Structural and functional studies of colibactin

大肠杆菌素的结构和功能研究

• 批准号: 10467675
• 负责人: Seth B. Herzon
• 金额: $ 67.76万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-11 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467675
• 关键词: Achievement; Aerobic; Affinity; Alkylation; Amines; Anabolism; Bacteria; Binding; Biochemical; Biological; Cells; Chemistry; Clustered Regularly Interspaced Short Palindromic Repeats; Colorectal Cancer; Complex; Crystallography; Cyclization; DNA; DNA Binding; DNA Crosslinking; DNA Interstrand Crosslinking; DNA Repair Pathway; Degradation Pathway; Detection; Development; Diagnosis; Disease; Enzymatic Biochemistry; Enzymes; Epidemiology; Epithelial Cells; Escherichia coli; Evaluation; Funding; Future; Gene Cluster; Genes; Genetic; Genomics; Goals; Grant; Half-Life; Hand; Human; Hydrolysis; Imines; Immune response; Incubated; Induced Mutation; Intestines; Isomerism; Isotope Labeling; Lactams; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Mediator; Membrane Transport Proteins; Metabolic; Methods; Modeling; Molecular; Mutagens; Mutation; Mutation Analysis; Natural Source; Nucleotides; Organism; Pathway interactions; Periodicity; Phenotype; Physiological; Physiology; Play; Prevention; Protein Isoforms; Resistance; Role; Stable Isotope Labeling; Structure; Synthesis Chemistry; Testing; Time; Tumor Promotion; Work; adduct; carcinogenesis; carcinogenicity; chemical synthesis; colon cancer patients; colorectal cancer prevention; colorectal cancer treatment; crosslink; cyclopropane; design; genotoxicity; inhibitor; insight; microbiome; microbiome composition; microbiota; novel therapeutic intervention; oxidation; pathogenic Escherichia coli; pharmacophore; prevent; resistance mechanism; response; screening; small molecule; tandem mass spectrometry; tool; treatment response; tumorigenesis; whole genome

PROJECT SUMMARY/ABSTRACT: Bacteria on and within the body (the microbiota) influence human physiology, therapeutic responses, and dis- ease states. Many of these effects are mediated by small molecules, but determining the structures and func- tions of these small molecule mediators is challenging due to the complex metabolic and genetic background of the microbiota. Certain strains of E. coli in the human gut contain a gene cluster (referred to as “clb” or “pks”) that encodes the biosynthesis of a metabolite known as colibactin. Several independent studies have demon- strated that the clb cluster is epidemiologically-correlated with colorectal cancer (CRC) in humans, and recently a casual role for clb+ bacteria in tumorigenesis has been established. However, it has been impossible to link these effects directly to colibactin because the molecule is unstable and cannot be isolated from the bacteria. During the prior funding period we indirectly elucidated the overall structure of colibactin by a combination of chemical synthesis, genetics, stable isotope labeling, mass spectrometry (MS) and enzymology. This work es- tablished colibactin as a heterodimeric molecule capable of generating interstrand cross-links (ICLs) in DNA via two-fold nucleotide addition to electrophilic cyclopropane residues. Nonetheless, because the structure was based largely on MS analysis, alternative colibactin isomers are possible and we now have evidence that the right-hand warhead of colibactin undergoes ring–chain isomerization to form more complex isomers. Addition- ally, the central α-aminoketone of colibactin is unstable toward oxidative degradation and it is not yet known whether or not this residue is present at the time of DNA cross-linking. Here we will carry out the synthesis and biological evaluation of synthetic colibactins with the goal of resolving these final structural ambiguities. We have designed and will synthesize colibactin derivatives locked in the ring and chain isoforms and carry out studies to evaluate the genotoxicity of each of these compounds. We have successfully synthesized the α-aminoketone core of colibactin and will use this chemistry to prepare colibactin 771, the immediate product of the clb pathway. We will carry out crystallographic studies to determine the mode of binding of colibactin to DNA, and the mode of interaction of colibactin precursors with the maturation enzyme ClbP, the self-resistance enzyme ClbS, and the transmembrane transporter ClbM. Significantly, the synthetic chemistry we have developed allows us to probe for a causal link between colibactin and tumorigenesis through elucidation of the mutational signature triggered by the molecule. We will map out the functional host response to colibactin exposure using a high- throughput CRISPR genomic screen. This work is essential in that it provides the only means to study colibactin since it cannot be obtained from natural sources. It will test the hypothesis that colibactin is the agent underlying clb+ bacteria-associated CRC. Overall, these synthetic and enzymological/structural studies will guide future strategies to diagnose and prevent microbiome-associated cancers.

项目总结/摘要： 身体上和身体内的细菌（微生物群）影响人体生理学，治疗反应和疾病。 缓解状态。这些效应中的许多是由小分子介导的，但决定结构和功能的是， 这些小分子介体的选择是具有挑战性的，这是由于复杂的代谢和遗传背景， 微生物某些E.人肠道中的大肠杆菌含有一个基因簇（称为“clb”或“pks”）， 编码大肠杆菌素代谢物的生物合成几项独立研究表明， 研究表明，clb簇与人类结直肠癌（CRC）流行病学相关，最近 已经确定了CLB+细菌在肿瘤发生中的偶然作用。然而，它一直无法联系起来， 这些作用直接作用于大肠杆菌素，因为该分子不稳定，不能从细菌中分离出来。 在上一个资助期，我们通过以下方法的组合间接阐明了大肠杆菌素的整体结构： 化学合成、遗传学、稳定同位素标记、质谱（MS）和酶学。这项工作是- 建立了大肠杆菌素作为异二聚体分子，能够通过以下途径在DNA中产生链间交联（ICL）： 亲电子环丙烷残基的两倍核苷酸加成。尽管如此，由于结构是 主要基于MS分析，大肠杆菌素的替代异构体是可能的，我们现在有证据表明， 大肠杆菌素的右侧弹头经历环-链异构化以形成更复杂的异构体。附加- 同样，大肠杆菌素的中心α-氨基酮对氧化降解不稳定，目前尚不清楚 在DNA交联时是否存在该残基。在这里，我们将进行合成， 对合成大肠杆菌素进行生物学评价，目的是解决这些最终的结构模糊性。我们有 设计并将合成锁定在环和链异构体中的大肠杆菌素衍生物，并进行研究， 评估这些化合物的遗传毒性。我们成功地合成了α-氨基酮 并将使用这种化学方法制备colibactin 771，即clb途径的直接产物。 我们将进行晶体学研究，以确定大肠杆菌素与DNA的结合模式，以及 大肠杆菌素前体与成熟酶ClbP、自身抗性酶ClbS和 跨膜转运蛋白ClbM。值得注意的是，我们开发的合成化学使我们能够 通过阐明突变特征探讨大肠杆菌素与肿瘤发生之间的因果关系 由分子触发。我们将绘制出宿主对大肠杆菌素暴露的功能反应，使用高- 通过CRISPR基因组筛选。这项工作是必不可少的，因为它提供了研究大肠杆菌素的唯一手段 因为它不能从天然来源获得。它将检验大肠杆菌素是导致 clb+细菌相关CRC。总的来说，这些合成和酶学/结构研究将指导未来的研究。 诊断和预防微生物群相关癌症的策略。