Main properties of duck hepatitis B virus DNA polymerase: comparison with the human and woodchuck hepatitis B virus DNA polymerases

The main properties of the duck hepatitis B virus (DHBV) DNA polymerase have been studied and compared with those of the human hepatitis B virus (HBV) and of the woodchuck hepatitis virus (WHV) DNA polymerases. All 3 enzymes are active under high salt conditions in the presence of high magnesium concentration. DHBV DNA polymerase was found less sensitive to ethanol and to operate at higher optimal pH than the HBV and WHV DNA polymerases. Like the other two viral endogenous DNA polymerases, the DHBV enzyme was strongly inhibited by phosphonoformic acid but not by aphidicolin, sulfhydryl group blockers or phosphonoacetic acid. Inhibition of DHBV DNA polymerase by the triphosphate derivatives of several nucleoside analogs appeared similar to that reported for HBV or WHV endogenous polymerase. FIACTP was the most, and ACVTP the least effective inhibitor; BVdUTP was of intermediary potency; araCTP and araTTP had a greater inhibitory effect on DHBV DNA polymerase than HBV or WHV DNA polymerase. The similarities in the properties of DHBV and HBV DNA polymerase justify the use of the duck hepatitis B polymerase model for screening and evaluation of potentially active drugs against HBV infection.     

Assessment of the effect of phosphorylated metabolites of anti-human immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the behavior of human deoxycytidylate deaminase

Deoxycytidylate deaminase, catalyzing the conversion of dCMP to dUMP, is an important enzyme in the de novo synthesis of thymidine nucleotides. It also may be involved in the action, as well as the metabolism of anticancer agents. Recently, several L- and D-configuration pyrimidine deoxynucleoside analogs were found to be potent antiviral and antitumor agents. Their interaction with dCMP deaminase as a monophosphate or a triphosphate metabolite is not clear. These include D-nucleoside analogs such as beta-D-2',3'-dideoxycytidine (ddC), beta-2'-fluoro-5-methyl-arabinofuranosyluracil (FMAU), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-didehydro-2',3'-dideoxythymidine (D4T) as well as L-nucleoside analogs such as beta-L-dioxolane-cytidine (L-OddC), beta-L-2',3'-dideoxy-3'-thiacytidine, beta-L-2',3'-dideoxy-5'-fluoro-3'-thia-cytidine (L-FSddC), beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine, and L-FMAU. None of the L-deoxycytidine analog monophosphates act as substrates or inhibitors. Among these pyrimidine deoxynucleoside analog monophosphates, D-FMAU monophosphate (MP) is the most potent competitive inhibitor, whereas L-FMAUMP has no inhibitory activity. Interestingly, AZTMP and D4TMP also have potent inhibitory activities on dCMP deaminase. Among the dCTP and TTP analogs examined, D- and L-FMAUTP were the most potent inhibitors and had the same extent of inhibitory effect. These results suggest that a chiral specificity for the substrate-binding site may exist, but there is no chiral specificity for the regulator-binding site. This is also supported by the observation that L-OddC and L-FSddC have inhibitory activities as triphosphates but not as monophosphates. None of the D- and L-dCTP analogs activated dCMP deaminase as dCTP. The biological activities of AZT and D4T could be partially attributable to their inhibitory activity against dCMP deaminase by their phosphorylated metabolites, whereas that of ddC and the L-deoxycytidine analogs may not involve dCMP deaminase directly.     

Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine.

The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.     

Interaction of deoxyguanosine nucleotide analogs with human telomerase

To maintain the telomeres at the ends of the chromosomes, telomerase in human cells adds a repeating sequence of nucleotides (TTAGGG) to the 3'-end of each chromosome using an RNA component of the enzyme as the template for DNA synthesis. Because of the selective expression of this enzyme in cancer cells, we have evaluated the interaction of human telomerase with several deoxyguanosine nucleotides of clinical importance. 2',3'-dideoxyguanosine 5'-triphosphate, 6-thio-2'-deoxyguanosine 5'-triphosphate (T-dGTP), carbovir 5'-triphosphate, and D-carbocyclic-2'-deoxyguanosine 5'-triphosphate (D-CdG-TP) inhibited telomerase activity by 50% when these analogs were present at only 2 to 9 times the dGTP concentration. The L-enantiomer of CdG-TP was far less inhibitory, thereby demonstrating the stereoselectivity of telomerase for nucleotide substrates. T-dGTP was incorporated into the DNA by telomerase in the absence of dGTP, but unlike dGTP there was little extension of the DNA chain after its incorporation. These results indicate that the metabolites of three clinically useful agents (6-mercaptopurine, 6-thioguanine, and Abacavir) can inhibit human telomerase activity, and it is possible that the effect of these nucleotides on telomerase activity or telomere function could contribute to the mechanism of action of these agents.