High-throughput quantification of ochronotic pigment formation in Escherichia coli to evaluate the potency of human 4-hydroxyphenylpyruvate dioxygenase inhibitors
Scope of the method
- Human health
- Basic Research
- In vitro - Ex vivo
- Other (e.g. bacteria)
- high throughput
- bacterial cells
- 4-hydroxyphenylpyruvate dioxygenase
- Screening assay
- in vitro
- Cell culture
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.
- - Biosafety Cabinet ;
- - 96-multiwell plates (flat bottom, V-bottom) ;
- - LB medium ;
- - Multichannel pipettes ;
- - Shaker incubator ;
- - Erlenmeyer culture flasks.
- Published in peer reviewed journal
Pros, cons & Future potential
- - Reliable and robust (Z' = 0.87) ;
- - Specific and sensitive readout in short measurement time ;
- - Cost-effective.
Method can be miniaturised to 384 and 1536-well format using adjusted equipement including liquid-handling robotics.
References, associated documents and other information
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).
Contact personJessie Neuckermans
OrganisationsVrije Universiteit Brussel
Institute of Biotechnology