the inhibitor concentration was no longer linear. Similar methods were applied for pol γ, pol ɛ, and pol β. The Km values for dCTP and L-OddCTP are presented in Table 2. In the evaluation of Table 2, it should be emphasized that the kinetic values for the incorporation of natural nucleotides or analogs could be dependent on the nucleotide sequence of template-primer used(31Ricchetti M. Buc H. EMBO J. 1990; 9: 1583-1593Google Scholar). However, the ratio of the Km value of any nucleotide analog to Km value of the natural nucleotide should give a relative measure of the ability of that particular analog to be a substrate of DNA polymerase in comparison with the natural nucleotide.Figure 4:Concentration-dependent incorporation of L-OddCTP into DNA by pol α (lanes 1-7) and pol δ (lanes 8-14) into the 3′-end of 5′-32P-15-mer primer annealed with M13 mp10 phage DNA. DNA primer extension was carried out as described under “Experimental Procedures” with the exception that incubation time was 10 min, and 0.2 unit of pol α and 0.25 unit of pol δ were used to assure that less than 30% of the primer was consumed during experiments. A, incorporation of 2.5 uM dCTP (lanes 1 and 8); 0.2, 0.5, 1, 2, 5, and 10 uML-OddCTP (lanes 2-7 and lanes 9-14, respectively. B, the intensity of each track from A was quantitated by computer densitometry and plotted as 1/V versus 1/[S] for the reactions catalyzed by DNA pol α (-+-) and pol δ (-▵-). 1/V is presented as relative value of density.View Large Image Figure ViewerDownload (PPT)Tabled 1 Open table in a new tab As previously shown(6Chang C.N. Skalski V. Zhou J.H. Cheng Y. J. Biol. Chem. 1992; 267: 22414-22420Google Scholar, 20Hart G.J. Orr D.C. Penn C.R. Figueiredo H.T. Gray N.M. Boehme R.E. Cameron J.M. Antimicrob. Agents Chemother. 1992; 36: 1688-1694Google Scholar), L-SddCTP was a weak inhibitor of pol α and pol δ and was not a substrate for either enzyme. We compared the ability of L-SddCTP and L-OddCTP to be incorporated at standing and running points of a DNA chain by pol α under similar conditions. As one can see in Fig. 5, L-SddCTP is incorporated neither into the first nor into the eighth position of an elongated primer. Conversely, L-OddCTP is a good substrate for pol α, and the DNA fragments terminated by L-OddCMP are accumulated in both cases.Figure 5:Incorporation of L-OddCTP (A, lane 2, and B, lanes 1 and 2) and L-SddCTP (A, lanes 3 and 4, and B, lanes 3-5) in standing (A) and running (B) points of 15-mer primer (A) or 14-mer primer (B) annealed to M13 mp10 phage DNA by pol α. Panel A, lane 1, shows the incorporation of dCTP. The reactions were performed as described under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT)Chain Termination of DNA Synthesis by L-OddCTP in the Presence of All Four dNTPsL-OddCTP was also assayed as a terminating substrate for pol α in the presence of all four dNTPs following Sanger's method(26Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar). The results are shown in Fig. 6. The comparison of the DNA sequence by Klenow fragment with D-ddCTP as a terminator are also presented. Synthesis was performed in the presence of M13 mp10 phage DNA annealed with 5′-32P-15-mer primer. DNA reaction products were analyzed using 12% polyacrylamide gel electrophoresis. The primer extension products of L-OddCTP were similar to the products observed for the reaction with D-ddCTP and Klenow fragment, as shown in lane 1 and lanes 2-5. These results indicate that L-OddCTP was incorporated into DNA at cytidine residue sites. Subsequent chase