Discovery, Structural Studies and Biosynthesis of Biologically Active Natural Products and Natural Product-Like Inhibitors

天然产物，也称为专门的代谢产物，是由微生物、真菌和无脊椎动物生物合成的小分子和肽。它们具有巨大的结构多样性，并表现出广泛的生物活性，因此对人类和兽医学以及农业需求至关重要。天然产物及其合成衍生物占目前临床上使用的大多数处方疗法，特别是在抗生素和抗肿瘤药物类别中。随着耐药性细菌感染的发生率不断增加，迫切需要进行旨在发现和开发新类别抗生素和新的抗菌靶标的基础和临床研究。重点是海洋无脊椎动物和未充分研究的环境细菌作为天然产物源生物，我们开展跨学科研究，包括抗生素发现，确定作用机制和完整的结构测定。我们还使用基因组挖掘技术来识别，表征和生产新类别的核糖体编码的天然产物，称为RIPPs（核糖体后修饰肽）。一些有代表性的项目和调查结果包括以下内容。在最近的工作中，我们已经使用了X射线晶体学，圆二色谱技术和计算ECD实验，以确定的绝对配置的双茚满天然产物haliclonadiamine和papuamine和展示意想不到的手性性质，其中大环二烯占的旋光和棉花效应的小分子，而不是手性中心的迹象。这项工作被扩展为人工智能方法的模型，通过振动圆二色性来验证绝对构型。 通过生态偏向的微生物收集和基因组挖掘，我们发现，表征并产生了一类新的核糖体编码的多聚肽天然产物类命名的chryseoviridins。黄绿青霉素前体肽CdnA 3包含前导序列和核心序列，并且不寻常之处在于后前导序列包含多个核心基序。大环化由ATP依赖性连接酶CdnC和CdnB催化。使用X-射线晶体学和质谱（MS），我们确定了CdnA 3的肽大环化的结构基础，并证明了连接酶在底物结合和释放后经历了主要的构象变化，以适应单个多肽前体上连续几轮的环化，并证明了多核环化发生在N-末端到C-末端的方向。这项工作对RiPP天然产物草肽家族的工程具有重要意义，并有可能获得不同类型的蛋白酶抑制剂。 我们的工作还旨在确定新的抗生素和抗病毒药。最近，我们已经确定了抑制野生型和耐药病原体生长的次级代谢产物的新结构类别，包括耐甲氧西林金黄色葡萄球菌和耐万古霉素屎肠球菌。体外酶测定、体内抗菌测定和耐药变体的基因组测序揭示了不同的机制和靶点。这些包括通过抑制细胞骨架蛋白FtsZ破坏细胞分裂，细胞壁生物合成与细胞分裂偶联的抑制剂，以及发现通过输出者诺拉的新的输入机制，用于一些含有核苷核心的天然产物。正在进行的工作包括表征一种新的聚酮特特拉姆酸（专利申请中）。

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 and are therefore critical to human and veterinary medicine and agricultural needs. 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. This work was expanded as a model for an artificial-intelligence approach to validate absolute configurations by vibrational circular dichroism. 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).