Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

ABSTRACT Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screenin...

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Autores principales:	Arielle Butts, Christian DeJarnette, Tracy L. Peters, Josie E. Parker, Morgan E. Kerns, Karen E. Eberle, Steve L. Kelly, Glen E. Palmer
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Candida albicans antifungal agents chemical genetics drug screening Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0ef5acc8ba6b48e0acfad3d99008315a
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spelling	oai:doaj.org-article:0ef5acc8ba6b48e0acfad3d99008315a2021-11-15T15:22:05ZTarget Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes10.1128/mSphere.00379-172379-5042https://doaj.org/article/0ef5acc8ba6b48e0acfad3d99008315a2017-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00379-17https://doaj.org/toc/2379-5042ABSTRACT Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound.Arielle ButtsChristian DeJarnetteTracy L. PetersJosie E. ParkerMorgan E. KernsKaren E. EberleSteve L. KellyGlen E. PalmerAmerican Society for MicrobiologyarticleCandida albicansantifungal agentschemical geneticsdrug screeningMicrobiologyQR1-502ENmSphere, Vol 2, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Candida albicans antifungal agents chemical genetics drug screening Microbiology QR1-502
spellingShingle	Candida albicans antifungal agents chemical genetics drug screening Microbiology QR1-502 Arielle Butts Christian DeJarnette Tracy L. Peters Josie E. Parker Morgan E. Kerns Karen E. Eberle Steve L. Kelly Glen E. Palmer Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes
description	ABSTRACT Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound.
format	article
author	Arielle Butts Christian DeJarnette Tracy L. Peters Josie E. Parker Morgan E. Kerns Karen E. Eberle Steve L. Kelly Glen E. Palmer
author_facet	Arielle Butts Christian DeJarnette Tracy L. Peters Josie E. Parker Morgan E. Kerns Karen E. Eberle Steve L. Kelly Glen E. Palmer
author_sort	Arielle Butts
title	Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes
title_short	Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes
title_full	Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes
title_fullStr	Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes
title_full_unstemmed	Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes
title_sort	target abundance-based fitness screening (tafis) facilitates rapid identification of target-specific and physiologically active chemical probes
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/0ef5acc8ba6b48e0acfad3d99008315a
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Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

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