containing 100 μM of four dNTPs (lane 4), which failed to extend the 3′-L-OddCMP terminated products, showed the ability of pol α to incorporate L-OddCTP into the growing DNA chain in the presence of dCTP. An accumulation of DNA fragments was observed at a length expected for the chain terminator with cytidine as a base.DISCUSSIONRecently, nucleoside derivatives with the unnatural L-configuration were evaluated against a broad spectrum of viruses, and some of these analogs proved to be very potent against hepatitis B virus and HIV(6Chang C.N. Skalski V. Zhou J.H. Cheng Y. J. Biol. Chem. 1992; 267: 22414-22420Google Scholar, 7Coates J.A.V. Cammack N. Junkinson H.J. Mutton I.M. Pearson B.A. Storer R. Cameron J.M. Penn C.R. Antimicrob. Agents Chemother. 1992; 36: 202-205Google Scholar, 8Shinazi R.F. Chu C.K. Peck A. McMillan A. Mathis R. Cannon D. Jeong L.S. Beach J.W. Choi W.B. Yeola S. Liotta D.C. Antimicrob. Agents Chemother. 1992; 36: 672-676Google Scholar, 9Beach J.W. Jeong L.S. Alves A.J. Pohl D. Kim H.O. Chang C.N. Doong S.L. Schinazi R.F. Cheng Y.C. Chu C.K. J. Org. Chem. 1992; 57: 2217-2219Google Scholar, 10Lin T.S. Luo M.Z. Liu M.C. Pai B. Dutschman G.E. Cheng Y.C. Biochem. Pharmacol. 1994; 47: 171-174Google Scholar, 11Lin T.S. Luo M.Z. Liu M.C. Pai B. Dutschman G.E. Cheng Y.C. J. Med. Chem. 1994; 37: 798-803Google Scholar, 12Schinazi R.F. Gosselin G. Faraj A. Korba B.E. Liotta D.C. Chu C.K. Mathe C. Imbach J.L. Sommadossi J.P. Antimicrob. Agents Chemother. 1994; 38: 2172-2174Google Scholar). At the same time, differences in their cellular toxicities were noticed. In contrast to L-SddC or L-ddC, both L- and D-enantiomers of FOddC and OddC were shown to be very cytotoxic(13Kim H.O. Shanmuganathan K. Alves A.J. Jeong L.S. Beach J.W. Shinazi R.F. Chang C.N. Cheng Y.C. Chu C.K. Tetrahedron Lett. 1992; 33: 6899-6902Google Scholar, 14Grove K.L. Guo X. Liu S.H. Gao Z. Chu C.K. Cheng Y.C. Cancer Res. 1995; 55: 3008-3011Google Scholar). The interaction of their 5′-triphosphate metabolites with cellular DNA polymerases may be one of the key factors in their varying toxicities. This possibility prompted us to evaluate the 5′-triphosphates of L- and D-enantiomers of dioxolane-cytidine as potential inhibitors or substrates of human DNA polymerases. For comparison, nucleoside 5′-triphosphates with the natural D-configuration were included in the study. The results presented here show that L-OddCTP and its 5-fluoro-derivative are potent inhibitors and chain terminators of cellular DNA polymerases including pol α, pol ɛ, and pol δ. The inhibition of polymerase activity is due to the incorporation of L-OddCTP into the DNA chain at cytidine residue sites but not to the inhibition of the rate of incorporation of other dNTPs. If the inhibition of synthesis was due to a dual mechanism including both incorporation and interference with the incorporation of other nucleosides, we would expect to see DNA fragments of a size inconsistent with DNA products terminated at cytidine residues (pauses) (Fig. 6). The interaction of L-OddCTP and L-FOddCTP with human DNA polymerases is the first example of the lack of enantioselectivity described for human replicative DNA polymerases. This phenomenon was described for HIV reverse transcriptase, pol γ, and pol β with respect to the L- and D-enantiomers of SddCTP (6Chang C.N. Skalski V. Zhou J.H. Cheng Y. J. Biol. Chem. 1992; 267: 22414-22420Google Scholar, 20Hart G.J. Orr D.C. Penn C.R. Figueiredo H.T. Gray N.M. Boehme R.E. Cameron J.M. Antimicrob. Agents Chemother. 