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.
Highlights
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For this high-throughput screening (HTS) system we rely on the capability of recombinant E. coli that express human hydroxyphenylpyruvate dioxygenase (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
That is used to treat tyrosine-related inherited metabolic disorders (TIMD), is nitisinone [2-(2-nitro-4-trifluoromethylbenzoyl—1,3-cyclohexadione; NTBC], a member of the β-triketone family of herbicides but many other inhibitors are being explored that may pave the way to more TIMD treatment options
Summary
General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Highthroughput quantification of ochronotic pigment formation in Escherichia coli to evaluate the potency of human 4hydroxyphenylpyruvate dioxygenase inhibitors in multi-well format.
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