Fluorescent retinoic acid analogues as probes for biochemical and intracellular characterization of retinoid signalling pathways

Chisholm, DR; Tomlinson, CWE; Zhou, G-L; Holden, C; Affleck, V; Lamb, R; Newling, K; Ashton, P; Valentine, R; Redfern, C; Erostyak, J; Makkai, G; Ambler, CA; Whiting, A; Pohl, E

Abstract

Retinoids, such as all-trans-retinoic acid (ATRA), are endogenous signalling molecules derived from Vitamin A that influ-ence a variety of cellular processes through mediation of transcription events in the cell nucleus. Due to these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they con-trol. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluo-rescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quanti-fy their binding to their cellular targets, including Cellular Retinoid Binding Protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding (Kd = 34.0 ± 2.5 nM) and we further used X-ray crystallography to solve the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid-binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behaviour of the com-pounds in cultured human epithelial cells, highlighting a fascinating nuclear localisation, and used RNA sequencing to con-firm that the compounds regulate similar cellular processes to ATRA. We anticipate that the unique properties of these fluo-rescent retinoids can now be used to shed new light on the vital and highly complex retinoid signalling pathway.

Citation

Chisholm, D., Tomlinson, C., Zhou, G., Holden, C., Affleck, V., Lamb, R., …Pohl, E. (2019). Fluorescent retinoic acid analogues as probes for biochemical and intracellular characterization of retinoid signalling pathways. ACS Chemical Biology, 14(3), 369-377. https://doi.org/10.1021/acschembio.8b00916

• Journal Article Type: Article
• Acceptance Date: Jan 18, 2019
• Online Publication Date: Feb 1, 2019
• Publication Date: Mar 15, 2019
• Deposit Date: Jan 22, 2019
• Publicly Available Date: Feb 14, 2019
• Journal: ACS Chemical Biology
• Print ISSN: 1554-8929
• Electronic ISSN: 1554-8937
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 14
• Issue: 3
• Pages: 369-377
• DOI: https://doi.org/10.1021/acschembio.8b00916