1992; 36: 1688-1694Google Scholar, 32Wilson J.E. Martin J.L. Borroto-Esoda K. Hopkins S. Painter G. Liotta D.S. Furman P.A. Antimicrob Agents Chemother. 1993; : 1720-1722Google Scholar) and to carbovir triphosphate(33Miller W.H. Daluge S.M. Garvey E.P. Hopkins S. Reardon J.E. Boyd F.L. Miller R.L. J. Biol. Chem. 1992; 267: 21220-21224Google Scholar). It should be mentioned that replicative DNA polymerases are believed to be more sensitive to changes in the conformation of substrates. Indeed, this is true for SddCTP whose L-isomer is not a substrate for replicative DNA polymerases. However, substitution of sulfur for oxygen at the 3′-position of the ribose residue leads to the opposite effect. L-OddCTP is a good terminating substrate for replicative DNA polymerases, inhibiting them with Ki values in the pharmacologically relevant concentration range.The amount of L-OddCMP present at the DNA terminus depends not only on the efficiency of incorporation of L-OddCTP by polymerases but also on the rate of excision by 3′ⅉ 5′-exonucleases(34Burgers P.M.J. Prog. Nucleic Acid Res. Mol. Biol. 1989; 37: 235-380Google Scholar). The ability of 3′ⅉ 5′-exonucleases associated with pol δ and pol ɛ are currently evaluated. The anticancer activity of L-OddC may be a result of termination of DNA synthesis after L-OddCTP incorporation into proliferating cells coupled with inefficient excision of incorporated L-OddCMP from DNA.In the present study, we also made an attempt to address the impact of the substitution of fluorine for hydrogen at the 5-position of cytidine 5′-triphosphate analogs on their ability to serve as substrates for cellular DNA polymerases. As shown in Table 1, both L- and D-enantiomers of ddCTP and FddCTP were not substrates for pol α, pol ɛ, and pol δ and inhibited pol β and pol γ at the same range of concentrations. Similar results were obtained for the L-enantiomers of OddCTP and FOddCTP. No significant differences were seen between L-OddCTP and its 5-fluoro-derivatives in terms of their interaction with DNA polymerases. Both L-enantiomers were equally potent inhibitors for these DNA polymerases. With respect to pol β, L-OddCTP was 6 times more potent than its 5-fluoro-analog. At present, we cannot explain the differences in the interaction of L-OddCTP and its 5-fluoro-derivative with pol β.In summary, L-OddC is the first L-nucleoside shown to have potent antitumor activity. This activity could be related to the ability of L0OddCTP to be utilized as a substrate by human replicative DNA polymerases. Surprisingly, this property is not shared with L-SddC and L-ddC, which are relatively noncytotoxic. The discovery that the L-enantiomers of chain-terminating nucleotides can be incorporated by replicative DNA polymerases could lead to the development of a new class of anticancer compounds INTRODUCTIONSeveral 2′,3′-dideoxynucleoside analogs have been approved for the treatment of patients with AIDS(1Mitsuya H. Broder S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1911-1915Google Scholar, 2Mitsuya H. Weinhold K.J. Furman P.A. Clair St., M.H. Lehrman S.N. Gallo R.C. Bolognesi D. Barry D.W. Broder S. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7096-7100Google Scholar). Among them, ddC 1The abbreviations used are: ddC, ddCMP, and ddCTP2′,3′-dideoxycytidine and its mono- and triphosphates, respectively, all other nucleosides and their 5′-triphosphates have the same abbreviations throughout the paper. pol α, pol δpol β pol γ, and pol ɛDNA polymerases α, δ, β, γ, and ɛ, respectivelydNTP2′-deoxynucleoside 5′-triphosphateHIVhuman immunodeficiency virusssDNAsingle-stranded DNABuAdATP2[p-(n-butyl)anilino]dATP. Abbreviations of nucleotide analogs used are shown in Fig. 