Optimization of the Antifungal Properties of the Lanthipeptide Pinensin and Search for Naturally Occurring Homologs

天然产物是新的抗感染药物和新的药物先导的重要来源。在这些次生代谢物中，核糖体合成和翻译后修饰多肽(RIPPS)代表着一个新兴的超家族，由于它们有趣的生物活性、结构多样性和显著的序列可塑性，使它们能够用于结构-活性-关系研究和作为生物工程支架。由于抗生素耐药性的增加，开发新的抗感染药物是一个日益紧迫的问题，而RIPP往往具有良好的抗感染活性，这就需要对它们进行更仔细的研究，以寻找抗击传染病的新药线索。RIPP前体是遗传编码的，这使得通过在合适的异源生产系统中通过简单突变前体编码基因来实现化合物快速多样化，极大地促进了这种努力。拟议研究项目的主要目的是从天然产物Pinensin作为先导分子开始开发新的抗真菌药物先导化合物。I类羊硫肽Pinensin是文献报道的第一个也是迄今为止唯一报道的抗真菌羊硫肽。它对病原真菌表现出广泛的活性，尽管有细胞毒性副作用，到目前为止还阻碍了它在临床上的使用。由于对新的抗真菌药物的需求很高，以及之前的研究表明，如何成功地优化具有类似初始问题的其他化合物以获得新的抗真菌药物，计划中的项目是及时的，具有很高的意义。其目的是通过结构-活性-关系研究来提高铅分子的治疗指数，为其潜在的治疗用途铺平道路。结构-活性-关系研究将得到基因组挖掘的补充，目标是分离密切相关的自然发生的同系物，以寻找更好的铅结构。初步的基因组挖掘数据已经表明，某些Pinensin同系物可能作为具有抗生素耐药性的临床相关病原体的毒力因子发挥作用，这提供了额外的一层，突显了计划中的项目的重要性。在这项提案的准备过程中，已经成功地建立了一个能够在大肠杆菌中异源生产Pinensin的系统。今后，该项目的工作将从进一步优化所述生产系统以最大化化合物产量开始。这反过来将有助于产生一个广泛的Pinensin变异体文库，旨在优化抗真菌相对于细胞毒活性的选择性。此外，还计划进行作用模式研究，利用耐药突变株的产生和测序、下拉试验和基于荧光显微镜的定位研究。

Natural products are an important source of new anti-infectives and novel drug leads. Amongst these secondary metabolites, ribosomally synthesized and post-translationally modified peptides (RiPPs) represent an emerging superfamily that stirs up increasing interest due to their intriguing bioactivities, structural diversity, and remarkable sequence plasticity that facilitates their use for structure-activity-relationship studies and as bioengineering scaffolds. The development of new anti-infective agents is an increasingly urgent matter due the rise of antibiotic resistances and RiPPs often exhibit promising anti-infective activities, which warrants to take a closer look at them in the search for novel drug leads in the fight against infectious diseases. Such efforts are greatly facilitated by the fact that RiPP precursors are genetically encoded, which enables rapid compound diversification by simple mutation of the precursor-encoding gene in a suitable heterologous production system. The main aim of the proposed research project is the development of new antifungal drug leads starting from the natural product pinensin as lead molecule. The class I lanthipeptide pinensin is the first and so far only antifungal lanthipeptide reported in the literature. It exhibits a broad activity against pathogenic fungi, albeit with cytotoxic side effects that so far have prevented its use in a clinical setting. Due to the high demand for new antifungal drugs and previous studies demonstrating how other compounds with similar initial problems could be optimized successfully to obtain new antifungal agents, the planned project is timely and of high significance. The goal is to improve the therapeutic index of the lead molecule through structure-activity-relationship studies for paving the way for its potential use as a therapeutic. The structure-activity-relationship studies will be complemented by genome mining targeted at isolating closely related, naturally occurring homologs in search for superior lead structures. Preliminary genome mining data already suggests that certain pinensin homologs might play roles as virulence factors of clinically relevant pathogens with antibiotic resistances, which provides an additional layer that underlines the significance of the planned project. In preparation of this proposal, a system that enables the heterologous production of pinensin in E. coli has already been successfully established. Henceforth, the work on this project will begin with the further optimization of said production system for maximizing compounds yields. This in turn will facilitate the generation of an extensive library of pinensin variants aiming at the optimization of the selectivity of the antifungal versus the cytotoxic activities. In addition, mode-of-action studies are planned that will be utilizing the generation and sequencing of resistant mutants, pull-down assays, and fluorescence microscopy based localization studies.