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

Arielle Butts, Christian DeJarnette, Tracy L. Peters, Josie E. Parker, Morgan E. Kerns, Karen E. Eberle, Steve L. Kelly, Glen Palmer

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere00379-17
JournalmSphere
Volume2
Issue number5
DOIs
StatePublished - Sep 1 2017

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Proteins
Lanosterol
Tetrahydrofolate Dehydrogenase
Drug Discovery
Candida albicans
Growth
Pharmaceutical Preparations
Population
Therapeutics

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

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.

In: mSphere, Vol. 2, No. 5, e00379-17, 01.09.2017.

Research output: Contribution to journalArticle

Butts, Arielle ; DeJarnette, Christian ; Peters, Tracy L. ; Parker, Josie E. ; Kerns, Morgan E. ; Eberle, Karen E. ; Kelly, Steve L. ; Palmer, Glen. / Target abundance-based fitness screening (TAFiS) facilitates rapid identification of target-specific and physiologically active chemical probes. In: mSphere. 2017 ; Vol. 2, No. 5.
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