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

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。环调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，并可能是遗传毒性的。人体肠道中的某些大肠杆菌菌株 含有一个基因簇(称为CLB)，它编码小分子环调蛋白，称为Pre colibac- 罐头。有证据表明，前大肠埃希菌毒素是一种前体药物，可通过一种新的代谢途径转化为细胞毒素(大肠杆菌毒素)。 Cated多肽酶(Colibactin多肽酶，ClbP)。Clb+E.Coli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(通过一种未知的机制)运输到真核细胞， 并在使用偶氮甲烷治疗的结肠炎易感小鼠中启动肿瘤形成。几个独立的种马- IES已经证明，CLB聚集性与人类结直肠癌的流行病学相关。AS 大肠杆菌是不稳定的，所有的分离工作都使用了clbP缺失菌株来促进细菌的积累 结肠杆菌前体素越稳定。我们开发了收敛的高产率的线性前结合蛋白生物合成。 合成前体，并显示它们在ClbP去酰化后转化为不饱和亚胺；这些亚胺 通过在亲电的环丙烷上加核苷酸来使DNA烷化。建立了结构-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，ClbP的使用 缺失菌株会导致产生替代的、非遗传毒性的结构，如前大肠杆菌A-C。 Precolibactin-886是已知的最复杂的CLB分离物，也是第一个含有α-氨基丙酸根的分离物。 DUE，这被认为是重要的细胞病变效应。我们假设这种不同寻常的大循环 前结合蛋白-886的结构也来自于使用clbP缺失菌株。为了测试这一点，我们将预先 Pare precolibactin-886和关键的合成衍生物/生物合成前体，并阐明它们的化学。我们 将确定前粘连蛋白-886的线性前体的脱酰是否会导致类似的电- 亲水性的亚胺类。我们将评估合成的Colibac的效力、细胞周期影响和DNA损伤能力- 斑马鱼模型中的罐头和控件。利用酶学、基因缺失研究和X射线结晶学， 我们将阐明ClbL、ClbO、ClbM和ClbS酶的作用，这些酶在CLB簇中编码，但 没有明确定义的职能角色。后两种酶在表型上对结节蛋白反应有贡献。 抗性和他们的研究可能会阐明抑制CLB+大肠杆菌相关性结直肠癌的方法。这笔赠款 雇佣了四名在化学合成、天然产物生物合成方面具有非重叠专业知识的研究人员 以及CLB+大肠杆菌的分离、体外和体内的临床前研究以及酶学和蛋白质结晶学。 这项工作将建立一个机制模型，解释所有已知的前大肠杆菌素，定义分子 某些大肠杆菌诱导致癌的机制和抑制CLB+大肠杆菌驱动的策略 肿瘤发生学。这些研究将为深入了解非蛋白生成环调蛋白的功能作用提供依据。

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.