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 (3Belleau, B., Dixit, D., Nguyen-Ba, N., Kraus, J. L., (1989) Fifth International Conference on AIDS, Montreal, Canada, June 4-9, 1989, p. 515.Google Scholar, 4Soudeyns H. Yao X.J. Gao Q. Belleau B. Kraus J.L. Nguyen-Ba N. Spira B. Wainberg M.A. Antimicrob. Agents Chemother. 1991; 35: 1386-1390Google Scholar) and hepatitis B virus (5Doong S.L. Tsai C.H. Schinazi R.F. Liotta D.C. Cheng Y.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8495-8499Google Scholar) in cell culture. It was subsequently shown that the stereoisomer L-SddC (3TC) was responsible for the antiviral effect, while cytotoxicity was associated mainly with the D-enantiomer(6Chang C.N. Skalski V. Zhou J.H. Cheng Y. J. Biol. Chem. 1992; 267: 22414-22420Google Scholar, 7Coates J.A.V. Cammack N. Junkinson H.J. Mutton I.M. Pearson B.A. Storer R. Cameron J.M. Penn C.R. Antimicrob. Agents Chemother. 1992; 36: 202-205Google Scholar, 8Shinazi R.F. Chu C.K. Peck A. McMillan A. Mathis R. Cannon D. Jeong L.S. Beach J.W. Choi W.B. Yeola S. Liotta D.C. Antimicrob. Agents Chemother. 1992; 36: 672-676Google Scholar, 9Beach J.W. Jeong L.S. Alves A.J. Pohl D. Kim H.O. Chang C.N. Doong S.L. Schinazi R.F. Cheng Y.C. Chu C.K. J. Org. Chem. 1992; 57: 2217-2219Google Scholar). L-ddC and L-FddC were also found to be active against hepatitis B virus and HIV in culture without much toxicity(10Lin T.S. Luo M.Z. Liu M.C. Pai B. Dutschman G.E. Cheng Y.C. Biochem. Pharmacol. 1994; 47: 171-174Google Scholar, 11Lin T.S. Luo M.Z. Liu M.C. Pai B. Dutschman G.E. Cheng Y.C. J. Med. Chem. 1994; 37: 798-803Google Scholar, 12Schinazi R.F. Gosselin G. Faraj A. Korba B.E. Liotta D.C. Chu C.K. Mathe C. Imbach J.L. Sommadossi J.P. Antimicrob. Agents Chemother. 1994; 38: 2172-2174Google Scholar). 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 (13Kim H.O. Shanmuganathan K. Alves A.J. Jeong L.S. Beach J.W. Shinazi R.F. Chang C.N. Cheng Y.C. Chu C.K. Tetrahedron Lett. 1992; 33: 6899-6902Google Scholar). 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(14Grove K.L. Guo X. Liu S.H. Gao Z. Chu C.K. Cheng Y.C. Cancer Res. 1995; 55: 3008-3011Google Scholar). 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 5′-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 5′-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 5′-triphosphates of D- and L-enantiomers of 2,3-dideoxycytidine and 1,3-dioxolane-cytidine as well as their 5-fluoro-derivatives with human pol α, pol β, pol ɛ, pol δ, and pol γ is reported." @default.
• W2024382021 created "2016-06-24" @default.
• W2024382021 creator A5000085395 @default.
• W2024382021 creator A5001115779 @default.
• W2024382021 creator A5015683860 @default.
• W2024382021 creator A5032991382 @default.
• W2024382021 creator A5037409604 @default.
• W2024382021 creator A5076513120 @default.
• W2024382021 date "1995-09-01" @default.
• W2024382021 modified "2023-09-29" @default.
• W2024382021 title "L- and D-Enantiomers of 2′,3′-Dideoxycytidine 5′-Triphosphate Analogs as Substrates for Human DNA Polymerases" @default.
• W2024382021 cites W1490124511 @default.
• W2024382021 cites W1502628406 @default.
• W2024382021 cites W1510860856 @default.
• W2024382021 cites W1512123554 @default.
• W2024382021 cites W1546382247 @default.
• W2024382021 cites W1557880692 @default.
• W2024382021 cites W1582508691 @default.
• W2024382021 cites W1597138031 @default.
