Fluorescence-Quenching Screening of Protein Kinase C Ligands with an Environmentally Sensitive Fluorophore

Nomura, Wataru; Ohashi, Nobukimi; Okuda, Yoshiaki; Narumi, Tetsuo; Ikura, Teikichi; Ito, Nobuyasu; Tamamura, Hirokazu

doi:10.1021/bc100567k

Cited by 14 publications

(8 citation statements)

References 40 publications

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“…The reaction mixture was concentrated under reduced pressure, followed by purification by silica gel column chromatography (hexane/EtOAc=5 : 1) to give the enone 4 (E isomer, 155 mg, 46% yield for two steps) as an orange solid. The E isomer was exclusively obtained, which was based on tentative assignment 7,29) : Rf 0.43 (hexane/EtOAc=3 : …”

Section: Methodsmentioning

confidence: 99%

“…Previously, we described fluorescence-based ligand screening assays as an alternative to radioisotope assays. 6,7) Protein kinase C (PKC) isozymes, which are classified as Ser/Thr kinases, play a critical role in cellular signaling pathways related to proliferation, 8,9) differentiation, 10) and apoptosis. 11,12) PKC has 11 isozymes which are classified into three subtypes: conventional PKC (cPKC; α, β I/II , γ), novel PKC (nPKC; δ, ε, η, τ) and atypical PKC (aPKC; ζ, λ, ι).…”

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confidence: 99%

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Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

Ohashi

Nomura

Minato

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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Protein kinase C (PKC) is correlated with cell signaling pathways and also receives attention as a therapeutic target for cancer and Alzheimer-type dementia. The application of Förster/fluorescence resonance energy transfer (FRET) phenomena to detect binding between proteins and small molecules, for example, PKC and its ligands, underlies a fluorescence-based assay method suitable for high-throughput screening. To accelerate studies on PKC functions in processing signals using small molecules and the development of drugs that target PKC, novel methods for the assessment of the PKC binding affinity of compounds are necessary. We previously developed solvatochromic fluorophore-based methods for that assessment. In this study, a novel method for a FRET-based PKC binding assay was developed and is expected to overcome the limitations of solvatochromic fluorophores.Key words protein kinase C; protein kinase C ligand; ligand screening; Förster resonance energy transfer; fluorescence resonance energy transfer Förster/fluorescence resonance energy transfer (FRET) is the transfer of excitation energy from a fluorescence donor to an acceptor. FRET efficiency and sensitivity depend on the distance between the donor and the acceptor. The FRET phenomenon can be applied as a spectroscopic measure and a probe of conformational change of macro-biomolecules [1][2][3] and a FRET-mediated competitive assay can be used for studies of the binding between two molecules. 4,5) Radioisotopebased methods are extremely sensitive and are widely used for ligand binding assays, but fluorescent-based assays, which are suitable for high-throughput screening are free of hazards associated with radioactivity. Previously, we described fluorescence-based ligand screening assays as an alternative to radioisotope assays. 6,7)Protein kinase C (PKC) isozymes, which are classified as Ser/Thr kinases, play a critical role in cellular signaling pathways related to proliferation, 8,9) differentiation, 10) and apoptosis.11,12) PKC has 11 isozymes which are classified into three subtypes: conventional PKC (cPKC; α, β I/II , γ), novel PKC (nPKC; δ, ε, η, τ) and atypical PKC (aPKC; ζ, λ, ι). The binding of diacylglycerols (DAG) to the C1 domain of PKC is an important step in an activation process except in the case of aPKC which contains an atypical C1 domain which fails to bind to DAG.13) Various synthetic PKC ligands targeting the C1 domain have been developed as chemical probes or drug candidates by radioisotope assays.14-17) We have previously developed a fluorescent assay for PKC-ligand binding affinity using a synthetic PKC δC1b domain labeled with a solvatochromic fluorophore on the edge of its ligand-binding pocket. This can detect ligand binding through changes in the surrounding environment.18) However, this method would not be suitable for high-throughput screening. The reason might be due to several possibilities: For instance, the change of fluorescence intensity is affected by properties of test compounds. It would be concerned that a fluorescen...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

Ohashi

Nomura

Minato

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…A fluorescence-quenching screening system for PKC ligands based on a reporter compound containing a solvatochromic fluorophore was developed. 35 In this assay, ligand binding to the C1b domain is detected by fluorescence-quenching, and IC 50 values against a reporter compound can be determined by titration experiments. When a reporter compound binds to the C1b domain, fluorescence of a fluorophore is enhanced because the environment around the fluorophore is more hydrophobic in the binding pocket than in the surrounding environment.…”

Section: Dag-lactone-based Ligand Screening Of Pkcmentioning

confidence: 99%

“…A fluorescence‐quenching screening system for PKC ligands based on a reporter compound containing a solvatochromic fluorophore was developed . In this assay, ligand binding to the C1b domain is detected by fluorescence‐quenching, and IC 50 values against a reporter compound can be determined by titration experiments.…”

Section: Dag‐lactone‐based Ligand Screening Of Pkcmentioning

confidence: 99%

Peptide‐based ligand screening and functional analysis of protein kinase C

et al. 2013

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Protein kinase C (PKC) plays an important role in cell signaling pathways and is implicated in disorders ranging from cancer to Alzheimer's disease. Highly potent PKC ligands as therapeutic drugs have not been developed to date and useful methodologies for controlling PKC activation in defined areas are necessary to analyze precise PKC functions in cells. Studies focused on the development of PKC ligand screening systems and methods for regulation of PKC activation have been performed in our laboratory. In this review, our ligand screening methods involving synthetic peptides and solvatochromic fluorescent dye-labeled small compounds are summarized and the technique of spatio-temporal manipulation of PKC activation by caging strategies is introduced.

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“…In contrast, coumarins are small, and have a solvatochromic fluorescence property that increases fluorescence intensity in hydrophobic environments and decreases it in hydrophilic environments [24]. Compounds containing coumarin were developed to detect ligand binding to target proteins [25].…”

Section: Introductionmentioning

confidence: 99%

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay

Yoshikawa¹,

Ishida²,

Ohashi³

et al. 2021

IJMS

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Peroxisome proliferator-activated receptor γ (PPARγ) is a molecular target of metabolic syndrome and inflammatory disease. PPARγ is an important nuclear receptor and numerous PPARγ ligands were developed to date; thus, efficient assay methods are important. Here, we investigated the incorporation of 7-diethylamino coumarin into the PPARγ agonist rosiglitazone and used the compound in a binding assay for PPARγ. PPARγ-ligand-incorporated 7-methoxycoumarin, 1, showed weak fluorescence intensity in a previous report. We synthesized PPARγ-ligand-incorporating coumarin, 2, in this report, and it enhanced the fluorescence intensity. The PPARγ ligand 2 maintained the rosiglitazone activity. The obtained partial agonist 6 appeared to act through a novel mechanism. The fluorescence intensity of 2 and 6 increased by binding to the ligand binding domain (LBD) of PPARγ and the affinity of reported PPARγ ligands were evaluated using the probe.

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Fluorescence-Quenching Screening of Protein Kinase C Ligands with an Environmentally Sensitive Fluorophore

Cited by 14 publications

References 40 publications

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

Peptide‐based ligand screening and functional analysis of protein kinase C

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay

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