Showing papers by "Yung-Chi Cheng published in 2002"

An exonucleolytic activity of human apurinic/apyrimidinic endonuclease on 3′ mispaired DNA

Abstract: Human apurinic/apyrimidinic endonuclease (APE1) is an essential enzyme in DNA base excision repair that cuts the DNA backbone immediately adjacent to the 5' side of abasic sites to facilitate repair synthesis by DNA polymerase beta (ref. 1). Mice lacking the murine homologue of APE1 die at an early embryonic stage. Here we report that APE1 has a DNA exonuclease activity on mismatched deoxyribonucleotides at the 3' termini of nicked or gapped DNA molecules. The efficiency of this activity is inversely proportional to the gap size in DNA. In a base excision repair system reconstituted in vitro, the rejoining of nicked mismatched DNA depended on the presence of APE1, indicating that APE1 may increase the fidelity of base excision repair and may represent a new 3' mispaired DNA repair mechanism. The exonuclease activity of APE1 can remove the anti-HIV nucleoside analogues 3'-azido-3'-deoxythymidine and 2',3'-didehydro-2', 3'-dideoxythymidine from DNA, suggesting that APE1 might have an impact on the therapeutic index of antiviral compounds in this category.

Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates.

Abstract: Pyrimidine nucleoside monophosphate kinase [UMP/CMP kinase (UMP/CMPK);EC 2.7.4.14] plays a crucial role in the formation of UDP, CDP, and dCDP, which are required for cellular nucleic acid synthesis. Several cytidine and deoxycytidine analogues are important anticancer and antiviral drugs. These drugs require stepwise phosphorylation to their triphosphate forms to exert their therapeutic effects. The role of UMP/CMPK for the phosphorylation of nucleoside analogues has been indicated. Thus, we cloned the human UMP/CMPK gene, expressed it in Escherichia coli , and purified it to homogeneity. Its kinetic properties were determined. UMP and CMP proved to be far better substrates than dCMP. UMP/CMPK used all of the nucleoside triphosphates as phosphate donors, with ATP and dATP being the best donors and CTP being the poorest. Furthermore, UMP/CMPK was able to phosphorylate all of the deoxycytidine analogue monophosphates that we tested. The relative efficiency was as follows: arabinofuranosyl-CMP > dCMP > β-l-2′,3′-dideoxy-3′-thia-CMP > Gemcitabine monophosphate > β-d-2′,3′-dideoxy-CMP; β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluoro-CMP; β-l-2′,3′-dideoxy-5-fluoro-3′-thia-CMP > β-l-2′,3′-dideoxy-CMP > β-l-dioxolane-CMP. By comparing the relative V max / K m values of d- and l-form dideoxy-CMP, we showed that this kinase lacked stereoselectivity. Reducing agents, such as DTT, 2-mercaptoethanol, and thioredoxin, were able to activate this enzyme, suggesting that its activity may be regulated by redox potential in vivo . UMP/CMPK localized predominantly to the cytoplasm. In addition, 196-amino acid UMP/CMPK was the actual form of UMP/CMPK, rather than the 228-amino acid form as suggested before.

X-ray repair cross-complementing gene I protein plays an important role in camptothecin resistance.

Abstract: X-ray repair cross-complementing gene I protein (XRCC1) in complex with DNA polymerase β, DNA ligase III, and poly(ADP-ribose) polymerase is important in the base excision repair process. Previously, we isolated camptothecin (CPT)-resistant cell lines (KB100 and KB300) from the human epidermoid carcinoma cell line KB by exposure to CPT. From these CPT-resistant cell lines, their revertants (KB100rev and KB300rev), which lost most of their CPT-resistant phenotype during passage in the absence of CPT, were established. In this study, we found the expression levels of XRCC1 protein in KB100 and KB300 were ≥5-fold more than in their respective revertant cell lines, whereas there was no difference in the expression of XRCC1-associated proteins such as DNA polymerase β, DNA ligase III, poly(ADP-ribose) polymerase, and apurinic/apyrimidinic endonuclease. The degree of CPT resistance was relatively correlated with the XRCC1 protein amount. We also found XRCC1 gene amplification in CPT-resistant KB100 and KB300 cell lines. To confirm a correlation between overexpression of XRCC1 and CPT resistance, we transfected the XRCC1 gene into KB100rev and obtained two different transfected cell lines (clones 14 and 16). The expression levels of XRCC1 in the transfected cell lines were higher than in KB100rev but lower than in KB100 with no difference in XRCC1-associated protein expression levels. Resistance to CPT in transfected cell lines was 2–2.5-fold higher than in KB100rev in regard to growth inhibition and 4-fold higher with respect to clonogenicity. Transfected cell lines also showed increased resistance to other topoisomerase I poisons. However, the cytotoxicity of VP-16 and cisplatin was similar in both the transfected cells and KB100rev. Similar to our CPT-resistant cell lines, the resistance of transfected cell lines was reversed by treatment with 3-aminobenzamide. These results indicate that CPT resistance in our cells could be partly attributable to the overexpression of XRCC1.

