Cellular and Biophysical Pipeline for the Screening of Peroxisome Proliferator-Activated Receptor Beta/Delta Agonists: Avoiding False Positives

PPAR Research
Natália Bernardi VideiraAna Carolina Migliorini Figueira

Abstract

Peroxisome proliferator-activated receptor beta/delta (PPARß/δ) is considered a therapeutic target for metabolic disorders, cancer, and cardiovascular diseases. Here, we developed one pipeline for the screening of PPARß/δ agonists, which reduces the cost, time, and false-positive hits. The first step is an optimized 3-day long cellular transactivation assay based on reporter-gene technology, which is supported by automated liquid-handlers. This primary screening is followed by a confirmatory transactivation assay and by two biophysical validation methods (thermal shift assay (TSA) and (ANS) fluorescence quenching), which allow the calculation of the affinity constant, giving more information about the selected hits. All of the assays were validated using well-known commercial agonists providing trustworthy data. Furthermore, to validate and test this pipeline, we screened a natural extract library (560 extracts), and we found one plant extract that might be interesting for PPARß/δ modulation. In conclusion, our results suggested that we developed a cheaper and more robust pipeline that goes beyond the single activation screening, as it also evaluates PPARß/δ tertiary structure stabilization and the ligand affinity constant, sel...Continue Reading

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Citations

Jan 8, 2020·Clinical and Experimental Pharmacology & Physiology·Odair Alves SilvaGlória Pinto Duarte
Mar 19, 2020·Natural Product Reports·Brice A P WilsonBarry R O'Keefe
Apr 24, 2019·Cancer Chemotherapy and Pharmacology·Deysi Viviana Tenazoa WongRoberto César Pereira Lima-Júnior
Jan 1, 2018·Nuclear Receptor Research·Qinglin Yang, Qinqiang Long
Jun 22, 2020·The Journal of Nutritional Biochemistry·Anaysa Paola BolinRosemari Otton

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Methods Mentioned

BETA
thermal shift
transfection
Assay
PCR
Fluorescence
transfect
FRET
transfections

Software Mentioned

OriginPro8
OriginPro
GraphPad Prism

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