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

• Applicant: Dr. Julian Hegemann
• Subject Area: Biochemistry ; Biological and Biomimetic Chemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525824070

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.

• DFG Programme: Research Grants