The development of methodologies for high-throughput retinoic acid binding assays in drug discovery and beyond

Tomlinson, Charles W.; Whiting, Andrew

doi:10.1016/bs.mie.2020.02.008

Cited by 3 publications

(4 citation statements)

References 44 publications

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“…1 H-NMR (400 MHz, CDCl 3 ): δ 8.86 (1H, d, J = 2.1 Hz), 8.03 (1H, s), 7.96 (1H, dd, J = 8.5, 2.1 Hz), 6.71 (1H, s), 6.26 (1H, d, J = 8.5 Hz), 5.43 (2H, s), 3.80 (2H, d, J = 6.0 Hz), 3.51 (3H, s), 3.38 (1H, sep, J = 6.9 Hz), 2.17 (1H, sep, J = 6.7 Hz), 1.32−1.24 (11H, m), 1.08 (6H, d, J = 6.7 Hz). 13 (15). To a solution of 14 (100 mg, 0.22 mmol) in EtOH (5 mL) was added 10% activated Pd-C (catalytic amount).…”

Section: ■ Conclusionmentioning

confidence: 99%

“…14 Thus, there is a great need for a screening method that can detect rexinoids quickly using a standard plate reader, as is currently being done for RAR. 15 We have already applied a fluorescent rexinoid (4, CU-6PMN) for this purpose, 14 developing a rapid and nonhazardous assay method without the need for bound-free (B/F) separation. However, this suffers from the drawback that 4 binds not only at the ligand-binding domain (LBD) but also at the dimer− dimer interface of hRXRα, and we found that the interaction with the dimer−dimer interface induced a conformational change of the LBD, leading to a 1.5-fold increase of [ 3 H]9cis-RA binding.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Reporter gene assays and time-resolved fluorescence resonance energy transfer (TR-FRET) assays are widely used as screening methods for the identification of rexinoids, besides the standard radioisotope method using [ 3 H]9- cis -retinoic acid ([ 3 H]9 cis -RA). , These assays can easily measure the transcriptional activity of RXR. On the other hand, the reporter gene assay requires 3–4 days, the TR-FRET assay requires special equipment, and the [ 3 H]9 cis -RA assay also requires special precautions .…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, the reporter gene assay requires 3–4 days, the TR-FRET assay requires special equipment, and the [ 3 H]9 cis -RA assay also requires special precautions . Thus, there is a great need for a screening method that can detect rexinoids quickly using a standard plate reader, as is currently being done for RAR . We have already applied a fluorescent rexinoid ( 4 , CU-6PMN) for this purpose, developing a rapid and nonhazardous assay method without the need for bound-free (B/F) separation.…”

Section: Introductionmentioning

confidence: 99%

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Convenient Retinoid X Receptor Binding Assay Based on Fluorescence Change of the Antagonist NEt-C343

et al. 2020

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Retinoid X receptor (RXR) modulators (rexinoids) are considered to have therapeutic potential for multiple diseases, such as Alzheimer’s disease and Parkinson’s disease. To overcome various disadvantages of prior screening methods, we previously developed an RXR binding assay using a fluorescent RXR ligand, CU-6PMN (4). However, this ligand binds not only at the ligand-binding domain (LBD) but also at the dimer–dimer interface of hRXRα. Here, we present a new fluorescent RXR antagonist 6-[N-ethyl-N-(5-isobutoxy-4-isopropyl-2-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxamido)phenyl)amino]nicotinic acid (NEt-C343, 7), which emits strong fluorescence only when bound to the RXR-LBD. It allows us to perform a rapid, simple, and nonhazardous binding assay that does not require bound/free separation and uses a standard plate reader. The obtained Ki values of known compounds were correlated with the Ki values obtained using the standard [3H]9cis-retinoic acid assay. This assay should be useful for drug discovery as well as for research on endocrine disruptors, functional foods, and natural products.

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Section: ■ Conclusionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Convenient Retinoid X Receptor Binding Assay Based on Fluorescence Change of the Antagonist NEt-C343

et al. 2020

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Determination of pharmacological inhibition of ALDH2 by ethanol clearance in mice

Haenisch,

Paik,

Kim

et al. 2024

Toxicology and Applied Pharmacology

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Structure–functional relationship of cellular retinoic acid-binding proteins I and II interacting with natural and synthetic ligands

Tomlinson

Cornish

Whiting

et al. 2021

Acta Cryst Sect D Struct Biol

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A detailed understanding of the interactions between small-molecule ligands and their proposed binding targets is of the utmost importance for modern drug-development programs. Cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) facilitate a number of vital retinoid signalling pathways in mammalian cells and offer a gateway to manipulation of signalling that could potentially reduce phenotypes in serious diseases, including cancer and neurodegeneration. Although structurally very similar, the two proteins possess distinctly different biological functions, with their signalling influence being exerted through both genomic and nongenomic pathways. In this article, crystal structures are presented of the L29C mutant of Homo sapiens CRABPI in complex with naturally occurring fatty acids (1.64 Å resolution) and with the synthetic retinoid DC645 (2.41 Å resolution), and of CRABPII in complex with the ligands DC479 (1.80 Å resolution) and DC645 (1.71 Å resolution). DC645 and DC479 are two potential drug compounds identified in a recent synthetic retinoid development program. In particular, DC645 has recently been shown to have disease-modifying capabilities in neurodegenerative disease models by activating both genomic and nongenomic signalling pathways. These co-crystal structures demonstrate a canonical binding behaviour akin to that exhibited with all-trans-retinoic acid and help to explain how the compounds are able to exert an influence on part of the retinoid signalling cascade.

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The development of methodologies for high-throughput retinoic acid binding assays in drug discovery and beyond

Cited by 3 publications

References 44 publications

Convenient Retinoid X Receptor Binding Assay Based on Fluorescence Change of the Antagonist NEt-C343

Convenient Retinoid X Receptor Binding Assay Based on Fluorescence Change of the Antagonist NEt-C343

Determination of pharmacological inhibition of ALDH2 by ethanol clearance in mice

Structure–functional relationship of cellular retinoic acid-binding proteins I and II interacting with natural and synthetic ligands

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