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Kai Hilpert, Christoph Rumancev, Jurnorain Gani, Dominic W.P. Collis, Paula Matilde Lopez-Perez, Vasyl M. Garamus, Ralf Mikut, Axel Rosenhahn

• The opportunistic yeast \(\textit {Candida albicans}\) is the most common cause of candidiasis. With only four classes of antifungal drugs on the market, resistance is becoming a problem in the treatment of fungal infections, especially in immunocompromised patients. The development of novel antifungal drugs with different modes of action is urgent. In 2016, we developed a groundbreaking new medium-throughput method to distinguish the effects of antibacterial agents. Using small-angle X-ray scattering for biological samples (BioSAXS), it is now possible to screen hundreds of new antibacterial compounds and select those with the highest probability for a novel mode of action. However, yeast (eukaryotic) cells are highly structured compared to bacteria. The fundamental question to answer was if the ultrastructural changes induced by the action of an antifungal drug can be detected even when most structures in the cell stay unchanged. In this exploratory work, BioSAXS was used to measure the ultrastructural changes of \(\textit {C. albicans}\) that were directly or indirectly induced by antifungal compounds. For this, the well-characterized antifungal drug Flucytosine was used. BioSAXS measurements were performed on the synchrotron P12 BioSAXS beamline, EMBL (DESY, Hamburg) on treated and untreated yeast \(\textit {C. albicans}\). BioSAXS curves were analysed using principal component analysis (PCA). The PCA showed that Flucytosine-treated and untreated yeast were separated. Based on that success further measurements were performed on five antifungal peptides {1. Cecropin A-melittin hybrid [CA (1–7) M (2–9)], KWKLFKKIGAVLKVL; 2. Lasioglossin LL-III, VNWKKILGKIIKVVK; 3. Mastoparan M, INLKAIAALAKKLL; 4. Bmkn2, FIGAIARLLSKIFGKR; and 5. optP7, KRRVRWIIW}. The ultrastructural changes of \(\textit {C. albicans}\) indicate that the peptides may have different modes of action compared to Flucytosine as well as to each other, except for the Cecropin A-melittin hybrid [CA (1–7) M (2–9)] and optP7, showing very similar effects on \(\textit {C. albicans}\). This very first study demonstrates that BioSAXS shows promise to be used for antifungal drug development. However, this first study has limitations and further experiments are necessary to establish this application.