Accessing Natural Products from Silent Biosynthetic Pathways

描述(由申请人提供)：真菌是一种特殊的结构独特和生物活性的小分子来源，其中许多已经成为当前和不断发展的治疗药物(例如青霉素、棘球菌素、环孢菌素、麦角生物碱和他汀类)开发的鼓舞人心的先导。尽管真菌前景看好，但由于次生代谢物编码基因的广泛沉默，真菌已被证明是一组具有挑战性的生物。表观遗传过程被认为是真菌主动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，表观遗传靶标的化学操作是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。这一假说是基于我们团队发表的研究和强有力的初步数据证明，化学表观遗传学方法是一种实用和基于理性的方法，可以转录激活沉默的生物合成途径，并确保各自的小分子天然产物的安全。我们的化学表观遗传学方法获得真菌隐蔽的次生代谢物提供了几个明显的优势，与目前的系统相比，因为它的简单，普遍适用，并能够很容易地纳入现代微生物筛选计划。美国国立卫生研究院的这一路线图倡议为检验中心假说和解决天然产物研究人员对进入沉默生物合成途径的有效范例的需求提供了一个独特的机会。以下两个具体目标将作为我们研究的重点。具体目标1涉及RFA-RM-09-005的关键方法发展部分。为此，我们将使用实时qRT-PCR和CHIP-SEQ来确定次生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洞察，这将使我们能够进一步完善我们团队的化学表观遗传技术，用于原位开采真菌中神秘的天然产物。特殊目的2是对化学表观遗传学方法从系统发育多样化的真菌中产生新代谢物的广谱能力的实验评估，并将导致产生一系列结构独特的天然产品，这些产品将提交给分子库小分子储存库(MLSMR)，以便在NIH赞助的分子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，在原位合理地操纵真菌沉默的生物合成途径的表达。这些结果预计将对天然产品领域产生积极和深远的影响，1)提供系统地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/医药应用的新化合物。 与公共卫生相关：真菌是一组高度多样化的生物(全球约有150万种)，负责生产一些人类已知的最重要的药物，包括抗生素(青霉素和环孢菌素)、降胆固醇药物(他汀类)和抗偏头痛药物(麦角胺)。真菌产生新的治疗线索的潜力是巨大的，但事实证明，真菌很难与之合作，因为它们能够在实验室培养条件下阻止天然产品的生产。我们的团队将测试一种基于表观遗传诱导的新方法，以定位参与天然产品生物合成的真菌基因的激活。这种方法有望让研究人员直接和立即获得大量新化合物，这些化合物有可能帮助研究和治疗许多人类疾病。

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.