High-throughput quantification of ochronotic pigment formation in Escherichia coli to evaluate the potency of human 4-hydroxyphenylpyruvate dioxygenase inhibitors in multi-well format

Jessie Neuckermans, Sien Lequeue, Alan Mertens, Steven Branson, Ulrich Schwaneberg, Joery De Kock

Research output: Contribution to journalArticle

Abstract

4-hydroxyphenylpyruvate dioxygenase (HPD) is a key enzyme in the catabolism of tyrosine and therefore of great importance as a drug target to treat tyrosine-related inherited metabolic disorders (TIMD). Inhibition of this enzyme is therapeutically applied to prevent accumulation of toxic metabolites in TIMD patients. Nowadays an ex-herbicide, nitisinone, is used for this purpose and many more inhibitors are being explored and need to be tested. Here, we describe a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of new human HPD inhibitors in a high-throughput and robust fashion. For this high-throughput screening (HTS) system we rely on the capability of recombinant E. coli that express human HPD, to generate a brown ochronotic pigment after the addition of tyrosine, whereafter this brown pigment can be quantified in a very specific and sensitive way by spectrophotometric analysis. Altogether, this robust and simple HTS screening system can be described as non-harmful, non-laborious and cost-effective with the aim to identify and evaluate novel therapeutic human HPD inhibitors for the treatment of TIMD.•This robust high-throughput screening system enables rapid identification and evaluation of potential inhibitors of human 4-hydroxyphenylpyruvate dioxygenase.•Simple and fast colorimetric quantification of the formation of ochronotic pigment.

Original languageEnglish
Article number101181
Number of pages11
JournalMethodsX
Volume8
DOIs
Publication statusPublished - Jan 2021

Bibliographical note

© 2020 The Authors. Published by Elsevier B.V.

Keywords

  • 4-Hydroxyphenylpyruvate dioxygenase
  • Bacterial cell culture
  • Colorimetric
  • High-throughput screening
  • In Vitro
  • Inhibitor testing
  • Ochronotic pigment
  • Tyrosinemia

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