TY - JOUR T1 - The Effects of 6-Chloro-8-aza-9-cyclopentylpurine on Nucleic Acid and Protein Synthesis in <em>Escherichia coli</em> JF - Molecular Pharmacology JO - Mol Pharmacol SP - 271 LP - 285 VL - 5 IS - 3 AU - JAMES M. JOHNSON AU - RAYMOND W. RUDDON AU - MORRIS S. ZEDECK AU - ALAN C. SARTORELLI Y1 - 1969/05/01 UR - http://molpharm.aspetjournals.org/content/5/3/271.abstract N2 - The purine deoxyribonucleoside analogue 6-chloro-8-aza-9-cyclopentylpurine has been found to inhibit DNA, RNA, and protein synthesis independently in Escherichia coil. The mechanism by which it inhibits DNA synthesis apparently involves a site of action in the pathway for the formation of thymine nucleotides, since the conversion of uracil or cytosine to thymine nucleotide derivatives in whole cells was completely abolished by 0.5 mM 6-chloro-8-aza-9-cyclopentylpurine within minutes after addition of drug to a growing culture. In vitro studies demonstrated that concentrations of the analogue as high as 2 mM had no effect on thymidylate synthetase activity; therefore, blockade of thymine nucleotide formation is apparently produced at an earlier step. The conversion of uracil or cytosine to nucleotide derivatives other than thymine derivatives was decreased 50-60% by 6-chloro-8-aza-9-cyclopentylpurine (0.5 mM) in whole cells, suggesting that inhibition of the formation of ribonucleotides contributes to the blockade of both DNA and RNA synthesis observed in vivo. Preliminary incubation of partially purified polymerase preparations with the analogue (1 mM) resulted in marked inhibition of RNA polymerase activity, with only slight effects on DNA polymerase. The template function of purified DNA, however, was not affected by incubation of DNA with drug for up to 16 hr prior to assay of template activity. Inhibition of protein formation was also observed after incubation of components of the cell-free protein-synthesizing system in vitro. The effect of the drug on protein synthesis appeared to be due to an action on the 100,000 x g supernatant enzymes involved in the polymerization of amino acids into polypeptide linkage. Neither aminoacyl-tRNA synthetase activity nor the ability of the ribosomes to support protein synthesis was affected by 6-chloro-8-aza-9-cyclopentylpurine. The inhibitory effects of the drug on the activity of enzymes involved in the formation of nucleic acids and protein observed in vitro required prior incubation with the drug and were irreversible. The data suggest that a portion of the actions of 6-chloro-8-aza-9-cyclopentylpurine are due to a site-directed alkylation of enzymes, leading to a blockade of enzymatic activity. ACKNOWLEDGMENTS The authors wish to thank Miss Linda Galligher and Miss Bonnie Wachter for their excellent technical assistance. ER -