Puvvada M S, Forrow S A, Hartley J A, Stephenson P, Gibson I, Jenkins T C, Thurston D E
Gene Targeted Drug Design Research Group, School of Pharmacy and Biomedical Science, University of Portsmouth, U.K.
Biochemistry. 1997 Mar 4;36(9):2478-84. doi: 10.1021/bi952490r.
The interactions of several pyrrolo[2, 1-c][1,4]benzodiazepine (PBD) antitumor antibiotics with linearized plasmid pGEM-2-N-ras DNA have been analyzed by quantitative in vitro transcription (QIVT) and in vitro transcription footprinting (IVTF) methods. A concentration-dependent inhibitory effect of the PBDs on transcription is observed using both techniques. The rank order for overall inhibition of transcription by the QIVT method is found to be: sibiromycin > tomaymycin > anthramycin > DC-81 > neothramycin, whereas the IVTF experiments show a different ranking: sibiromycin > anthramycin > neothramycin > tomaymycin. In addition, stimulation of transcription was observed at low PBD concentrations in both the QIVT and IVTF experiments. These results demonstrate unequivocally that the formation of PBD-DNA adducts at AGA-5' base sequences on the transcribed strand results in transcription blockage for all PBDs examined. Furthermore, the sequence of flanking base pairs appears to influence the degree of blocking, with the sequences ACAGAAA-5', AAAGATG-5', AGAGATA-5', and CAAGAAC-5' providing the most pronounced blocks for all PBDs studied in this system. Neothramycin and tomaymycin cause additional blocks at some GGA-5' and TGA-5' sequences. Parallel MPE-Fe(II) footprinting studies have revealed PBD binding sites on both the transcribing and nontranscribing strands, although all transcription blocks determined from the IVTF assays are due to drug bound on the transcribing DNA template strand.
通过定量体外转录（QIVT）和体外转录足迹法（IVTF）分析了几种吡咯并[2,1 - c][1,4]苯并二氮杂卓（PBD）抗肿瘤抗生素与线性化质粒pGEM - 2 - N - ras DNA的相互作用。使用这两种技术均观察到PBD对转录具有浓度依赖性抑制作用。通过QIVT方法对转录总体抑制的排序为：西比霉素＞托马霉素＞蒽环类抗生素＞DC - 81＞新蒽环霉素，而IVTF实验显示出不同的排序：西比霉素＞蒽环类抗生素＞新蒽环霉素＞托马霉素。此外，在QIVT和IVTF实验中，在低PBD浓度下均观察到转录的刺激作用。这些结果明确表明，在所研究的所有PBD中，转录链上AGA - 5'碱基序列处PBD - DNA加合物的形成导致转录阻断。此外，侧翼碱基对的序列似乎影响阻断程度，对于该系统中研究的所有PBD，序列ACAGAAA - 5'、AAAGATG - 5'、AGAGATA - 5'和CAAGAAC - 5'产生最明显的阻断。新蒽环霉素和托马霉素在一些GGA - 5'和TGA - 5'序列处引起额外的阻断。平行的MPE - Fe(II)足迹研究揭示了转录链和非转录链上的PBD结合位点，尽管从IVTF分析确定的所有转录阻断都是由于药物结合在转录DNA模板链上。
