Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Chan Zuckerberg Biohub 499 Illinois Street San Francisco California 94158 United States

Department of Biochemistry and Biophysics University of California San Francisco UCSF Genentech Hall 600 16th St Rm N374 San Francisco California 94158 United States

Department of Biochemistry Faculty of Science Charles UniversityHlavova 8 Prague 2 12843 Czech Republic

Department of Chemical Biology and Bioimaging Wroclaw University of Science and Technology Wybrzeze Wyspianskiego 27 Wroclaw 50 370 Poland

Department of Pharmaceutical Chemistry University of California San Francisco UCSF Genentech Hall 600 16th St Rm S512 San Francisco California 94158 United States

Department of Physical and Macromolecular Chemistry Faculty of Science Charles UniversityHlavova 8 Prague 2 12843 Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 Prague 4 14220 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo n 2 Prague 6 16610 Czech Republic

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Advances in Antifungal Drug Development: An Up-To-Date Mini Review