Development and validation of enzyme-based CYP26A1 inhibition assay in a high-throughput screening platform

Authors

• S Sakamuru
• D Ma
• JD Pierro
• NC Baker
• N Kleinstreuer
• JJ Cali
• TB Knudsen
• M Xia

Abstract

All-trans retinoic acid (ATRA), an endogenous ligand for retinoic acid receptor (RAR) that heterodimerizes with retinoid X receptor, is essential for embryonic development and tissue homeostasis. ATRA is locally generated from vitamin A metabolism and degraded by cytochrome p450 family 26 (CYP26) enzymes. Differential expression of the enzymes involved in retinol metabolism locally determine the kinematics of ATRA signaling gradients that are further resolved into nuclear delivery by several molecular transporters. Tox21 assays have enabled high-throughput screening for RAR-agonist and various CYP biochemical activities, although the CYP26 family is not currently represented in this portfolio. The CYP26 family consists of three subtypes (A1, B1, C1) that are differentially expressed during development. In this study, we developed a HTS luminescent P450-Glo assay using both the human recombinant enzyme product and a specific substrate to quantify chemical effects on CYP26A1 biochemical activity and compare these effects with other CYP or retinoid-related bioactivities in the Tox21 portfolio. The assay performed well with regards to signal to background ratio (25.7), Coefficient of variation (8.9%) and Z factor (0.7). To validate this assay, we tested a subset of 39 compounds including well-known CYP26 inhibitors, retinoids and selective RAR agonists, as well as possible negative control compounds including caffeine inferred from the literature. This initial screen identified 33 compounds including AM580, ketoconazole, R115866, raloxifene, SR11237, and tamibarotene, that inhibit CYP26A1 activity, while the known negative controls showed no activity. This assay can be used to identify CYP26A1 inhibitors, which may represent potential developmental toxicants.

Scientific Focus Area: Developmental Biology

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