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.

• MeSH
• Antifungal Agents chemistry   metabolism   pharmacology   MeSH
• Aspartic Acid Proteases antagonists & inhibitors   genetics   metabolism   MeSH
• Cryptococcus neoformans enzymology   MeSH
• Fungal Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• HIV Protease chemistry   metabolism   MeSH
• HIV enzymology   MeSH
• Fungi drug effects   MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Drug Evaluation, Preclinical MeSH
• Recombinant Proteins biosynthesis   chemistry   isolation & purification   MeSH
• Molecular Dynamics Simulation MeSH
• Substrate Specificity MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Antifungal Agents MeSH
• Aspartic Acid Proteases MeSH
• Fungal Proteins MeSH
• HIV Protease MeSH
• Recombinant Proteins MeSH

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors.

• MeSH
• Aspartic Acid Proteases metabolism   MeSH
• Cryptococcus neoformans enzymology   pathogenicity   MeSH
• Virulence Factors metabolism   MeSH
• Fungal Proteins metabolism   MeSH
• Mass Spectrometry MeSH
• Immunoblotting MeSH
• Hydrogen-Ion Concentration MeSH
• Cryptococcosis enzymology   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Disease Models, Animal MeSH
• Mice MeSH
• Peptide Hydrolases metabolism   MeSH
• Proteomics MeSH
• Gene Expression Profiling MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aspartic Acid Proteases MeSH
• Virulence Factors MeSH
• Fungal Proteins MeSH
• Peptide Hydrolases MeSH