SYNTHESIS AND MECHANISM OF DNA--CROSSLINKING OF FR900482

本研究的主要目的是研究 具有临床意义的新型抗肿瘤药物FR 900482（1a）、FK 973的作用 (1b)和FR 66979（1c）（FK 973，1b是第一个衍生物， 临床试验）。 FK 973已被证明可交联双链DNA 并将DNA交联到L1210细胞中的DNA结合蛋白。 努力将 旨在阐明完整的机械细节，精确的 FR 900482的体外还原活化机制，导致 DNA的共价修饰。 在上一个资助期内， 确定了DNA交联形成的序列特异性， FR 900482和天然还原产物FR 66979，并已确定 这些物质的减少导致线粒体的产生， 优选地在5 '-dC-dG 3'盒处交联DNA的衍生物。 的 FK 973与与各种DNA结合蛋白复合DNA的相互作用 也将被详细审查。 合成DNA底物将被 构建并与它们各自的DNA结合蛋白一起孵育， 存在FK 793（或FR 900482）;随后酶消化 将检查交联核苷酸-药物-氨基酸加合物以分离 并表征共价交联的结构。 合成 将利用第一个供资周期制定的方法， 完成FR 900482（1a）和FR 66979（2）的全合成; 技术将被应用于合成同位素标记的 用于阐明DNA-药物的结构的药物形式， 蛋白质交联 一种新的“潜伏”触发的有丝分裂素将是 合成并用作潜在的新型抗癌药物和探针， 大分子交联。

The primary objective of the proposed research is to study the mechanism of action of clinically significant new anti-tumor FR900482 (1a), FK973 (1b) and FR66979 (1c) (FK973, 1b was the first derivative to go to clinical trials). FK973 has been shown to cross-link double-stranded DNA and cross-link DNA to DNA-binding proteins in L1210 cells. Efforts will be directed at elucidating in complete mechanistic detail, the precise mechanism of the in vitro reductive activation of FR900482 that results in covalent modification of DNA. In the previous funding period, we have determined the sequence specificity of DNA cross-link formation by FR900482 and the natural reduction product FR66979 and have determined that reduction of these substances leads to the production of a mitosene derivative that preferentially cross-links DNA at 5'-dC-dG3' boxes. The interaction of FK973 with DNA complexed to various DNA-binding proteins will also be examined in detail. Synthetic DNA substrates will be constructed and incubated with their respective DNA-binding proteins in the presence of FK793 (or FR900482); subsequent enzymatic digestion of the cross-linked nucleotide-drug-amino acid adduct will be examined to isolate and characterize the structure of the covalent cross-link. Synthetic methodology developed during the first funding cycle will be utilized to complete a total synthesis of FR900482 (1a) and FR66979 (2); this technology will be applied to the synthesis of an isotopically-labeled form of the drug for use in elucidating the structure of the DNA-drug- protein cross-link. A new class of "latent" triggered mitosenes will be synthesized and utilized as potential new anti-cancer drugs and probes for the macromolecular cross-links.