Discovery of Natural Products and Natural Product-Like Inhibitors

天然产物，也被称为特化代谢物，是由微生物、真菌和无脊椎动物生物合成的小分子和多肽。它们具有巨大的结构多样性，并表现出广泛的生物活性。天然产物及其合成衍生物占目前临床上使用的大部分处方治疗药物，特别是抗生素和抗肿瘤药物。随着耐药细菌感染的发病率不断上升，迫切需要进行旨在发现和开发新型抗生素和新的抗菌靶点的基础和临床研究。重点是海洋无脊椎动物和未被充分研究的环境细菌作为天然产品来源生物，我们开展跨学科研究，包括抗生素的发现，作用机制的确定，以及完整的结构确定。我们还使用基因组挖掘技术来鉴定、表征和生产新型核糖体编码的天然产物，称为RiPPs（核糖体翻译后修饰肽）。以下是一些具有代表性的项目和研究结果。在最近的工作中，我们使用x射线晶体学、圆二色技术和计算ECD实验来确定双吲哚类天然产物盐氯二胺和木瓜胺的绝对构型，并展示了意想不到的手性性质，其中大环二烯在小分子而不是手性中心中占旋光和棉花效应的标志。通过生态偏向的微生物收集和基因组挖掘，我们发现，鉴定并产生了一类新的核糖体编码的多环肽天然产物，命名为黄绿蛋白。黄绿蛋白前体肽CdnA3由前导和核心序列组成，其不寻常之处在于后先导序列包含多个核心基序。大环化是由atp依赖性连接酶CdnC和CdnB催化的。利用x射线晶体学和质谱（MS），我们确定了CdnA3肽大环化的结构基础，并证明了连接酶在底物结合和释放时经历了主要的构象变化，以适应单个多肽前体的连续几轮环化，并证明了多核环化发生在n端到c端方向。这项工作对RiPP天然产物的草肽家族的工程设计具有重要意义，并有可能获得不同类别的蛋白酶抑制剂。

Natural products, also referred to as specialized metabolites, are small molecules and peptides that are biosynthesized by microorganisms, fungi and invertebrates. They possess enormous structural diversity and exhibit a broad range of biological activities. Natural products and their synthetic derivatives account for most of the prescribed therapeutics currently used in the clinic, especially among the antibiotic and antitumor drug classes. With the growing incidence of drug-resistant bacterial infections, there is a critical need for basic and clinical research aimed at the discovery and development of new classes of antibiotics and new antimicrobial targets. With an emphasis on marine invertebrates and understudied environmental bacteria as natural product source organisms, we carry out interdisciplinary research that includes antibiotic discovery, determination of mechanisms of action, and complete structural determination. We are also using genome mining techniques to identify, characterize and produce new classes of ribosomally-encoded natural products known as RiPPs (Ribosomal Post-translationally modified Peptides). A few representative projects and findings include the following. In recent work we have used X-ray crystallography, circular dichroism techniques and computational ECD experiments to determine the absolute configuration of the bis-indane natural products haliclonadiamine and papuamine and demonstrate unexpected chiroptical properties where a macrocyclic diene accounts for the sign of the optical rotation and Cotton effect in a small molecule rather than the chiral centers. Through ecology-biased microbial collections and genome mining we discovered, characterized and produced a new class of ribosomally-encoded multicycle peptide natural product class names the chryseoviridins. The chryseoviridin precursor peptide CdnA3 comprises a leader and core sequence and is unusual in that the post-leader sequence contains multiple core motifs. Macrocyclization is catalyzed by the ATP-dependent ligases CdnC and CdnB. Using X-ray crystallography and Mass Spectrometry (MS) we determined the structural basis for peptide macrocyclization of CdnA3 and demonstrated that the ligase undergoes a major conformational change upon substrate binding and release to accommodate successive rounds of cyclization on a single polypeptide precursor and demonstrated that multi-core cyclization takes place in an N-terminal to C-terminal direction. This work has important implications in the engineering of the graspetide family of RiPP natural products and has the potential to obtain diverse classes of protease inhibitors. Our work also aims to identify new antibiotics and antivirals. Recently we have identified new structural classes of secondary metabolites that inhibit growth of wild type and drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. In vitro enzyme assays, in vivo antibacterial assays, and genome sequencing of resistant variants has revealed diverse mechanisms and targets. Those include disruption of cell division through inhibition of the cytoskeletal protein FtsZ, inhibitors of cell wall biosynthesis coupled with cell division, and discovery of a novel import mechanism via the exporter NorA for some natural products containing a nucleoside core. Ongoing work includes characterization of a novel polyketide tetramic acid (patent pending).