Characterization of YcaO-Dependent Natural Product Biosynthetic Pathways

 描述(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的确定。毋庸置疑，NPs及其简单的衍生品一直是制药业历史上最重要的药物先导来源。除了在医学上的应用，NPs还启发了几代合成化学家，并提供了揭开细胞生物学基本方面的化学工具。了解NP的哪些部分可以产生特定的性质，可以合理地增强靶标结合能力、药代动力学参数、活性光谱等。传统的合成方法建立构效关系(SAR)对于人工挑战NP支架通常是困难的。相反，一条设计得当的生物合成路线可以代表一种在操作上更好的方法来建立SAR并加速药物先导的引入。该项目由三个相关但完全独立的具体目标组成。每个拟议的目标都以一种独特的、结构复杂的NP支架为目标，该支架源于核糖体前体多肽。对于每个目标，我们的目标是在体外重建生物合成途径，并通过突变前体多肽基因来生成用于SAR目的的类似物。还将对关键生物合成酶的机理特征进行评估。目的研究富含噻唑的硫肽类抗生素，其成员对细菌翻译有不同的抑制作用。噻唑类化合物是由一种环脱水酶作用形成的，我们对该酶有相当的专业知识。形成噻唑的环脱水酶属于神秘命名的“YcaO”超家族，我们已经证明了该超家族利用三磷酸腺苷来磷酸化酰胺基氧。这种直接的酰胺主链活化机理促进了环脱水反应。硫肽由一个中心吡啶或脱氢哌啶大环进一步定义，该大环由两个脱水Ser残基的预测和化学上令人着迷的[4+2]环加成而产生，本项目也将充分描述这一点。目的II研究硫代病毒胺的体外生物合成，硫代病毒胺是一种含硫代酰胺的细胞凋亡激活素NP。AIM III致力于含有大分子嘧啶和噻唑的肉霉素，它们代表着一类未开发的细菌翻译抑制物。所有这三个生物合成的基因簇都编码YcaO酶(两个用于博特霉素)，我们暗示这些酶的作用不仅仅是环状脱水。在硫代病毒胺的情况下，YcaO被预测参与硫代酰胺的形成，而博特霉素中的YcaO被怀疑参与了噻唑和大脒的形成，所有这些都是通过依赖于ATP的酰胺羰基激活机制来实现的。该项目将极大地促进我们对NP生物合成和机械酶学的总体理解，同时也将建立三种NP途径的酶耐受性。通过我们提出的异源和化学酶方法将额外的化学多样性赋予已知的NP支架，为揭示新的药物线索并最终扩大我们的药物馆藏带来了巨大的希望。

 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.