Discovery of Fungus-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination
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Evaluation Summary:
This study is an extension of previous work by the same authors, which established a two step high-throughput screening approach to monitor germination and growth of fungal spores of the human pathogen Cryptococcus neoformans and identified an FDA-approved drug with antifungal activity (DOI: 10.1128/AAC.00994-19). The current work extends this approach to three libraries of drug-like molecules comprising 75,000 candidate compounds and employs automated image analysis methods to identify classes of inhibition phenotypes. The key result of this work is the identification of 191 inhibitors, of which 76 could be grouped in to 8 classes based on chemical structure - inhibitors that share structural similarities tend to share phenotypic impact.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)
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Abstract
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.
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Evaluation Summary:
This study is an extension of previous work by the same authors, which established a two step high-throughput screening approach to monitor germination and growth of fungal spores of the human pathogen Cryptococcus neoformans and identified an FDA-approved drug with antifungal activity (DOI: 10.1128/AAC.00994-19). The current work extends this approach to three libraries of drug-like molecules comprising 75,000 candidate compounds and employs automated image analysis methods to identify classes of inhibition phenotypes. The key result of this work is the identification of 191 inhibitors, of which 76 could be grouped in to 8 classes based on chemical structure - inhibitors that share structural similarities tend to share phenotypic impact.
(This preprint has been reviewed by eLife. We include the public reviews from …
Evaluation Summary:
This study is an extension of previous work by the same authors, which established a two step high-throughput screening approach to monitor germination and growth of fungal spores of the human pathogen Cryptococcus neoformans and identified an FDA-approved drug with antifungal activity (DOI: 10.1128/AAC.00994-19). The current work extends this approach to three libraries of drug-like molecules comprising 75,000 candidate compounds and employs automated image analysis methods to identify classes of inhibition phenotypes. The key result of this work is the identification of 191 inhibitors, of which 76 could be grouped in to 8 classes based on chemical structure - inhibitors that share structural similarities tend to share phenotypic impact.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This manuscript is centered around first a high-throughput screen with a nano-luciferase readout for spore germination. Hits from the initial screen are then validated using a quantitative germination assay (QGA) based on cell shape (spores are elongated, yeast cells are spherical). This combination of primary and secondary screens is a very powerful and rigorous approach for identifying small molecule inhibitors of germination. Subsequent experiments focus on the precise phenotypic nature of germination inhibition by different categories of germination behavior and elucidation of the molecular target/molecular mechanism of each validated QGA hit.
The data in Figure 2, demonstrating that 76 of the 191 germination inhibitors can be grouped into conserved structural categories. This suggests that there are a …
Reviewer #1 (Public Review):
This manuscript is centered around first a high-throughput screen with a nano-luciferase readout for spore germination. Hits from the initial screen are then validated using a quantitative germination assay (QGA) based on cell shape (spores are elongated, yeast cells are spherical). This combination of primary and secondary screens is a very powerful and rigorous approach for identifying small molecule inhibitors of germination. Subsequent experiments focus on the precise phenotypic nature of germination inhibition by different categories of germination behavior and elucidation of the molecular target/molecular mechanism of each validated QGA hit.
The data in Figure 2, demonstrating that 76 of the 191 germination inhibitors can be grouped into conserved structural categories. This suggests that there are a limited number of ways to inhibit germination and supports, at least to this reviewer, the hypothesis that spore germination is a promising target for antifungal therapies and/or prophylaxis.
The Seahorse data nicely show that several group B small molecules inhibit oxygen consumption and thus likely the electron transport chain, either directly or indirectly. The B2 profile closely resembles the known complex II inhibitor furcarbanil, but the B5 profile does not. It is not clear to me why the authors attribute B5-induced germination inhibition to targeting complex II, as despite the structural similarity to known complex II inhibitors.
Overall, this is an exciting and rigorous paper. While inhibiting spore germination is unlikely to treat systemic fungal infections, as many patients present in clinical with an advanced infection, the authors address this in their discussion. The idea of using spore germination inhibitors as a prophylactic treatment is reasonable, and the authors include a measured discussion of overall impact. The QGA is an impressive technique with potential application to other morphological transitions in fungi. The use of robust and fundamentally different primary and secondary screens make this work an exemplary chemical/phenotypic genomics paper. Too many secondary assays end up looking for hits with the same mechanism as the primary assay. Here, the use of a primary reporter assay and secondary germination assays are very well done.
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Reviewer #2 (Public Review):
Ortiz et al. previously established a two step high-throughput screening approach to monitor germination and growth of Cryptococcus neoformans spores and identified an FDA-approved drug with antifungal activity (DOI: 10.1128/AAC.00994-19).
In the current work, they use the same techniques, but apply this pipeline to three libraries of drug-like molecules comprising 75,000 candidate compounds. Using automated image analysis methods, they identify classes of inhibition phenotype. They identify 191 inhibitors, of which 76 could be grouped in to 8 classes based on chemical structure, and provide these structures as part of the supplemental data, creating a rich dataset for future investigations. Within these classes, they show that shared structure is associated with shared phenotypic impact. Finally, the …
Reviewer #2 (Public Review):
Ortiz et al. previously established a two step high-throughput screening approach to monitor germination and growth of Cryptococcus neoformans spores and identified an FDA-approved drug with antifungal activity (DOI: 10.1128/AAC.00994-19).
In the current work, they use the same techniques, but apply this pipeline to three libraries of drug-like molecules comprising 75,000 candidate compounds. Using automated image analysis methods, they identify classes of inhibition phenotype. They identify 191 inhibitors, of which 76 could be grouped in to 8 classes based on chemical structure, and provide these structures as part of the supplemental data, creating a rich dataset for future investigations. Within these classes, they show that shared structure is associated with shared phenotypic impact. Finally, the authors use structural similarity with a known inhibitor of mitochondrial Complex II succinate dehydrogenase to raise and test the hypothesis that compounds that break germination synchrony target respiration.
The authors previously developed a medium-throughput germination assay based on microscopic live cell imaging and automated analysis to measure changes in aspect ratio and area as small oval spores germinate to larger round yeast. They validate the capacity of their germination assay at a range of physiologically relevant drug concentrations using a protein synthesis inhibitor, cyclohexamide. A strength of the work, they calculate the IC50 and observe that increasing concentrations cause a dose-dependent delay in germination. They also observe that treatment of spores with other protein synthesis inhibitors induce a similar "slow down" germination phenotype. This raises the hypothesis that shared phenotype may indicate a similar mode of action.
The authors then build on this hypothesis to identify novel compounds with anti-germination activity and reveal biological processes central to germination, an understudied process. By using a high-throughput nanoluciferase reporter assay for germination as a primary screen and the medium through-put germination assay as a secondary screen, the authors identify classes of chemical structures that have similar phenotypic consequences. They further examine two of the 8 classes, showing that all compounds in one class, and the majority of compounds in the second class, have similar phenotypic impacts. By searching for compounds with known modes of action, they identify a succinate dehydrogenase inhibitor that is structurally similar to one of their classes of compounds. Treatment of spores with this compound or with compounds in the related class disrupt oxygen consumption as measured by seahorse assay, suggesting shared mode of action and revealing mitochondrial activity as a key biological process underpinning germination synchrony.
A weakness of the work is the limited analysis of outlier structures and phenotypes and the limited analysis how the different compounds affect fungal growth beyond germination or how these chemotherapeutics may reduce pathogenesis.
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