ABSTRACT
The syntheses of several novel halogenated bithiazoles structurally related to the bithiazole moiety of bleomycin A(5) are described. Also described is the ability of these compounds to mediate photoactivated DNA cleavage. Chlorinated bithiazole analogues were shown to be much more active than an analogous brominated derivative. DNA strand scission activity was strictly light dependent and was accompanied by dechlorination of the bithiazole nucleus, apparently in a stoichiometric fashion. Inhibition of DNA cleavage in the presence of DMSO, as well as photoaddition to 1-octene by both brominated and chlorinated bithiazole derivatives, suggest strongly that the initial step in photoactivated DNA cleavage involves homolysis of the thiazole carbon-halogen bond. The chlorinated bithiazoles were found to mediate sequence selective cleavage of a (32)P-end labeled DNA, although the selectivity observed was not the same as that of bleomycin itself. The implications of this observation are discussed.
Subject(s)
Bleomycin/analogs & derivatives , DNA/metabolism , Photolysis/drug effects , Thiazoles/pharmacology , Alkenes/chemistry , Antineoplastic Agents/chemistry , Base Sequence , Binding Sites , Bleomycin/chemistry , Bromine , Chlorine , DNA/chemistry , DNA/radiation effects , Hydrolysis , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/metabolismABSTRACT
The ends of eukaryotic chromosomes are specialized sequences, called telomeres comprising tandem repeats of simple DNA sequences. Those sequences are essential for preventing aberrant recombination and protecting genomic DNA against exonucleolytic DNA degradation. Telomeres are maintained at a stable length by telomerase, an RNA-dependent DNA polymerase. Recently, human telomerase has been recognized as a unique diagnostic marker for human tumors and is potentially a highly selective target for antitumor drugs. In this study, we have examined the major factors affecting the catalytic activity and processivity of human telomerase. Specifically, both the catalytic activity and processivity of human telomerase were modulated by temperature, substrate (dNTP and primer) concentration, and the concentration of K+. The catalytic activity of telomerase increased as temperature (up to 37 degrees C), concentrations of dGTP, primer, and K+ were increased. However, the processivity of human telomerase decreased as temperature, primer concentration, and K+ were increased. Our results support the current model for human telomerase reaction and strengthen the hypothesis that a G-quadruplex structure of telomere DNA plays an important role in the regulation of the telomerase reaction.
Subject(s)
Protein Processing, Post-Translational , Telomerase/chemistry , Telomerase/metabolism , Catalysis , DNA Primers/chemistry , DNA Primers/metabolism , Deoxyguanine Nucleotides/chemistry , Deoxyguanine Nucleotides/metabolism , Enzyme Activation/genetics , HeLa Cells , Humans , Models, Biological , Models, Chemical , Potassium/chemistry , Potassium/metabolism , Protein Processing, Post-Translational/genetics , Telomerase/geneticsABSTRACT
The design and syntheses of functional models for bleomycin in which AMPHIS, a simplified model of the metal-chelating subunit of bleomycin is connected to distamycin analogs with a series of linkers, are described. Kinetic studies and DNA cleavage assay show that 1,2-trans-disubstituted cyclopropane units are the best linkers within this series. Study of selective DNA cleavage on high resolution polyacrylamide sequencing gels indicates that the linker modified hybrids generally cleave selectively at the 5' end of poly T sites and at the 3' end of poly A sites. Cleavage activity is enhanced for most of the compounds related to those with shorter linkers, previously reported, (Huang, L.; Quada, Jr J. C.; Lown, J. W. Bioconjugate Chem. 1995, 6, 21, Ref. 1) probably as a result of the linker allowing the active complex to approach the target deoxyribose more closely and efficiently. Certain of the compounds, ones containing a (S)-methyl in the linker and the (S,S)-cyclopropyl linker, exhibit unique cleavage sites, indicating that these linkers allow the hybrids to locate novel, individual DNA binding sites.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Bleomycin/pharmacology , Cyclopropanes/chemistry , DNA/drug effects , Antibiotics, Antineoplastic/chemistry , Base Sequence , Bleomycin/chemistry , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/pharmacology , Distamycins/chemistry , Drug Design , Histidine/analogs & derivatives , Histidine/chemistry , Histidine/pharmacology , Kinetics , Models, Molecular , Molecular Sequence Data , Pyrroles/chemistry , Pyrroles/pharmacology , Structure-Activity RelationshipABSTRACT
The syntheses of functional models for bleomycin, which are composed of a simple analog of the metal-complexing moiety of bleomycin and oligo-N-methylpyrrole peptide DNA-binding moieties, are described. The extent and the relative rate of their cleavage of DNA in the presence of reductants were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The rate of DNA cleavage increases with the number of N-methylpyrrole units in the carrier moiety. The sequence selectivity of DNA cleavage was demonstrated by polyacrylamide sequencing gel electrophoresis of cleavage reactions on two 5'-32P labeled restriction fragments: a 158 bp GC-rich fragment from pBR322 and a 241 bp AT-rich fragment fragment from SV40. Comparison of the sequence selectivity with that of bleomycin A2 indicates that the poly-N-methylpyrrole moiety directs the hybrid compounds to AT-rich sequences of DNA. High-resolution denaturing gel electrophoresis of DNA cleaved by the model compounds reveals that 3' phosphate is produced exclusively, indicating that the chemistry of DNA cleavage differs from that of bleomycin.
