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A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors. / Neuckermans, Jessie; Mertens, Alan; Schwaneberg, Ulrich; De Kock, Joery.

In: Scientific Reports, Vol. 9, No. 1, 02.10.2019, p. 14145.

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Neuckermans, Jessie ; Mertens, Alan ; Schwaneberg, Ulrich ; De Kock, Joery. / A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors. In: Scientific Reports. 2019 ; Vol. 9, No. 1. pp. 14145.

BibTeX

@article{245938c63a83431182082a32e98fd0ab,
title = "A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors",
abstract = "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.",
author = "Jessie Neuckermans and Alan Mertens and Ulrich Schwaneberg and {De Kock}, Joery",
year = "2019",
month = "10",
day = "2",
doi = "10.1038/s41598-019-50533-1",
language = "English",
volume = "9",
pages = "14145",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",

}

RIS

TY - JOUR

T1 - A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

AU - Neuckermans, Jessie

AU - Mertens, Alan

AU - Schwaneberg, Ulrich

AU - De Kock, Joery

PY - 2019/10/2

Y1 - 2019/10/2

N2 - 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.

AB - 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.

U2 - 10.1038/s41598-019-50533-1

DO - 10.1038/s41598-019-50533-1

M3 - Article

VL - 9

SP - 14145

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

ER -

ID: 47605521