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Target abundance-based fitness screening (TAFiS) facilitates rapid identification of target-specific and physiologically active chemical probes

Butts, Arielle, DeJarnette, Christian, Peters, Tracy L., Parker, Josie E., Kerns, Morgan E., Eberle, Karen E., Kelly, Steve L., Palmer, Glen E. and Alspaugh, J. Andrew 2017. Target abundance-based fitness screening (TAFiS) facilitates rapid identification of target-specific and physiologically active chemical probes. mSphere 2 (5) , e00379-17. 10.1128/mSphere.00379-17

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: American Society for Microbiology
ISSN: 2379-5042
Date of First Compliant Deposit: 8 July 2022
Date of Acceptance: 14 September 2017
Last Modified: 26 May 2023 04:02

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