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.-L., Holden, C., Affleck, V., Lamb, R., Newling, K., Ashton, P., Valentine, R., Redfern, C., Erostyak, J., Makkai, G., Ambler, C., Whiting, A., & 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
• Public URL: https://durham-repository.worktribe.com/output/1309757