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Tsuji K, Ishii T, Kobayakawa T, et al. Fluorescence resonance energy transfer-based screening for protein kinase C ligands using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones. Org Biomol Chem. 2021;19(38):8264-8271doi: 10.1039/d1ob00814e.
Tsuji, K., Ishii, T., Kobayakawa, T., Ohashi, N., Nomura, W., & Tamamura, H. (2021). Fluorescence resonance energy transfer-based screening for protein kinase C ligands using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones. Organic & biomolecular chemistry, 19(38), 8264-8271. https://doi.org/10.1039/d1ob00814e
Tsuji, Kohei, et al. "Fluorescence resonance energy transfer-based screening for protein kinase C ligands using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones." Organic & biomolecular chemistry vol. 19,38 (2021): 8264-8271. doi: https://doi.org/10.1039/d1ob00814e
Tsuji K, Ishii T, Kobayakawa T, Ohashi N, Nomura W, Tamamura H. Fluorescence resonance energy transfer-based screening for protein kinase C ligands using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones. Org Biomol Chem. 2021 Oct 06;19(38):8264-8271. doi: 10.1039/d1ob00814e. PMID: 34338277.
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Org Biomol Chem. 2021 Oct 06;19(38):8264-8271. doi: 10.1039/d1ob00814e.

Fluorescence resonance energy transfer-based screening for protein kinase C ligands using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones.

Organic & biomolecular chemistry

Kohei Tsuji, Takahiro Ishii, Takuya Kobayakawa, Nami Ohashi, Wataru Nomura, Hirokazu Tamamura

Affiliations

  1. Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan. [email protected].

PMID: 34338277 DOI: 10.1039/d1ob00814e

Abstract

Protein kinase C (PKC) is associated with a central cellular signal transduction pathway and disorders such as cancer and Alzheimer-type dementia and is therefore a target for the treatment of these diseases. The development of simple methods suitable for high-throughput screening to find potent PKC ligands is desirable. We have developed an assay based on fluorescence-quenching screening with a solvatochromic fluorophore attached to a competitive probe and its alternative method based on Förster/fluorescence resonance energy transfer (FRET) phenomena. Here, an improved FRET-based PKC binding assay using a diacylglycerol (DAG) lactone labeled with a donor fluorescent dye, 6-methoxynaphthalene (6MN), was developed. The 6MN-labeled DAG-lactone has a higher binding affinity for the PKCδ C1b domain and the fluorescent PKCδ C1b domain labeled by fluorescein as an acceptor fluorescent dye (Fl-δC1b) than the diethylaminocoumarin (DEAC)-labeled DAG-lactone. The combination of the 6MN-labeled DAG-lactone and Fl-δC1b showed a change in fluorescence response larger than that of the DEAC-labeled DAG-lactone and Fl-δC1b. The IC

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