Accessing Natural Products from Silent Biosynthetic Pathways

描述（由申请人提供）：真菌是结构独特和具有生物活性的小分子的特殊来源，其中许多已经成为当前和不断发展的治疗剂（例如，青霉素、棘白菌素、环孢菌素、麦角生物碱和他汀类药物）开发的灵感来源。尽管真菌具有巨大的前景，但由于次级代谢物编码基因的广泛沉默，真菌已被证明是一种具有挑战性的生物探索群体。表观遗传过程被认为是真菌积极抑制参与天然产物生物合成的基因转录的重要手段。我们假设表观遗传靶点的化学操作是一种从沉默的生物合成途径获得结构独特的天然产物的有效技术。这一假设是基于我们小组发表的研究和强有力的初步数据，表明化学表观遗传学方法是一种实用的、合理的方法，可以激活沉默的生物合成途径，并确保它们各自的小分子天然产物。我们的化学表观遗传学方法获得真菌隐次生代谢物，由于其简单，普遍适用性和易于纳入现代微生物筛选程序的能力，与当前系统相比具有几个明显的优势。美国国立卫生研究院路线图倡议提出了一个独特的机会来测试中心假设，并解决天然产物研究人员需要一个有效的范式来访问沉默的生物合成途径。以下两个具体目标将成为我们研究的重点。具体目标1涉及RFA-RM-09-005的关键方法开发组成部分。为此，我们将使用实时qRT-PCR和ChIP-Seq来确定次级代谢物编码基因抑制的表观遗传学基础。这有望为表观遗传过程在沉默生物合成途径的转录抑制中所起的作用提供新的机制见解，这将使我们进一步完善我们小组的化学表观遗传技术，用于真菌中隐藏天然产物的原位开采。Specific Aim 2作为化学表观遗传学方法广谱能力的实验评估，从系统发育不同的真菌中产生新的代谢物，并将产生一系列结构独特的天然产物，这些产物将提交给分子文库小分子库（MLSMR），以便在美国国立卫生研究院资助的分子文库探针生产中心网络（MLPCN）中进行高通量筛选。我们的方法是高度创新的，因为它利用了一种独特的基于表观遗传学的技术来合理地操纵真菌沉默生物合成途径的原位表达。这些结果预计将对天然产物领域产生积极而深远的影响：1)提供系统探索真菌沉默生物合成途径所需的关键研究工具；2)提供具有重要生物医学/制药应用的新化合物。

DESCRIPTION (provided by applicant): Fungi are an exceptional source of structurally unique and biologically active small molecules, many of which have served as inspirational leads for the development of current and evolving therapeutic agents (e.g., penicillins, echinocandins, cyclosporins, ergot alkaloids, and statins). Despite their tremendous promise, fungi have proven to be a challenging group of organisms to explore due to extensive silencing of secondary- metabolite-encoding genes. Epigenetic processes are proposed to be an important means by which fungi actively suppress the transcription of genes involved in natural product biosynthesis. We hypothesize that chemical manipulation of epigenetic targets is an effective technique for accessing structurally-unique natural products from silent biosynthetic pathways. This hypothesis is based on our group's published studies and strong preliminary data demonstrating that a chemical epigenetic approach is a practical and rationally-based method for transcriptionally activating silent biosynthetic pathways and securing their respective small- molecule natural products. Our chemical epigenetic approach to accessing fungal cryptic secondary metabolites offers several distinct advantages over current systems due to its simplicity, universal applicability, and ability to be readily incorporated into modern microbial screening programs. This NIH Roadmap initiative presents a unique opportunity for testing the central hypothesis and addressing the need of natural products researchers for an effective paradigm to access silent biosynthetic pathways. The following two specific aims will serve as the focus of our studies. Specific Aim 1 addresses the key methodological development component of RFA-RM-09-005. For this aim, we will determine the epigenetic underpinnings of secondary- metabolite-encoding gene suppression using real-time qRT-PCR and ChIP-Seq. This is expected to provide novel mechanistic insight into the role that epigenetic processes play in the transcriptional suppression of silent biosynthetic pathways, which will enable us to further refine our group's chemical epigenetic technique for the in situ mining of cryptic natural products from fungi. Specific Aim 2 serves as an experimental assessment of the broad-spectrum capacity of the chemical epigenetic methodology to yield novel metabolites from phylogenetically diverse fungi and will result in the generation of a series of structurally unique natural products that will be submitted to the Molecular Libraries Small Molecule Repository (MLSMR) for high throughput screening throughout the NIH-sponsored Molecular Libraries Probe Production Centers Network (MLPCN). Our methodology is highly innovative because it utilizes a unique epigenetic-based technique for rationally manipulating the expression of fungal silent biosynthetic pathways in situ. These results are expected to have a positive and far-reaching impact on the field of natural products by 1) providing a critical research tool that is needed to systematically explore fungal silent biosynthetic pathways and 2) affording new compounds with important biomedical/pharmaceutical applications. PUBLIC HEALTH RELEVANCE: Fungi are a highly diverse group of organisms (approximately 1.5 million species worldwide) that are responsible for producing some of the most important drugs known to humankind including antibiotics (penicillins and cyclosporins), cholesterol-lowering agents (statins), and antimigraine drugs (ergotamine). The potential for fungi to yield new therapeutic leads is tremendous, but fungi have proven difficult to work with due to their ability to block the production of natural products under laboratory culture conditions. Our group will test a new methodology based on epigenetic induction for targeting the activation of fungal genes involved in natural product biosynthesis. This method is expected to give researchers direct and immediate access to a wealth of new compounds that have the potential to aid in the study and treatment of numerous human diseases.