Topoisomerase I-directed Anticancer Drugs

人DNA拓扑异构酶I（hTOP 1）是喜树碱等抗癌药物的一个新的高效分子靶点。 CPT通过捕获共价反应中间体（三元TOP 1可裂解复合物）来抑制（毒害）TOP 1。 这些TOP 1可切割复合物代表了一种新型的细胞损伤，在杀死肿瘤细胞方面非常有效。 除了CPT，越来越多的化合物（TOP 1毒物）（例如吲哚并咔唑、两面针碱、saintopin、吗啉多柔比星、阿克拉霉素A、茚并[1，2-c]异喹啉、诺加霉素、放线菌素D、原小檗碱、二苯并[c，h]噌啉、2-苯基苯并咪唑和三苯并咪唑）已被鉴定为诱导TOP 1可裂解复合物。 尽管TOP 1可切割复合物在肿瘤细胞杀伤中的重要性明确，但TOP 1毒物杀伤肿瘤细胞的分子机制仍不清楚。 我们的初步研究表明，至少有两种不同的分子机制可能参与捕获TOP1裂解复合物，这些不同的TOP1毒药。 CPT，原型TOP1毒物，通过特异性抑制TOP1催化的断裂/再连接反应的再连接步骤来捕获TOP1可切割复合物，推测是通过在DNA切割位点结合TOP1- DNA复合物（再连接阻断剂）。相比之下，诺加霉素，一种具有插入和小沟定向相互作用模式的DNA结合剂，通过诱导有利于TOP 1结合/切割的远端DNA弯曲（切割增强剂）来捕获TOP 1可切割复合物。 通过不同的TOP1毒物鉴定两种不同的TOP1中毒模式可能为药物设计和开发提供新的机会。 在当前的应用中，我们计划表征这两种模式的TOP1中毒，并探测不同的TOP1毒物诱导的差异生物反应。 具体目标是（1）以诺加霉素为模型建立TOP1切割增强剂的DNA曲率模型;（2）探索不同TOP1毒物诱导的TOP1可切割复合物的蛋白质构象和细胞修饰。

Human DNA topoisomerase I (hTOP1) is a highly effective new molecular target for anticancer drugs such as camptothecins (CPTs). CPTs inhibit (poison) TOP1 by trapping a covalent reaction intermediate, the ternary TOP1 cleavable complex. These TOP1 cleavable complexes represent a new type of cellular lesion highly effective in killing tumor cells. In addition to CPTs, a growing list of compounds (TOP1 poisons) (e.g. indolocarbazoles, nitidines, saintopin, morpholinyl doxorubicin, aclacinomycin A, indeno[1,2-c]isoquinolines, nogalamycin, actinomycin D, protoberberines, dibenzo[c,h]cinnolines, 2-phenylbenzimidazoles and terbenzimidazoles) have been identified to induce TOP1 cleavable complexes. Despite the clear importance of TOP1 cleavable complexes in tumor cell killing, the molecular mechanism underlying tumor cell killing by TOP1 poisons remains unclear. Our preliminary studies have suggested that at least two distinct molecular mechanisms may be involved in trapping TOP1 cleavable complexes by these different TOP1 poisons. CPTs, the prototypic TOP1 poisons, trap TOP1 cleavable complexes by specifically inhibiting the religation step of the TOP1-catalyzed breakage/religation reaction, presumably by binding to the TOP1- DNA complex at the site of DNA cleavage (religation blockers). By contrast, nogalamycin, a DNA binder with both intercalative and minor groove-directed modes of interactions, traps TOP1 cleavable complexes by inducing a distal DNA bend which favors TOP1 binding/cleavage (cleavage enhancers). The identification of two distinct modes of TOP1 poisoning by different TOP1 poisons may offer new opportunities for drug design and development. In the current application, we plan to characterize these two modes of TOP1 poisoning and to probe the differential biological responses induced by different TOP1 poisons. The specific aims are (1) to establish a DNA curvature model for TOP1 cleavage enhancers using nogalamycin as a model, and (2) to probe the protein conformation and cellular modification of TOP1 cleavable complexes induced by different TOP1 poisons.