Structural and functional studies of colibactin

项目摘要/摘要： 人体内和体内的细菌(微生物区系)影响人类的生理、治疗反应和疾病。 放松状态。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 由于复杂的代谢和遗传背景，这些小分子介质的检测是具有挑战性的 微生物区系。人体肠道中的某些大肠杆菌菌株含有一个基因簇(称为“CLB”或“PKS”)。 编码一种代谢物的生物合成，这种代谢物被称为Colibactin。几项独立研究都有恶魔- 研究表明，CLB簇与人类结直肠癌(CRC)的流行病学相关，最近 CLB+细菌在肿瘤发生中的偶然作用已经确定。然而，它一直无法联系到 这些作用直接作用于丛生肌动蛋白，因为该分子不稳定，无法从细菌中分离出来。 在之前的资助期间，我们通过以下组合间接阐明了Colibactin的整体结构 化学合成、遗传学、稳定同位素标记、质谱学和酶学。这项工作是- 作为一种能够在DNA中产生链间交链(ICL)的异源二聚体分子 亲电的环丙烷残基加成两倍核苷酸。尽管如此，因为这个结构是 在很大程度上基于MS分析，替代的结节蛋白异构体是可能的，我们现在有证据表明 结节蛋白的右侧弹头发生环链异构化，形成更复杂的异构体。添加- 此外，结节蛋白的中心α-氨基酮对氧化降解不稳定，目前尚不清楚。 在DNA交联时该残基是否存在。在这里，我们将进行综合和 对合成大肠埃希菌毒素进行生物学评价，以解决这些最终的结构模糊问题。我们有 设计并将合成锁定在环状和链状异构体中的粘连蛋白衍生物，并开展研究 评估每种化合物的遗传毒性。我们成功地合成了α-氨基酮 并将使用这种化学方法来制备CLB途径的直接产物--Colibactin 771。 我们将进行结晶学研究，以确定结缔组织蛋白与DNA的结合方式，以及 结节蛋白前体与成熟酶ClbP，自抗性酶ClbS，以及 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结节蛋白与肿瘤发生之间的因果联系 由分子触发。我们将用一种高密度的 吞吐量CRISPR基因组筛选。这项工作是必不可少的，因为它提供了研究结缔组织蛋白的唯一手段 因为它不能从自然来源获得。它将检验这样一种假设，即大肠杆菌是潜在的病原体。 CLB+细菌相关性结直肠癌。总体而言，这些合成和酶/结构研究将指导未来 诊断和预防微生物组相关癌症的战略。

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.