Characterization of YcaO-Dependent Natural Product Biosynthetic Pathways

 描述（由申请人提供）：我们的团队专注于新天然产物（NP）的发现，生物合成和作用模式测定。毋庸置疑，NP及其简单衍生物一直是制药工业药物先导物的最重要的历史来源。除了在医学上的应用，纳米粒子还启发了几代合成化学家，并提供了化学工具来解开细胞生物学的基本问题。了解NP的哪些部分产生特定性质使得能够合理地增强靶标结合效力、药代动力学参数、活性谱等。传统的用于建立构效关系（SAR）的合成方法通常难以合成具有挑战性的NP支架。相比之下，适当设计的生物合成途径可能是建立SAR和加速药物先导物引入的操作优选方法。该项目由三个相关但完全独立的具体目标组成。每一个提出的目标都针对一个独特的，结构复杂的NP支架，该支架来源于核糖体前体肽。对于每个目标，我们的目标是在体外重建生物合成途径，并通过突变前体肽基因产生用于SAR目的的类似物。还将评估关键生物合成酶的机理表征。目的研究一类富含噻唑的硫肽类抗生素，其成员抑制细菌翻译的不同方面。噻唑是通过环己基转移酶的作用而形成的，对此我们有相当多的专业知识。噻唑形成环己基转移酶属于神秘命名的“YcaO”超家族，我们已经证明其在酰胺羰基氧的磷酸化中利用ATP。这种直接的酰胺骨架活化机制促进了环化脱水反应。硫肽由中心吡啶或脱氢哌啶大环进一步定义，产生于两个脱水Ser残基的预测和化学上迷人的[4+2]环加成，该项目也将充分表征。目的II靶向体外生物合成含硫代酰胺的硫代维瑞胺，一种具有抗肿瘤活性的NP。目的III是致力于macroamidine和噻唑含有bottromycins，这代表了一个未开发的类细菌翻译抑制剂。所有这三个生物合成基因簇编码YcaO酶（两个用于bottromycin），我们暗示其作用超出了环化脱水。在thioviridamide的情况下，YcaO被预测为参与硫代酰胺的形成，而在bottromycin中的YcaO怀疑参与噻唑和大环脒的形成，所有通过ATP依赖性酰胺羰基活化机制。该项目将显著推进我们对NP生物合成和机制酶学的总体理解，同时还建立了三种NP途径的酶耐受性。通过我们提出的异源和化学酶促方法将额外的化学多样性引入已知的NP支架中，为揭示新药线索并最终扩大我们的制药设备带来了巨大的希望。

 DESCRIPTION (provided by applicant): Our group has focused on the discovery, biosynthesis, and mode of action determination for novel natural products (NPs). It is beyond contestation that NPs, and simple derivatives thereof, have been the most historically significant source of drug leads for the pharmaceutical industry. Beyond their use in medicine, NPs have inspired generations of synthetic chemists and provided the chemical tools to unravel fundamental aspects of cell biology. Understanding what moieties of a NP give rise to a specific property enables the rational enhancement of target-binding potency, pharmacokinetic parameters, spectrum of activity, among others. Traditional synthetic approaches for establishing structure-activity relationships (SAR) are often intractable for synthetically challenging NP scaffolds. In contrast, a properly engineered biosynthetic route could represent an operationally preferable method to establish SAR and accelerate the introduction of drug leads. This project is comprised of three related, yet fully independent, specific aims. Each proposed aim targets a unique, architecturally-complex NP scaffold that originates from a ribosomal precursor peptide. For each tar- get, we aim to reconstitute the biosynthetic pathway in vitro and generate analogs for SAR purposes by mutation of the precursor peptide gene. The mechanistic characterization of key biosynthetic enzymes will also be evaluated. Aim I focuses on the thiazole-rich thiopeptide antibiotics, whose members inhibit different aspects of bacterial translation. The thiazoles are formed by the action of a cyclodehydratase, of which we have considerable expertise. Thiazole-forming cyclodehydratases belong to the cryptically named "YcaO" superfamily, which we have shown to utilize ATP in the phosphorylation of amide carbonyl oxygens. This direct amide backbone activation mechanism facilitates the cyclodehydration reaction. Thiopeptides are further defined by a central pyridine or dehydropiperidine macrocycle, arising from a predicted and chemically fascinating [4+2] cycloaddition of two dehydrated Ser residues, which this project will also fully characterize. Aim II targets the in vitro biosynthesis of thioviridamide, a thioamide-containing, apoptosis-activatin NP. Aim III is dedicated to the macroamidine- and thiazole-containing bottromycins, which represent an undeveloped class of bacterial translation inhibitor. All three of these biosynthetic gene clusters encode YcaO enzymes (two for bottromycin), which we implicate in roles beyond cyclodehydration. In the case of thioviridamide, the YcaO is predicted to be involved in thioamide formation while the YcaOs in bottromycin have suspected involvement in thiazole and macroamidine formation, all via an ATP-dependent amide carbonyl activation mechanism. This project will significantly advance our general understanding of NP biosynthesis and mechanistic enzymology, while also establishing the enzymatic tolerance of three NP pathways. Imparting additional chemical diversity into known NP scaffolds by our proposed heterologous and chemoenzymatic approaches holds enormous promise for revealing new drug leads and eventually expanding our pharmaceutical armamentarium.