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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.
Abstract:
All-trans retinoic acid (atRA) is an endogenous ligand of the retinoic acid receptors, which heterodimerize with retinoid X receptors. AtRA is generated in tissues from vitamin A (retinol) metabolism to form a paracrine signal and is locally degraded by cytochrome P450 family 26 (CYP26) enzymes. The CYP26 family consists of three subtypes: A1, B1, and C1, which are differentially expressed during development. This study aims to develop and validate a high throughput screening assay to identify CYP26A1 inhibitors in a cell-free system using a luminescent P450-Glo assay technology. The assay performed well with a signal to background ratio of 25.7, a coefficient of variation of 8.9%, and a Z-factor of 0.7. To validate the assay, we tested a subset of 39 compounds that included known CYP26 inhibitors and retinoids, as well as positive and negative control compounds selected from the literature and/or the ToxCast/Tox21 portfolio. Known CYP26A1 inhibitors were confirmed, and predicted CYP26A1 inhibitors, such as chlorothalonil, prochloraz, and SSR126768, were identified, demonstrating the reliability and robustness of the assay. Given the general importance of atRA as a morphogenetic signal and the localized expression of Cyp26a1 in embryonic tissues, a validated CYP26A1 assay has important implications for evaluating the potential developmental toxicity of chemicals.
Insights
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.
