Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands

Ziemek, Ralf; Brennauer, Albert; Schneider, Erich H.; Cabrele, Chiara; Beck‐Sickinger, Annette G.; Bernhardt, Günther; Buschauer, Armin

doi:10.1016/j.ejphar.2006.08.075

Cited by 36 publications

(74 citation statements)

References 34 publications

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“…Additionally, in the same work the binding of fluorescently labeled DUP-1 was observed qualitatively using confocal microscopy. To further demonstrate the reliability of the proposed screening technique, we performed a competitive binding assay on cells in suspension based on this work, using fluorescently labeled probes and FACS analysis [13]. In this assay, unlabeled molecules to be tested at different concentrations and fluorescently labeled DUP-1 at a given concentration are added to the cells and their fluorescence intensity, representing the amount of the known molecule bound, is tested by flow cytometry.…”

Section: Validation Studiesmentioning

confidence: 99%

“…Each molecule can bind to multiple cellular targets, some of them might be common, and if this is the case, competition binding can be qualitatively evaluated. We have developed an independent validation assay in our laboratory based on previous works [1,13]. Candidate ligands identified by the screening were individually synthesized and characterized in solution.…”

Section: Validation Studiesmentioning

confidence: 99%

“…A nonconfocal DIC transmitted light image was also acquired. The (FITC- 13 Lys)DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) , (FITC-13 Lys) DUP-1 (1-12) 2 Ala and non-treated cells images were acquired under the same acquisition conditions. All image processing was done in Image J [6].…”

Section: Validation Studiesmentioning

confidence: 99%

“…(FITC-13 Lys)DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) standard peptide was incubated with PC-3 cells at a concentration of 1µM in the presence of different concentrations of non-labeled DUP-1, negative control peptide 15-mer or ligand hits as previously shown [1]. The cells were analyzed by flowcytometry and the affinity was determined using the geometric means obtained from FACS plots at the different concentrations of ligands [13]. The displacement obtained by the specific candidate ligand was compared to that of the standard DUP-1 peptide.…”

Section: Validation Studiesmentioning

confidence: 99%

“…On the day of experiment, the medium (RPMI 1640) was replaced with 2 ml of fresh medium. FITC- 13 Lys-DUP-1 or FITC- 13 Lys -DUP-1 (1-12) 2 Ala and Sulforhodamine B were dissolved in RPMI-1640 (without FCS) at 100 µM. 20 µL of each peptide was added to the cells to reach a final concentration of 1µM in the medium.…”

Section: -Cell Binding and Penetration Validation Of Dup-1 (1-12) 2 mentioning

confidence: 99%

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A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Khandadash¹,

Partouche²,

Weiss³

et al. 2011

TOOPTSJ

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Herein we present a new application of a recently demonstrated fully synthetic "phage-like" system for screening of combinatorial mixtures in a live cell assay. The new application includes the direct synthesis of peptides and combinatorial libraries on 2 µm cross-linked mono-dispersed microspheres bearing a panel of fluorescence tags. Their characterization using classical chemical analysis as well as biological recognition of the synthesized sequences on the microspheres by specific antibodies, demonstrate the robustness of the system. Two biased positional combinatorial peptide libraries derived from peptide DUP-1 were synthesized and screened for affinity to prostate cancer PC-3 cell line as compared to DUP-1, a peptide with good affinity for this cell line. The best sub-library was deconvoluted and mixtures of deconvoluted peptides on microspheres were screened for identifying the sequences with the best affinity for cells. The best peptide, DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala with high affinity for PC-3 cells, was individually synthesized and validated in an independent binding assay. Finally, the cellular fate of DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala in PC-3 cell line is demonstrated using confocal laser scanning microscopy. Overall, these results demonstrate that the synthetic phage-like system recently assessed for screening mixtures of small organic molecules, can be also used for synthesis and screening of combinatorial libraries of peptides in live cell assays.

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Section: Validation Studiesmentioning

confidence: 99%

Section: Validation Studiesmentioning

confidence: 99%

Section: Validation Studiesmentioning

confidence: 99%

Section: Validation Studiesmentioning

confidence: 99%

Section: -Cell Binding and Penetration Validation Of Dup-1 (1-12) 2 mentioning

confidence: 99%

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A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Khandadash¹,

Partouche²,

Weiss³

et al. 2011

TOOPTSJ

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Toward Labeled Argininamide‐Type NPY Y₁ Receptor Antagonists: Identification of a Favorable Propionylation Site in BIBO3304

Keller

Schindler

Bernhardt

et al. 2015

Archiv der Pharmazie

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Aiming at molecular tools for the neuropeptide Y Y1 receptor (Y1 R), three types of derivatives of the argininamide-type Y1 R antagonist BIBO3304 were prepared by (i) propionylation at the guanidine group (3), (ii) substitution at the urea moiety with a propionamidobutyl residue (4), and (iii) replacement of ureidomethyl by a propionylaminomethyl group (5). With Ki and Kb values in the range of 1.5-4.3 nM, determined in binding and functional assays, and high selectivity for the Y1 R over the Y2 R, Y4 R, and Y5 R, compounds 4 and 5 were identified as promising candidates for radiolabeling by [(3) H]propionylation according to established protocols.

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Synthesis and Characterization of the First Fluorescent Nonpeptide NPY Y₁ Receptor Antagonist

et al. 2007

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Cyanine-5-labelled neuropeptide Y (NPY) was demonstrated to be an ideal universal fluorescent ligand for the combined investigation of NPY Y(1), Y(2) and Y(5) receptors. With respect to improved stability, detection of receptor subtypes in cells and tissues, and prevention of receptor internalization, small nonpeptidic fluorescent antagonists should be superior. Here we present a set of four fluorescent nonpeptide NPY Y(1) receptor (Y(1)R) antagonists. The highest affinity was obtained by labelling an N(G)-(6-aminohexanoyl)argininamide derived from the Y(1)R antagonist BIBP 3226, with Py-1, a small pyrylium dye. The fluorescent pyridinium-type Y(1)R antagonist, compound 4 had K(i) values of 29 nM and 2.7 nM, which were determined by radioligand binding and flow cytometry under equilibrium conditions, respectively; 4 had a K(b) value of 0.6 nM (Ca(2+) assay). The large Stoke's shift (541 vs. 615 nm) in buffer (PBS, pH 7.4) in the presence of 1% BSA and the red emission (quantum yield 56%) are advantageous with respect to the signal-to-noise ratio. The new probe was successfully used in fluorescence-based binding experiments evaluated by flow cytometry and confocal microscopy; this demonstrates the potential of pyrylium dyes for the preparation of fluorescent ligands that are applicable for the study of G protein-coupled receptors on living cells.

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Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands

Cited by 36 publications

References 34 publications

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Toward Labeled Argininamide‐Type NPY Y₁ Receptor Antagonists: Identification of a Favorable Propionylation Site in BIBO3304

Synthesis and Characterization of the First Fluorescent Nonpeptide NPY Y₁ Receptor Antagonist

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Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands

Cited by 36 publications

References 34 publications

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Toward Labeled Argininamide‐Type NPY Y1 Receptor Antagonists: Identification of a Favorable Propionylation Site in BIBO3304

Synthesis and Characterization of the First Fluorescent Nonpeptide NPY Y1 Receptor Antagonist

Contact Info

Product

Resources

About

Toward Labeled Argininamide‐Type NPY Y₁ Receptor Antagonists: Identification of a Favorable Propionylation Site in BIBO3304

Synthesis and Characterization of the First Fluorescent Nonpeptide NPY Y₁ Receptor Antagonist