Structural Biochemistry of DNA Dealkylation

描述(申请人提供)：O6-烷基鸟嘌呤-DNA烷基转移酶(AGT)不仅是保护人类免受烷化细胞毒素和致癌物侵害的关键蛋白质，而且也是限制烷化化疗药物抗肿瘤效果的主要因素。在人类中，AGT是一种独特的、直接的DNA损伤逆转蛋白，通过选择性地将O6-烷基加合物转移到内部半胱氨酸残基，在一步中修复O6-烷基鸟嘌呤位点。该项目解决了在分子水平上表征AGT活性的挑战，该活性与其在基因组完整性中的作用以及AGT抑制剂和抗药蛋白的基于知识的设计有关。这项工作将利用和整合研究人员及其机构现有的研究优势和计划，以促进、开发和测试对AGT蛋白质和新型抑制剂的统一理解。该项目的四个具体目标将1)确定作用于DNA结合和构象变化的AGT残基和基序，2)建立作用于损伤特异性和脱烷基的结构化学，3)通过对关键AGT家族成员的深入比较结构分析，确定AGT活性的基本结构原理，以及4)创建一个基于这些耦合的结构、突变、生化和计算结果的综合抑制剂设计周期，以开发和测试新型AGT抑制剂。通过X射线衍射、生物物理方法以及泰纳实验室的计算分析和设计对AGT结构和蛋白质抑制物复合体的定量表征将与PEGG实验室的详细的体外和体内生化和突变结果相协调。总体而言，这些结果将提供对AGT的统一理解，这与表征其在遗传完整性和抗癌治疗中的作用有关。此外，将这种详细的分子信息与新的抑制剂相结合，作为敲除细胞中AGT的强大工具，将促进与癌症治疗改进直接相关的基于知识的进展。

DESCRIPTION (provided by applicant): O6-Alkylguanine-DNA alkyltransferase (AGT) is not only a key protein in protecting humans from alkylating cytotoxins and carcinogens, but it is also a predominant factor limiting the anti-tumor effectiveness of alkylating chemotherapeutic agents. In humans, AGT is a unique, direct DNA damage reversal protein that repairs O6-alkylguanine sites in a single step by selectively transferring the O6-alkyl adduct to an internal cysteine residue. This Project addresses the challenge of characterizing at the molecular level the activities of AGT relevant to its roles in genome integrity and to the knowledge-based design of AGT inhibitors and inhibitor-resistant proteins. This work will leverage and integrate the existing research strengths and programs of the investigators and their institutions to promote, develop, and test a unified understanding of AGT proteins and of novel inhibitors. The Project's four Specific Aims will 1) determine the AGT residues and motifs acting in DNA binding and conformational change, 2) establish the structural chemistry acting in damage specificity and dealkylation, 3) identify fundamental structural principles for AGT activities by in-depth comparative structural analyses of key AGT family members, and 4) create an integrated inhibitor design cycle that builds upon these coupled structural, mutational, biochemical, and computational results to develop and test new classes of AGT inhibitors. Quantitative characterization of AGT structures and protein-inhibitor complexes by X-ray diffraction, biophysical methods, and computational analyses and design in the Tainer lab will be coordinated with detailed in vitro and in vivo biochemical and mutational results from the Pegg lab. Overall, these results will provide a unified understanding of AGT relevant to characterizing its role in genetic integrity and resistance to cancer therapies. Moreover, the combination of this detailed molecular information with new inhibitors as powerful tools to knock out AGT in cells will promote knowledge-based advances directly relevant to improvements in cancer therapies.