• W2024382021 cites W1977521763 @default.
• W2024382021 cites W1989341969 @default.
• W2024382021 cites W2007423548 @default.
• W2024382021 cites W2026570309 @default.
• W2024382021 cites W2032026161 @default.
• W2024382021 cites W2040962830 @default.
• W2024382021 cites W2047432961 @default.
• W2024382021 cites W2054979333 @default.
• W2024382021 cites W2065280709 @default.
• W2024382021 cites W2080750598 @default.
• W2024382021 cites W2085459671 @default.
• W2024382021 cites W2086186867 @default.
• W2024382021 cites W2095592056 @default.
• W2024382021 cites W2137956846 @default.
• W2024382021 cites W2138270253 @default.
• W2024382021 cites W2166336747 @default.
• W2024382021 cites W2950946613 @default.
• W2024382021 cites W935686051 @default.
• W2024382021 cites W95412636 @default.
• W2024382021 cites W959939080 @default.
• W2024382021 doi "https://doi.org/10.1074/jbc.270.39.23055" @default.
• W2024382021 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7559445" @default.
• W2024382021 hasPublicationYear "1995" @default.
• W2024382021 type Work @default.
• W2024382021 sameAs 2024382021 @default.
• W2024382021 citedByCount "83" @default.
• W2024382021 countsByYear W20243820212012 @default.
• W2024382021 countsByYear W20243820212013 @default.
• W2024382021 countsByYear W20243820212014 @default.
• W2024382021 countsByYear W20243820212015 @default.
• W2024382021 countsByYear W20243820212016 @default.
• W2024382021 countsByYear W20243820212021 @default.
• W2024382021 crossrefType "journal-article" @default.
• W2024382021 hasAuthorship W2024382021A5000085395 @default.
• W2024382021 hasAuthorship W2024382021A5001115779 @default.
• W2024382021 hasAuthorship W2024382021A5015683860 @default.
• W2024382021 hasAuthorship W2024382021A5032991382 @default.
• W2024382021 hasAuthorship W2024382021A5037409604 @default.
• W2024382021 hasAuthorship W2024382021A5076513120 @default.
• W2024382021 hasBestOaLocation W20243820211 @default.
• W2024382021 hasConcept C185592680 @default.
• W2024382021 hasConcept C2756471 @default.
• W2024382021 hasConcept C552990157 @default.
• W2024382021 hasConcept C55493867 @default.
• W2024382021 hasConcept C71240020 @default.
• W2024382021 hasConcept C82381507 @default.
• W2024382021 hasConceptScore W2024382021C185592680 @default.
• W2024382021 hasConceptScore W2024382021C2756471 @default.
• W2024382021 hasConceptScore W2024382021C552990157 @default.
• W2024382021 hasConceptScore W2024382021C55493867 @default.
• W2024382021 hasConceptScore W2024382021C71240020 @default.
• W2024382021 hasConceptScore W2024382021C82381507 @default.
• W2024382021 hasIssue "39" @default.
• W2024382021 hasLocation W20243820211 @default.
• W2024382021 hasOpenAccess W2024382021 @default.
• W2024382021 hasPrimaryLocation W20243820211 @default.
• W2024382021 hasRelatedWork W1835778803 @default.
• W2024382021 hasRelatedWork W1996781975 @default.
• W2024382021 hasRelatedWork W2011868437 @default.
• W2024382021 hasRelatedWork W2015629286 @default.
• W2024382021 hasRelatedWork W2019375035 @default.
• W2024382021 hasRelatedWork W2020827133 @default.
• W2024382021 hasRelatedWork W2080778966 @default.
• W2024382021 hasRelatedWork W2087714034 @default.
• W2024382021 hasRelatedWork W2142087835 @default.
• W2024382021 hasRelatedWork W2401460060 @default.
• W2024382021 hasVolume "270" @default.
• W2024382021 isParatext "false" @default.
• W2024382021 isRetracted "false" @default.
• W2024382021 magId "2024382021" @default.
• W2024382021 workType "article" @default.