Design and Characterization of DNA Interactive Agents

描述（申请人提供）：拟议的项目加入了克尔温集团的合成和药理学专业知识（药学院药物化学部），拥有Brodbelt集团的分析专业知识（化学和生物化学系）和艾灵顿小组的分子生物学专业知识（化学和生物化学系，细胞和分子生物学研究所），以提供一个策略的设计和表征的DNA相互作用剂。该项目的总体目标是开发电喷雾电离质谱（ESI-MS）的能力，用于表征金属介导的药物/DNA和药物/四链体相互作用沿着快速体外选择方法，以深入了解药物-金属和药物-药物缔合影响药物/DNA结合、选择性和酶抑制的过程。具体目标包括：1）金属介导的DNA相互作用化合物和G-四链体DNA结合化合物的设计和合成，重点在于解决金属介导的药物/DNA结合和药物/G-四链体DNA结合的特定结构方面，并用作基于特定ESI-MS的筛选应用的定向文库。采用溶液法和固相法制备了Mg 2+离子介导的DNA结合抗肿瘤剂UK-1和A-62176、二萘嵌苯二酰亚胺以及苯并环化的UK-1和A-62176类似物的化合物和类似物库。2)ESI-MS和体外筛选方法在金属介导的药物/DNA复合物和药物/四链体复合物研究中的发展和应用。将进行结合亲和力和选择性的ESI-MS和分光光度/等温量热测量。能量可变的碰撞活化解离方法将用于研究药物/DNA复合物的碎片化，并将开发一系列新的气相足迹法来绘制结合位点。将开发基于亲和捕获和释放的快速选择方法，包括固定化DNA或药物。3)表征药物/DNA和药物/G-四链体复合物的金属介导的DNA结合的后果，以建立生物相关系统中DNA结合的功能相关性。 一方面涉及测定金属介导的DNA结合剂和G-四链体DNA配体抑制特异性DNA加工酶的能力。将对选定的DNA配体进行单独的SV 40大T抗原双链DNA和Gquadruplex DNA解旋酶抑制研究。将对UK-1和A-62176类似物进行人拓扑异构酶II抑制研究，以确定金属离子介导的DNA结合和酶抑制之间的关系。将使用DNA引物，用二萘嵌苯二酰亚胺类似物进行人端粒酶抑制测定。将在一系列人类癌细胞系以及革兰氏阴性和革兰氏阳性细菌中检查UK-1和A-62176类似物的抗癌和抗菌作用。

DESCRIPTION (provided by applicant): The proposed project joins the synthetic and pharmacological expertise of the Kerwin group (Division of Medicinal Chemistry, College of Pharmacy) with the analytical expertise of the Brodbelt group (Department of Chemistry and Biochemistry) and the molecular biology expertise of the Ellington group (Department of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology) to provide a strategy for the design and characterization of DNA-interactive agents. The overall goal of the project is to develop the capabilities of electrospray ionization mass spectrometry (ESI-MS) for characterization of metal-mediated drug/DNA and drug/quadruplex interactions along with rapid in vitro selection methods to provide insights into the processes by which drug-metal and drug-drug association affect drug/DNA binding, selectivity and enzyme inhibition. Specific objectives include: 1) the design and synthesis of metal-mediated DNA interactive compounds and G-quadruplex DNA binding compounds with a focus on addressing specific structural aspects of metal-mediated drug/DNA binding and drug/G-quadruplex DNA binding and for use as directed libraries for specific ESI-MS based screening applications. Individual compounds and libraries of analogs of the Mg 2+ ion-mediated DNA binding antitumor agents UK-1 and A62176, perylene diimides, and benzannulated UK-1and A-62176 analogs will be prepared by solution and solid-phase methods. 2) the development and application of ESI-MS and in vitro selection methods for investigation of metal-mediated drug/DNA complexes and drug/quadruplex complexes. ESI-MS and spectrophotometric/isothermal calorimetric measurements of binding affinities and selectivities will be undertaken. Energy-variable collision activated dissociation methods will be used to investigate the fragmentation of drug/DNA complexes, and an array of novel gas-phase footprinting methods will be developed to map the binding sites. Rapid selection methods based on affinity capture and release involving immobilized DNA or drugs will be developed. 3) the characterization of the consequences of metal-mediated DNA binding of drug/DNA and drug/G-quadruplex complexes to establish functional correlates of DNA binding in biologically relevant systems. One aspect involves determining the ability of the metal-mediated DNA binding agents and G-quadruplex DNA ligands to inhibit specific DNA processing enzymes. Separate SV40 large T antigen double-stranded DNA and Gquadruplex DNA helicase inhibition studies will be carried out on select DNA ligands. Human topoiomerase II inhibition studies will be carried out on the UK-1 and A-62176 analogs in order to establish the relationship between metal ion-mediated DNA binding and enzyme inhibition. Human telomerase inhibition assays will be performed with the perylene diimide analogs using DNA primers. The anticancer and antibacterial effects of UK-1 and A-62176 analogs will be examined in a range of human cancer cell lines and Gram negative and Gram positive bacteria.