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

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Other (e.g. bacteria)
Escherichia coli

Description

Method keywords
  • high throughput
  • bacterial cells
  • colorimetric
  • 4-hydroxyphenylpyruvate dioxygenase
  • Inhibitor
  • Screening assay
Scientific area keywords
  • biotechnology
  • in vitro
  • Cell culture
  • microbiology
Method description

This method is a straightforward, colorimetric, and inexpensive high-throughput screening system in bacteria which depends on the activity of human HPD. This screening assay is based on the formation and accumulation of a melanin-like ochronotic pigment which has a characteristic brown color. In the presence of an HPD-inhibitor this ochronosis process will be reduced or even prevented when the HPD activity is blocked by a human HPD inhibitor. The screening system will allow to identify new and human-specific HPD inhibitors and evaluate their therapeutic potential for the development of therapies for tyrosine-dependent inborn errors of metabolism.

Lab equipment
  • - Biosafety Cabinet ;
  • - 96-multiwell plates (flat bottom, V-bottom) ; 
  • - LB medium ;
  • - Multichannel pipettes ;
  • - Shaker incubator ;
  • - Erlenmeyer culture flasks.
Method status
  • Published in peer reviewed journal

Pros, cons & Future potential

Advantages
  • - Reliable and robust (Z' = 0.87) ;
  • - Specific and sensitive readout in short measurement time ;
  • - Cost-effective.
Modifications

Method can be miniaturised to 384 and 1536-well format using adjusted equipement including liquid-handling robotics.

References, associated documents and other information

References

Neuckermans, J., Mertens, A., De Win, D. et al. A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors. Sci Rep 9, 14145 (2019).

Associated documents
scientific reports.pdf
Links
A robust bacterial assay for high-throughput screening of human 4-hydroxyphenyl…

Contact person

Jessie Neuckermans

Organisations

Vrije Universiteit Brussel
IVTD
Belgium
Brussels Region

RWTH Aachen
Institute of Biotechnology
Germany

Partners

RWTH Aachen