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.

项目概要/摘要： 体内和体外的细菌（微生物群）影响人体生理、治疗反应和疾病。 缓解状态。许多这些效应是由小分子介导的，但决定结构和功能的 由于复杂的代谢和遗传背景，这些小分子介质的作用具有挑战性 微生物群。人类肠道中的某些大肠杆菌菌株含有基因簇（称为“clb”或“pks”） 它编码一种称为大肠杆菌素的代谢物的生物合成。多项独立研究表明 指出 clb 集群在流行病学上与人类结直肠癌 (CRC) 相关，最近 已确定 clb+ 细菌在肿瘤发生中的偶然作用。然而一直无法链接 这些影响直接作用于大肠杆菌素，因为该分子不稳定并且无法从细菌中分离出来。 在之前的资助期间，我们通过结合以下方法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 colibactin 作为一种异二聚体分子，能够在 DNA 中产生链间交联 (ICL) 亲电环丙烷残基上加成两倍核苷酸。尽管如此，由于结构 主要基于质谱分析，替代的大肠杆菌素异构体是可能的，我们现在有证据表明 大肠杆菌素的右侧弹头发生环链异构化，形成更复杂的异构体。添加- 盟友，colibactin 的中心 α-氨基酮对于氧化降解不稳定，目前尚不清楚 DNA 交联时是否存在该残基。在这里我们将进行合成和 对合成大肠杆菌素进行生物学评估，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colibactin衍生物，并进行研究 评估每种化合物的遗传毒性。我们成功合成了α-氨基酮 colibactin 的核心，并将利用这种化学方法制备 colibactin 771，这是 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，以及结合模式 大肠杆菌素前体与成熟酶 ClbP、自抗性酶 ClbS 的相互作用，以及 跨膜转运蛋白 ClbM。重要的是，我们开发的合成化学使我们能够 通过阐明突变特征来探究大肠杆菌素与肿瘤发生之间的因果关系 由分子触发。我们将使用高通量绘制出宿主对大肠杆菌素暴露的功能性反应。 高通量 CRISPR 基因组筛选。这项工作至关重要，因为它提供了研究大肠杆菌素的唯一方法 因为它不能从天然来源获得。它将检验大肠杆菌素是潜在药物的假设 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 诊断和预防微生物组相关癌症的策略。