Affiliation:
1. Division of Pre‐clinical Innovation National Center for Advancing Translational Sciences National Institutes of Health Rockville Maryland USA
2. Promega Corporation Madison Wisconsin USA
3. Center for Computational Toxicology and Exposure Office of Research and Development, United States Environmental Protection Agency Research Triangle Park North Carolina USA
4. Leidos Research Triangle Park North Carolina USA
5. National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods National Institute of Environmental Health Sciences, National Institutes of Health Research Triangle Park North Carolina USA
Abstract
AbstractAll‐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.