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

天然产物，也被称为特殊代谢物，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，因此对人类和兽医以及农业需求至关重要。目前临床上使用的处方疗法中，天然产物及其合成衍生物占大多数，特别是在抗生素和抗肿瘤药物类别中。随着耐药细菌感染的发病率不断增加，迫切需要进行基础和临床研究，以发现和开发新类别的抗生素和新的抗菌靶点。以海洋无脊椎动物和未被充分研究的环境细菌为天然产物来源生物，我们开展跨学科研究，包括抗生素的发现、作用机制的确定和完整的结构确定。我们还使用基因组挖掘技术来识别、表征和生产称为Ripps(核糖体翻译后修饰多肽)的新型核糖体编码天然产品。一些具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，我们使用了X射线结晶学、圆二色谱技术和计算ECD实验来确定双吲哚天然产物卤代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中解释了旋光和Cotton效应的符号，而不是手性中心。这项工作被扩展为一种人工智能方法的模型，通过振动圆二色谱来验证绝对构型。 通过生态偏向的微生物采集和基因组挖掘，我们发现、鉴定和产生了一类新的核糖体编码的多环肽天然产物类，命名为金绿素。金绿素前体多肽CDnA3由一个前导和核心序列组成，其不同寻常之处在于前导后序列包含多个核心基序。大环化是由依赖于ATP的连接酶CDnC和CDnB催化的。用X射线结晶学和质谱仪(MS)确定了多肽环化的结构基础，证明了连接酶在底物结合和释放时发生了主要的构象变化，以适应单一多肽前体上的连续几轮环化，并证明了多核环化是沿N端到C端的方向进行的。这项工作对RIPP天然产物GRAPID家族的工程具有重要意义，并有可能获得不同类别的蛋白水解酶抑制剂。 我们的工作还旨在确定新的抗生素和抗病毒药物。最近，我们发现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，包括耐甲氧西林的金黄色葡萄球菌和耐万古霉素的屎肠球菌。体外酶分析、体内抗菌试验和耐药变异体的基因组测序揭示了不同的机制和靶点。这些措施包括通过抑制细胞骨架蛋白FtsZ来破坏细胞分裂，抑制细胞壁生物合成与细胞分裂相结合，以及发现一些含有核苷核心的天然产物通过出口商Nora的新的进口机制。正在进行的工作包括表征一种新型聚酮四酸(正在申请专利)。

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).