Fluorophore Labeling Enables Imaging and Evaluation of Specific CXCR4−Ligand Interaction at the Cell Membrane for Fluorescence-Based Screening

Nomura, Wataru; Tanabe, Yasuaki; Tsutsumi, Hiroshi; Tanaka, Tomohiro; Ohba, Kihachiro; Yamamoto, Naoki; Tamamura, Hirokazu

doi:10.1021/bc800216p

Cited by 40 publications

(60 citation statements)

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“…[8,9] For CXCR4 targeting several peptidic probes have been reported, including the cyclic Ac-TZ14011 peptide, and also a chelate-based fluorescent probe. [10][11][12][13][14][15][16] Transition-metal complexes based on, for example, iridium-(III), rhenium(I) and ruthenium(II) are attaining significant interest for application in imaging. [17][18][19][20][21][22][23][24][25][26][27][28] These phosphorescent metal complexes often posses better photophysical properties than organic fluorophores, such as long luminescence lifetimes.…”

Section: Introductionmentioning

confidence: 99%

“…[17,21,22,39] However, a neutral Ir III complex functionalized with peptides for specific targeting of a cancer associated membrane receptor has not been reported yet. In this paper, we present CXCR4 targeting with three different Ac-TZ14011 peptide-conjugated Ir III complexes, Ir-(Ac-TZ14011) (8), Ir-(Ac-TZ14011) 2 (9) and Ir-(Ac-TZ14011) 3 (10).…”

Section: Introductionmentioning

confidence: 99%

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Peptide‐Functionalized Luminescent Iridium Complexes for Lifetime Imaging of CXCR4 Expression

et al. 2011

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The chemokine receptor 4 (CXCR4) is over-expressed in 23 types of cancer in which it plays a role in, among others, the metastatic spread. For this reason it is a potential biomarker for the field of diagnostic oncology. The antagonistic Ac-TZ14011 peptide, which binds to CXCR4, has been conjugated to luminescent iridium dyes to allow for CXCR4 visualization. The iridium dyes are cyclometalated octahedral iridium(III) 2-phenylpyridine complexes that can be functionalized with one, two or three targeting Ac-TZ14011 peptides. Confocal microscopy and fluorescence lifetime imaging microscopy (FLIM) showed that the peptide-iridium complex conjugates can be used to visualize CXCR4 expression in tumor cells. The CXCR4 receptor affinity and specific cell binding of the mono-, di- and trimeric peptide derivatives were assessed by using flow cytometry. The three derivatives possessed nanomolar receptor affinity and could distinguish between cell lines with different CXCR4 expression levels. This yields the first example of a neutral iridium(III) complex functionalized with peptides for FLIM-based visualization of a cancer associated membrane receptor.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Peptide‐Functionalized Luminescent Iridium Complexes for Lifetime Imaging of CXCR4 Expression

et al. 2011

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“…[8] There have been a few reports of optical imaging probes designed to target CXCR4; most involved fluorescently tagged peptides with high binding affinities. [9][10][11] In 2007, Archibald et al reported a small molecule with structural similarities to AMD3100 that also contained a fluorescent rhodamine attachment. [12] Whilst the uncomplexed compound did not inhibit the binding of CXCR4-specific monoclonal antibodies, the corresponding copper complex was shown to have more favourable binding properties.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a Fluorescent Derivative of AMD3100 and its Interaction with the CXCR4 Chemokine Receptor

et al. 2011

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AMD3100 is a potent and selective antagonist of the CXCR4 receptor; it has been shown to block the route of entry of HIV into host T-cells. This compound and its analogues have since been found to act as haematopoietic stem cell mobilisation agents and, more recently, as anti-cancer agents. Here, we have examined a fluorescent derivative of AMD3100, L(1), which offered the potential to assess the behaviour of AMD3100 at the cell surface by using optical imaging modalities. The binuclear Zn(II) , Cu(II) and Ni(II) complexes of L(1) have also been investigated as these metals have been previously shown to enhance the binding properties of AMD3100. Furthermore, Zn(II) and Cu(II) are known to enhance and quench, respectively, the fluorescence of similar anthracenyl-based ligands. Whilst L(1) demonstrates an ability to inhibit the binding of the anti-CXCR4 monoclonal antibody 12G5 (IC(50) =0.25-0.9 μM), the incorporation of an anthracenyl moiety resulted in a significantly reduced affinity for CXCR4 compared to AMD3100 (IC(50) =10 nM). We observed no significant increase in fluorescence intensity following incubation with murine pre-B cells overexpressing CXCR4 compared to a control cell line. This limits the usefulness of L(1) as a fluorescent imaging probe. Interestingly, the Zn(II) complex, which carries an overall +4 charge, revealed marginally higher specificity and reduced toxicity in vitro compared to the free ligand, albeit with reduced affinity for CXCR4 (IC(50) =1.8-5 μM). We suggest that the incorporation of an anthracenyl group contributes to the lipophilic character of the free ligand, thereby resulting in transport across the plasma membrane. This effect is seemingly diminished when the ligand is complexed to charged metal ions.

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“…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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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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Fluorophore Labeling Enables Imaging and Evaluation of Specific CXCR4−Ligand Interaction at the Cell Membrane for Fluorescence-Based Screening

Cited by 40 publications

References 24 publications

Peptide‐Functionalized Luminescent Iridium Complexes for Lifetime Imaging of CXCR4 Expression

Peptide‐Functionalized Luminescent Iridium Complexes for Lifetime Imaging of CXCR4 Expression

Evaluation of a Fluorescent Derivative of AMD3100 and its Interaction with the CXCR4 Chemokine Receptor

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

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