A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

Jessie Neuckermans, Alan Mertens, Ulrich Schwaneberg, Joery De Kock

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    13 Citaten (Scopus)
    45 Downloads (Pure)

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    Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the β-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human HPD inhibitors in a high-throughput and a robust fashion. The principle of our screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. With the aim to optimise the assay, we tested different E. coli strains, expression and reaction temperatures, and time-points for supplementing the substrate. We found that in our assay the addition of prototypical β-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. In addition, plate uniformity, signal variability and spatial uniformity assessment showed that we have developed a robust high-throughput screening assay that is simple to use, cost-effective and enables identification and evaluation of novel therapeutic human HPD inhibitors for the treatment of tyrosine-related metabolic disorders.
    Originele taal-2English
    Artikelnummer14145
    Pagina's (van-tot)14145
    Aantal pagina's11
    TijdschriftScientific Reports
    Volume9
    Nummer van het tijdschrift1
    DOI's
    StatusPublished - 2 okt 2019

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