Chemical approaches toward the identification, functional analysis, and biosynthesis of small molecule cyclomodulins

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。细胞周期调节蛋白是调节真核细胞周期进程的细菌毒素和效应物， 增殖、分化或凋亡，并且可能具有遗传毒性。某些E.大肠杆菌在人体肠道中 含有编码小分子环调节蛋白的基因簇（称为“clb”）， 罐头。有证据表明，前大肠杆菌素是前药，通过脱乙酰化酶转化为细胞毒素（大肠杆菌素）。 大肠杆菌素肽酶（colibactin peptidase，ClbP）。clb+ E.大肠杆菌诱导哺乳动物细胞DNA双链断裂 在体外和体内，表明这些分子被贩运（通过未知机制）到真核细胞， 并在用氧化偶氮甲烷处理的结肠炎易感小鼠中引发肿瘤形成。几个独立的螺柱- IE已经证明CLB簇与人类结肠直肠癌在流行病学上相关。作为 大肠杆菌素是不稳定的，所有的分离工作都采用clbP缺失菌株，以促进大肠杆菌素的积累。 更稳定的前乳酸菌素。我们开发了聚合高产率的线性前利巴菌素生物合成， 合成的前体，并表明它们在ClbP脱酰化后转化为不饱和亚胺;这些亚胺 通过核苷酸加成到亲电子环丙烷上使DNA烷基化。建立的结构-功能研究 不同的DNA识别和前药结构域。同样重要的是，我们的数据表明，使用clbP 缺失菌株导致产生替代的、非遗传毒性的结构，如前利巴菌素A-C。 preolibactin-886是已知的最复杂的clb分离物，是第一个含有α-氨基丙二酸酯的分离物， 由于，这被认为是重要的细胞病变效应。我们假设，不寻常的大环 前脂质体-886的结构也来源于clbP缺失菌株的使用。为了测试这一点，我们将预先- 帕雷precolibactin-886和关键合成衍生物/生物合成前体，并阐明其化学性质。我们 将确定前利巴菌素-886的线性前体的脱酰作用是否导致类似的电- 亲亚胺我们将评估合成大肠杆菌的效力、细胞周期效应和DNA损伤能力， 在斑马鱼模型中进行测试。利用酶学、基因缺失研究和X射线晶体学， 我们将阐明酶ClbL、ClbO、ClbM和ClbS的作用，这些酶在clb簇中编码，但 没有明确的职能。后两种酶的表型有助于大肠杆菌素的重新表达。 对clb+ E的研究为clb+ E的抑制提供了新的思路。大肠杆菌相关的结直肠癌。这笔赠款 拥有四名研究人员，他们在化学合成、天然产物生物合成 clb+ E的分离、临床前研究。大肠杆菌的体外和体内研究，以及酶学和蛋白质晶体学。 这项工作将建立一个机制模型，占所有已知的前利巴菌素，定义分子 某些E.大肠杆菌诱导的致癌作用，并提供了抑制clb+ E.大肠杆菌驱动的 肿瘤发生这些研究将为深入了解非蛋白源性cyclomodulins的功能作用提供帮助。

PROJECT SUMMARY/ABSTRACT. Bacteria on and within the body (the microbiota) influence human physiology, therapeutic responses, and dis- ease states. Cyclomodulins are bacterial toxins and effectors that modulate eukaryotic cell cycle progression, proliferation, differentiation, or apoptosis, and may be genotoxic. Certain strains of E. coli in the human gut contain a gene cluster (referred to as “clb”) that encodes small molecule cyclomodulins known as precolibac- tins. Evidence suggests precolibactins are prodrugs that are converted to cytotoxins (colibactins) by a dedi- cated peptidase (colibactin peptidase, ClbP). clb+ E. coli induce DNA double-strand breaks in mammalian cells in vitro and in vivo, suggesting these molecules are trafficked (by an unknown mechanism) to eukaryotic cells, and initiate tumor formation in colitis-susceptible mice treated with azoxymethane. Several independent stud- ies have demonstrated that the clb cluster is epidemiologically correlated with colorectal cancer in humans. As colibactins are unstable, all isolation efforts have employed clbP deletion strains to facilitate accumulation of the more stable precolibactins. We developed convergent high-yielding syntheses of linear precolibactin bio- synthetic precursors and showed they transform to unsaturated imines after ClbP deacylation; these imines alkylate DNA by nucleotide addition to an electrophilic cyclopropane. Structure–function studies established distinct DNA recognition and prodrug domains. Of equal significance, our data indicate that the use of clbP deletion strains results in the production of alternative, non-genotoxic structures, such as precolibactins A–C. Precolibactin-886 is the most complex clb isolate known and is the first that contains an α-aminomalonate resi- due, which is believed to be important for cytopathic effects. We hypothesize that the unusual macrocyclic structure of precolibactin-886 also derives from employment of a clbP deletion strain. To test this we will pre- pare precolibactin-886 and key synthetic derivatives/biosynthetic precursors and elucidate their chemistry. We will determine if deacylation of the linear precursor to precolibactin-886 leads to production of similar electro- philic imines. We will evaluate the potency, cell cycle effects, and DNA-damaging abilities of synthetic colibac- tins and controls in a zebrafish model. Using enzymology, genetic deletion studies, and X-ray crystallography, we will elucidate the roles of the enzymes ClbL, ClbO, ClbM and ClbS, which are encoded in the clb cluster but do not have well-defined functional roles. The latter two enzymes phenotypically contribute to colibactin re- sistance and their study may illuminate methods to inhibit clb+ E. coli-associated colorectal cancer. This grant employs four investigators with non-overlapping expertise in chemical synthesis, natural products biosynthesis and isolation, preclinical studies of clb+ E. coli in vitro and in vivo, and enzymology and protein crystallography. This work will establish a mechanistic model that accounts for all known precolibactins, define the molecular mechanisms by which certain E. coli induce carcinogenesis, and inform strategies to inhibit clb+ E. coli-driven tumorigenesis. These studies will provide insights into the functional roles of non-proteiogenic cyclomodulins.