BIOSYNTHESIS OF THE CYCLIC PEPTIDE TOXINS OF AMANITA MUSHROOMS

描述（由申请人提供）：迫切需要新的药剂来对抗各种各样的旧的和新出现的人类疾病，例如AIDS、抗药性病原体（例如结核分枝杆菌、金黄色葡萄球菌和铜绿假单胞菌）、真菌疾病和许多类型的癌症。然而，发现新型化合物的速度已经放缓。天然产品（由细菌和真菌等生物体产生的化学物质）历来是我们大多数药物的来源。自然界提供了具有医学相关生物活性的新型化学类别的多个实例。一个例子是鹅膏属真菌的环肽毒素。这些化合物在已知的天然产物中具有几种独特和理想的性质，使它们成为有前途的支架，在其上设计用于改善人类健康的新的治疗化合物。例如，它们是已知的在核糖体上合成的最小的肽，其中一些已经被认为具有强大和独特的生物靶标。拟议研究中使用的方法包括分子遗传学、下一代深度基因组测序、生物信息学、生物化学、化学以及针对靶生物和哺乳动物癌细胞系的生物测定。本建议的具体目的是，首先，确定鹅膏毒肽和鬼笔毒素生物合成中的所有步骤，预计这些步骤包括蛋白水解加工、环化、羟基化、合成独特的氨基酸交联桥（trypathiridine），以及在鬼笔毒素的情况下，将一个氨基酸从L转化为不寻常的D形式。第二个目的是研究帽盖菇中鹅膏毒素的生物合成，帽盖菇是一种与鹅膏无关的蘑菇，可以产生相同的化合物。对鹅膏菌和帽盖菌的研究将在几个方面相互补充，从而对生物合成途径进行更明确的描述。第三个目标是在另一种比鹅膏菌本身更易遗传的宿主生物中表达毒素生物合成途径。这将建立一个实验平台来详细剖析该途径，并以有益的方式对其进行修改。第四个目的是利用该实验系统在体内合成基于鹅膏毒素支架的新型环肽。鹅膏蕈碱有超过300万种可能的排列，预计这个新化合物库富含对治疗细菌和真菌疾病以及癌症具有潜在药物重要性的化学物质。 公共卫生相关性：我们目前使用的大多数抗生素和抗癌化合物来自天然来源，如细菌、真菌和植物。鹅膏属的某些蘑菇产生生物活性环肽，在生物医学研究和临床医学中具有一系列已知或潜在的用途。该提案描述了了解这类独特的天然产物是如何生物合成的基础研究，目的是生产具有医疗用途的新化合物家族。

DESCRIPTION (provided by applicant): There is a critical need for new pharmaceutical agents against a wide variety of old and emerging human diseases, such as AIDS, antibiotic-resistant pathogens such as Mycobacterium tuberculosis, Staphylococcus aureus, and Pseudomonas aeruginosa, fungal diseases, and many types of cancer. However, the rate at which new types of compounds are being discovered has slowed. Natural products (chemicals made by living organisms such as bacteria and fungi) have historically been the source of most of our medicines. The natural world provides multiple examples of novel chemical classes with medically relevant biological activities. One is exemplified by the cyclic peptide toxins of fungi in the genus Amanita. These compounds have several unique and desirable properties among known natural products that make them promising scaffolds on which to design new therapeutic compounds for improving human health. For example, they are the smallest known peptides synthesized on ribosomes, and some of them already are known to have strong and unique biological targets. Methods used in the proposed studies include molecular genetics, next-generation deep genome sequencing, bioinformatics, biochemistry, chemistry, and bioassays against target organisms and mammalian cancer lines. The specific aims of this proposal are, first, to identify all of the steps in the biosynthesis of the amatoxins and phallotoxins, which are predicted to include proteolytic processing, cyclization, hydroxylation, synthesis of a unique amino acid crossbridge (trypathionine), and, in the case of the phallotoxins, conversion of one amino acid from the L to the unusual D form. The second aim is to study the biosynthesis of the amatoxins in Galerina, a mushroom unrelated to Amanita that makes the same compounds. The work on Amanita and Galerina will complement each other in several ways, leading to a more definitive description of the biosynthetic pathway. The third aim is to express the toxin biosynthetic pathway in another host organism that is more genetically tractable than Amanita itself. This will establish an experimental platform to dissect the pathway in detail, and to modify it in beneficial ways. The fourth aim is to use this experimental system to synthesize in vivo new cyclic peptides based on the Amanita toxin scaffold. There are more than 3 million possible permutations of amanitin, and this library of novel compounds is predicted to be rich in chemicals of potential pharmaceutical importance for treatment of bacterial and fungal diseases, and cancer. PUBLIC HEALTH RELEVANCE: Most of our current antibiotics and anticancer compounds come from natural sources such as bacteria, fungi, and plants. Certain mushrooms in the genus Amanita produce bioactive cyclic peptides with a range of known or potential uses in biomedical research and clinical medicine. This proposal describes basic research on understanding how this unique class of natural products is biosynthesized, with the goal of producing families of new compounds of medical utility.