Deciphering the structure and dynamics of quadruplex DNA and DNA-ligand complexes

项目总结 拟议中的研究将通过贡献新的研究成果来提高抗癌疗法的选择性和有效性。 关于非规范G-四链(GQ)DNA结构的知识，以及GQ与小分子DNA的相互作用 分子配体。生物信息学研究已确定370,000个序列具有G-四链形成 人类基因组中的潜力。现在有令人信服的生物学证据表明GQ在体内形成，而且 这些结构调节各种与癌症相关的生物学过程，如端粒保护， 癌基因表达、表观遗传修饰和DNA修复。因此，GQ DNA被牢固地确立为 癌症的重要治疗靶点。选择性地与GQ DNA结合的小分子一直是 已被证实，其中一些已被证明能抑制肿瘤细胞的生长；然而， 这种抑制作用是未知的。这样的小分子配体最终可能成为 产生优于传统诱变疗法的新型选择性抗癌药物。不幸的是， 以DNA为中心的药物发现计划受到可用于GQS的结构信息有限的影响，尤其是 在配体存在的情况下。GQ的高度结构多样性使情况变得更加复杂，他们的 相互矛盾的生物学功能，以及我们针对特定GQ折叠拓扑的有限能力。 为了应对这些挑战，我们建议进行全面的结晶学研究 重点研究端粒和癌基因启动子G-四链，既有单独的，也有与多种 小分子配体。这项工作将伴随着光谱分析和量热研究 配体与GQ DNA结合的热力学参数(如化学计量比、亲和力、选择性、驱动力 力量)。GQS和GQ-配体络合物的静态结构图将由严格的动力学补充 配体辅助的Gq折叠和结构重排的研究。动态信息可以帮助我们识别 G-四链体形成的时间尺度以及因此而可能受存在的 这些结构。G-四链结合或结构所必需的配体的化学和结构特征 重排将从动力学和结构研究中确定。此外，我们建议使用化学物质 我们展示了对N-甲基中卟啉IX(NMM)支架的修饰，这是前所未有的 对平行GQ折叠的选择性，但具有适度的结合亲和力。这些修饰应该会产生新的配体 它们保留了这种选择性，但具有更好的结合亲和力。显示选择性GQ相互作用的配体 体外试验将在体内测试其生物学效应，并将确定基因组靶标。总体而言， 建议的工作将提高我们对GQ结构可塑性的理解，为药物提供坐标 发现平台，阐明特定DNA靶标的配体选择性的来源，并指导设计 新的高选择性抗癌疗法，同时为斯沃斯莫尔提供变革性培训 本科生。

PROJECT SUMMARY The proposed research will improve the selectivity and efficacy of anticancer therapies by contributing new knowledge about non-canonical G-quadruplex (GQ) DNA structure, and the interactions of GQs with small- molecule ligands. Bioinformatics studies have identified 370,000 sequences with G-quadruplex-forming potential in the human genome. There is now convincing biological evidence that GQs form in vivo and that these structures regulate a variety of cancer-related biological processes, such as telomere protection, oncogene expression, epigenetic modification, and DNA repair. Thus, GQ DNA has been firmly established as an important therapeutic target for cancer. Small molecules that bind selectively to GQ DNA have been identified, and some have been shown to inhibit tumor cells growth; however, exact mechanisms underlying this inhibition are not known. Such small-molecule ligands may ultimately become lead compounds for the generation of novel selective cancer drugs superior to conventional mutagenetic therapies. Unfortunately, DNA-centered drug discovery programs suffer from limited structural information available for GQs, especially in the presence of ligands. The situation is further complicated by high structural diversity of GQs, their contradictory biological functions, and our limited ability to target a specific GQ folding topology. To address these challenges, we propose to perform comprehensive crystallographic investigations focused on telomeric and oncogene promoter G-quadruplexes, both alone and in complex with a variety of small-molecule ligands. This work will be accompanied by spectroscopic and calorimetric studies of the thermodynamic parameters of ligand binding to GQ DNA (e.g., stoichiometry, affinity, selectivity, driving forces). The static structural view of GQs and GQ-ligand complexes will be complemented by rigorous kinetics studies of ligand-assisted GQ folding and structural rearrangements. Kinetic information can help us identify the timescale of G-quadruplex formation and thus biological processes that can be affected by the presence of these structures. Chemical and structural features of ligands essential for G-quadruplex binding or structural rearrangements will be identified from kinetics and structural studies. Furthermore, we propose chemical modification to the scaffold of N-methylmesoporphyrin IX (NMM), which we showed displays unprecedented selectivity for a parallel GQ fold, yet has modest binding affinity. The modifications should lead to new ligands that retain this selectivity but have improved binding affinity. Ligands that display selective GQ interactions in vitro will be tested in vivo for their biological effects, and genomic targets will be identified. Collectively, the proposed work will enhance our understanding of GQ structural plasticity, supply coordinates for drug discovery platforms, shed light on the origin of ligand selectivity for a specific DNA target, and guide the design of novel highly selective anticancer therapies while providing transformative training to Swarthmore undergraduate students.