Identification of compounds that modulate retinol signaling using a cell-based qHTS assay

Abstract

In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.

Molecular Image-Based Prediction Models of Nuclear Receptor Agonists and Antagonists Using the DeepSnap-Deep Learning Approach with the Tox21 10K Library

“…The chemicals were derived from the Tox21 10K library, which contains approximately 8900 unique compounds gathered from commercial sources, such as pesticides, industrial and environmental chemicals, natural dietary supplement products, food additives, and drugs, by the NTP, the National Center for Advancing Translational Sciences (NCATS), and the EPA ( Table 1 ) [ 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. These compounds were dissolved in dimethyl sulfoxide (DMSO) as stock solutions, and compound plates with the different concentrations were prepared in the 1536-well plate format [ 71 , 72 , 73 , 80 , 83 ].…”

“…These bioassay data consisted of quantitative HTS (qHTS) data derived from two cell-based reporter gene assays, including beta-lactamase or luciferase reporter genes. The activity of these reporter genes is controlled by the binding of transcriptional factors induced or suppressed by an agonist/antagonist with response elements ( The chemicals were derived from the Tox21 10K library, which contains approximately 8900 unique compounds gathered from commercial sources, such as pesticides, industrial and environmental chemicals, natural dietary supplement products, food additives, and drugs, by the NTP, the National Center for Advancing Translational Sciences (NCATS), and the EPA (Table 1) [71][72][73][74][75][76][77][78][79][80][81][82]. These compounds were dissolved in dimethyl sulfoxide (DMSO) as stock solutions, and compound plates with the different concentrations were prepared in the 1536-well plate format [71][72][73]80,83].…”

Identification of Compounds That Inhibit Estrogen-Related Receptor Alpha Signaling Using High-Throughput Screening Assays

“…RAR-C3H10T1/2 [ 88 ] and ShhGli1-3T3 cell lines were provided by Drs. Yanling Chen and David H. Reese (FDA).…”

Identification and Profiling of Environmental Chemicals That Inhibit the TGFβ/SMAD Signaling Pathway

“…The use of C3RL4 cells (passage 8) stably transfected with the retinoic acid response element (RARE) in qHTS screening was previously reported . The cells were seeded at 1000 cells/well/4 μL culture medium in 1536-well white-solid plates using a Multidrop Combi.…”

“…The use of C3RL4 cells (passage 8) stably transfected with the retinoic acid response element (RARE) in qHTS screening was previously reported. 29 The cells were seeded at 1000 cells/well/4 μL culture medium in 1536-well white-solid plates using a Multidrop Combi. After the assay plates were incubated at 37 °C and 5% CO 2 overnight, 23 nL of compounds was transferred to the assay plates by a Wako Pintool station, followed by addition of 1 μL of 1.0 μM retinol using a BioRaptr FRD.…”