Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination

ABSTRACT There is a critical need for new antifungal drugs; however, the lack of available fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungus-specific target...

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Autores principales:	Sébastien C. Ortiz, Mingwei Huang, Christina M. Hull
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	Cryptococcus antifungal agents fungi germination spores Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/28a3da15dde244bf9a4c1ef81f9345d7
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spelling	oai:doaj.org-article:28a3da15dde244bf9a4c1ef81f9345d72021-11-10T18:37:52ZDiscovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination10.1128/mBio.01672-212150-7511https://doaj.org/article/28a3da15dde244bf9a4c1ef81f9345d72021-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01672-21https://doaj.org/toc/2150-7511ABSTRACT There is a critical need for new antifungal drugs; however, the lack of available fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungus-specific targets. To capitalize on this possibility, we developed novel phenotypic assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we carried out a high-throughput screen of ∼75,000 drug-like small molecules and identified and characterized 191 novel inhibitors of spore germination, many of which also inhibited yeast replication and demonstrated low cytotoxicity against mammalian cells. Using an automated, microscopy-based, quantitative germination assay (QGA), we discovered that germinating spore populations can exhibit unique phenotypes in response to chemical inhibitors. Through the characterization of these spore population dynamics in the presence of the newly identified inhibitors, we classified 6 distinct phenotypes based on differences in germination synchronicity, germination rates, and overall population behavior. Similar chemical phenotypes were induced by inhibitors that targeted the same cellular function or had shared substructures. Leveraging these features, we used QGAs to identify outliers among compounds that fell into similar structural groups and thus refined relevant structural moieties, facilitating target identification. This approach led to the identification of complex II of the electron transport chain as the putative target of a promising structural cluster of germination inhibitory compounds. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics. IMPORTANCE Fungal pathogens cause 1.5 million deaths annually, and there is a critical need for new antifungal drugs. However, humans and fungi are very similar on a molecular level, and so many drugs that kill fungi also damage human cells, leading to extreme side effects, including death. The lack of fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a process absent in humans that could harbor fungus-specific targets. To capitalize on this possibility, we developed new assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we identified and characterized 191 novel inhibitors of spore germination. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics.Sébastien C. OrtizMingwei HuangChristina M. HullAmerican Society for MicrobiologyarticleCryptococcusantifungal agentsfungigerminationsporesMicrobiologyQR1-502ENmBio, Vol 12, Iss 4 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cryptococcus antifungal agents fungi germination spores Microbiology QR1-502
spellingShingle	Cryptococcus antifungal agents fungi germination spores Microbiology QR1-502 Sébastien C. Ortiz Mingwei Huang Christina M. Hull Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination
description	ABSTRACT There is a critical need for new antifungal drugs; however, the lack of available fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungus-specific targets. To capitalize on this possibility, we developed novel phenotypic assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we carried out a high-throughput screen of ∼75,000 drug-like small molecules and identified and characterized 191 novel inhibitors of spore germination, many of which also inhibited yeast replication and demonstrated low cytotoxicity against mammalian cells. Using an automated, microscopy-based, quantitative germination assay (QGA), we discovered that germinating spore populations can exhibit unique phenotypes in response to chemical inhibitors. Through the characterization of these spore population dynamics in the presence of the newly identified inhibitors, we classified 6 distinct phenotypes based on differences in germination synchronicity, germination rates, and overall population behavior. Similar chemical phenotypes were induced by inhibitors that targeted the same cellular function or had shared substructures. Leveraging these features, we used QGAs to identify outliers among compounds that fell into similar structural groups and thus refined relevant structural moieties, facilitating target identification. This approach led to the identification of complex II of the electron transport chain as the putative target of a promising structural cluster of germination inhibitory compounds. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics. IMPORTANCE Fungal pathogens cause 1.5 million deaths annually, and there is a critical need for new antifungal drugs. However, humans and fungi are very similar on a molecular level, and so many drugs that kill fungi also damage human cells, leading to extreme side effects, including death. The lack of fungus-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a process absent in humans that could harbor fungus-specific targets. To capitalize on this possibility, we developed new assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we identified and characterized 191 novel inhibitors of spore germination. These inhibitors showed high potency against Cryptococcus spore germination while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics.
format	article
author	Sébastien C. Ortiz Mingwei Huang Christina M. Hull
author_facet	Sébastien C. Ortiz Mingwei Huang Christina M. Hull
author_sort	Sébastien C. Ortiz
title	Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination
title_short	Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination
title_full	Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination
title_fullStr	Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination
title_full_unstemmed	Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination
title_sort	discovery of fungus-specific targets and inhibitors using chemical phenotyping of pathogenic spore germination
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/28a3da15dde244bf9a4c1ef81f9345d7
work_keys_str_mv	AT sebastiencortiz discoveryoffungusspecifictargetsandinhibitorsusingchemicalphenotypingofpathogenicsporegermination AT mingweihuang discoveryoffungusspecifictargetsandinhibitorsusingchemicalphenotypingofpathogenicsporegermination AT christinamhull discoveryoffungusspecifictargetsandinhibitorsusingchemicalphenotypingofpathogenicsporegermination
_version_	1718439760441114624

Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination

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