Animal models for the study of HBV infection and the evaluation of new anti‐HBV strategies

Abstract: Backgroud: Our aim was to evaluate the anti-HBV activity of a novel L-nucleoside analog, 2',3'-dideoxy-2',3'-didehydro-β-L-5-fluorocytidine (β-L-Fd4C), in study models of HBV infection. Method: Its mechanism of action was evaluated on the in vitro expressed duck HBV (DHBV) reverse transcriptase and in primary hepatocyte cultures of duck and human origin. The capacity of antiviral therapy to clear viral infection was analyzed in vivo in the duck and woodchuck models. Results: β-L-Fd4C-TP exhibited a more potent inhibitory effect on the RT activity of the DHBV polymerase than other cytidine analogs (lamivudine-TP, ddC-TP, β-L-FddC-TP). In primary duck hepatocyte cultures, β-L-Fd4C exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral cccDNA. In vivo treatment with β-L-Fd4C in infected ducklings and woodchucks, induced a greater suppression of viremia and intrahepatic viral DNA synthesis than with lamivudine. However, covalently closed circular DNA persistence explained the relapse of viral replication after treatment withdrawal. Viral spread was strongly reduced in the case of early therapeutical intervention, but the number of infected cells did not decline when therapy was started during chronic infection. Liver histology analysis showed a decrease in the inflammatory activity of chronic hepatitis while no ultrastructural modification of liver cells was observed in electron microscopy studies. Furthermore, in human primary hepatocyte cultures, β-L-Fd4C induced a significant inhibition of HBV DNA synthesis. Conclusion: β-L-Fd4C is a potent inhibitor of hepadnavirus RT and inhibits viral DNA synthesis in hepatocytes both in vitro and in vivo. These experimental studies allowed as to show that β-L-Fd4C is a promising anti-HBV agent. Combination therapy should be evaluated to eradicate viral infection.

Antiviral agents and methods of treating viral infections

Abstract: The present invention relates to methods of treating viral or fungal infections using 3-aminopyridine-2-carboxyaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) and its prodrug forms and to pharmaceutical compositions comprising these compounds.

Synthesis of Halogen‐Substituted 3‐Deazaadenosine and 3‐Deazaguanosine Analogues as Potential Antitumor/Antiviral Agents.

Abstract: Various 2-halogen-substituted analogues (38, 39, 43 and 44), 3-halogen-substituted analogues (51 and 52), and 2',3'-dihalogen-substituted analogues (57-60) of 3-deazaadenosine and 3-halogen-substituted analogues (61 and 62) of 3-deazaguanosine have been synthesized as potential anticancer and/or antiviral agents. Among these compounds, 3-deaza-3-bromoguanosine (62) showed significant cytotoxicity against L1210, P388, CCRF-CEM and B16F10 cell lines in vitro, producing IC50 values of 3, 7, 9 and 7 microM, respectively. Several 3-deazaadenosine analogues (38, 51, 57 and 59) showed moderate to weak activity against hepatitis B virus.

Nucleosides and related processes, pharmaceutical compositions and methods

Abstract: The invention provides novel nucleosides and related processes, pharmaceutical compositions, and methods. The novel nucleosides are useful in a wide variety of antiviral, antineoplastic, and antibacterial applications. Preferred embodiments of the instant invention include novel 2 halogen-substituted, 3 halogen-substituted, and 2′,3′dihalogen-substituted analogues of 3-deazaadenosine, and novel 3 halogen-substituted analogues of 3-deazaguanosine. Compounds of the instant invention, including 4-Amino-6-fluoro-1-(β-D-ribofuranosyl)imidazo[4,5-c]pyridine and 6-Amino-7-bromo-1,5-dihydro-1-β- D -ribofuranosylimidazo[4,5-c]pyridin-4-one, have exhibited potent antiviral and anticancer activity in vitro. The compounds are also useful in the concomitant treatment of bacterial infections associated with viral infections such as AIDS.