PROTOBERBERINES AS DUAL POISONS OF TOPOISOMERASES

描述：原黄连素代表了一种新的有机结构类别 对中枢神经系统癌细胞具有选择性细胞毒性的阳离子。他们的 细胞毒性机制已被证明包括人类中毒 DNA拓扑异构酶。使用纯化的拓扑异构酶，原黄连素 显示对拓扑异构酶I和II(TOP1和TOP2)都有毒害作用。这对双人 拓扑异构酶的毒物代表了一类新兴的抗癌药物 可能比TOP I或TOP2特定的药物活性更高的药物 毒药。这项提议的总体目标是理解分子 原黄连素不同程度毒害人上皮细胞的机制 TOP2。为了实现这一目标，已有100多种原黄连素 合成及门对拓扑异构酶的毒性和杀伤能力的评价 广泛的肿瘤细胞。除了这些生化和细胞 除了生物学研究，我们还启动了对 原黄连素与DNA的相互作用。初步研究表明 一种混合的DNA结合模式(结合了两种次要的沟槽定向 和插层相互作用)是双重中毒的原因 原黄连素的活性。我们假设未成年人 沟槽定向相互作用是TOP I中毒的关键，而 插层相互作用在TOP2中毒中起重要作用。具体目标 这一建议的目的是：(A)描述DNA的特征并将其相互关联 原黄连素的结合和拓扑异构酶毒化特性。这些 研究的目的是提供证据支持特定的 药物与DNA的相互作用(即小沟槽定向与嵌入性) 不同的拓扑异构酶中毒。(B)确定未成年人的角色 TOP I中毒中的沟槽定向相互作用。具体地说，我们将测试 小沟结合诱导DNA结构的可能性 导致TOP1介导的DNA切割被刺激的扰动。(C) 为了表征特定的药物-DNA和药物-酶的相互作用 使用各种生化方法的三元可裂解络合物，如 亲和标记和蛋白质修饰干扰。这些研究是 旨在评估支配环境的单个分子相互作用 三元可裂解络合物的形成和稳定。我们的建议 使用这种新型拓扑异构酶毒药的研究将增强我们的 对拓扑异构酶中毒的分子基础的了解 已被确定为与抗肿瘤有关的关键分子事件 广泛的天然化合物和合成化合物的活性。

DESCRIPTION: Protoberberines represent a new structural class of organic cations with selective cytotoxicity against CNS cancer cells. Their cytotoxic mechanisms have been demonstrated to involve poisoning of human DNA topoisomerases. Using purified topoisomerases, protoberberines are shown to poison both topoisomerases I and II (TOP1 and TOP2). These dual poisons of topoisomerases represent an emerging class of new anticancer agents with potential improved activity over either TOP I - or TOP2-specific poisons. The overall goal of this proposal is to understand the molecular mechanisms by which protoberberines differentially poison human TOP I and TOP2. Toward this goal, over one hundred protoberberines have been synthesized and evaluated for door ability to poison topoisomerases and kill a broad range of tumor cells. In addition to these biochemical and cell biological studies, we also have initiated biophysical studies of protoberberine interactions with DNA. Preliminary studies have suggested that a mixed mode of DNA binding (incorporating both minor groove-directed and intercalative interactions) is responsible for the dual poisoning activities of protoberberines. We hypothesize that the minor groove-directed interaction is critical for TOP I poisoning, while the intercalative interaction is important in TOP2 poisoning. The Specific Aims of this proposal are: (a) To characterize and cross-correlate the DNA binding and topoisomerase poisoning properties of protoberberines. These studies are designed to provide evidence supporting the role of specific drug-DNA interactions (i.e. minor groove-directed versus intercalative) in differential topoisomerase poisoning. (b) To determine the role of minor groove-directed interactions in TOP I poisoning. Specifically, we will test the possibility that minor groove binding induces a DNA structural perturbation that results in stimulation of TOP1-mediated DNA cleavage. (c) To characterize specific drug-DNA and drug-enzyme interactions within the ternary cleavable complex using various biochemical methods, such as affinity labeling and protein modification interference. These studies are designed to assess the individual molecular interactions that govern the formation and stabilization of the ternary cleavable complex. Our proposed studies using this new class of topoisomerase poisons will enhance our understanding of the molecular basis for topoisomerase poisoning, which has been established as the key molecular event responsible for the antitumor activity of a broad -range of naturally occurring and synthetic compounds.