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Development and validation of CYP26A1 inhibition assay for high-throughput screening
Srilatha Sakamuru1, Dongping Ma2, Jocylin D Pierro3
1Division of Pre-clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA.
Biotechnology Journal
|June 12, 2024
Summary
This study developed a reliable cell-free assay to identify inhibitors of CYP26A1, an enzyme crucial for all-trans retinoic acid (atRA) metabolism. The assay successfully identified known and potential CYP26A1 inhibitors, aiding in developmental toxicity assessments.
Area of Science:
- Biochemistry
- Toxicology
- Drug Discovery
Background:
- All-trans retinoic acid (atRA) is vital for embryonic development, regulated by its local degradation via cytochrome P450 family 26 (CYP26) enzymes.
- CYP26A1, a key subtype, plays a critical role in controlling atRA levels during development.
- Understanding CYP26A1 activity is crucial for assessing chemical developmental toxicity.
Purpose of the Study:
- To develop and validate a high-throughput screening assay for identifying CYP26A1 inhibitors.
- To establish a robust cell-free system for evaluating potential CYP26A1 modulators.
Main Methods:
- Utilized a luminescent P450-Glo assay technology for a cell-free screening system.
- Validated the assay's performance with metrics including signal-to-background ratio, coefficient of variation, and Z-factor.
- Tested a diverse set of 39 compounds, including known inhibitors and retinoids, for assay validation.
Main Results:
- The developed assay demonstrated excellent performance with a signal-to-background ratio of 25.7, CV of 8.9%, and Z-factor of 0.7.
- Successfully confirmed known CYP26A1 inhibitors and identified predicted inhibitors like chlorothalonil, prochloraz, and SSR126768.
- The assay proved reliable and robust in identifying CYP26A1 modulators.
Conclusions:
- A validated, high-throughput cell-free assay for CYP26A1 inhibition screening has been established.
- This assay provides a valuable tool for evaluating the developmental toxicity potential of various chemicals.
- The findings support the importance of CYP26A1 activity in developmental processes and chemical safety assessment.
