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EphB receptor-binding peptides

A technology of receptors and amino acids, applied in the direction of peptides, specific peptides, drug combinations, etc., can solve problems such as unclear how EphA2-binding peptides activate EphA2

Inactive Publication Date: 2008-03-19
THE BURNHAM INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is unclear how EphA2-binding peptides activate EphA2

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0188] Identification of peptides that bind to different EphB receptors

[0189] To identify EphB receptor-binding peptides, immunoscreens displaying random peptides 12 amino acids long based on EphB1, EphB2, or EphB4 ectodomains fused to human Fc and immobilized on nickel-coated wells by carboxy-terminal hexahistidine tagging M13 phage library (R&D Systems, Minneapolis, MN) (Figure 1). Anti-phage antibody conjugated with horseradish peroxidase (M13 Phage Detection Kit, Amersham Biosciences) was used with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as substrate Phage bound to Ni-NTA wells coated with 1 μg / ml Eph receptor Fc fusion protein was quantified. Background subtracted from assay values ​​was determined in the absence of EphB4Fc. Histidine-tagged EphB ectodomain Fc fusion protein was incubated overnight at 4°C in nickel-nitrilotriacetic acid (Ni-NTA)-coated ELISA wells in Tris-buffered saline (TBS) (150 mM NaCl , 50 mM Tris-HCl, pH 7.5) at a concentration o...

Embodiment 2

[0193] Different peptides compete with each other for binding to the EphB receptor for their isolation

[0194] To determine whether different phage clones target overlapping Eph receptor binding sites, EWLS EphB1-binding peptides and TNYL EphB4-binding peptides were chemically synthesized. These peptides were chosen because they contain motifs found in Ephrin and bind specifically to EphB1 or EphB4, respectively, when they are displayed on phage (Table 1). The peptide was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by high pressure liquid chromatography. Biotinylated peptides were synthesized with a carboxy-terminal GSGSK linker with biotin attached to the side chain of a lysine, or with a carboxy-terminal N-biotinyl-N'-Fmoc-ethylenediamine (Novabiochem ) carboxy-terminal GSGS sequence. Correct peptide synthesis and purity were confirmed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The stock solution o...

Embodiment 3

[0202] Peptides that selectively antagonize the binding of Ephrin to EphB1, EphB2, and EphB4 receptors

[0203] Peptides that bind to the same receptor region as Ephrin are expected to inhibit Eph receptor-Ephrin binding if they interact with sufficient affinity. A number of synthetic peptides were tested for their ability to inhibit the binding of alkaline phosphatase-tagged Ephrin-B2 (Ephrin-B2-AP) to the EphB receptor (Figure 3). Most peptides were synthesized biotin-labeled and immobilized on streptavidin plates to demonstrate their ability to capture the EphB ectodomain for their isolation.

[0204] Binding of Ephrin-B2AP to the indicated extracellular domains of immobilized EphB receptors was assayed in the presence of the indicated peptides and detected by measuring alkaline phosphatase activity. Among the peptides tested for their ability to inhibit the binding of Ephrin to the EphB receptor, EWLS and AHTF inhibited the binding of Ephrin to EphB1; SNEW (but not DHWRI,...

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Abstract

The application is related to the identification of peptides that selectively bind to Eph receptors of the B class. Also disclosed are uses of such peptides in the treatment of a variety of diseases. Additionally, imaging tumors in patients is described by administrating labeled peptides to patients and then obtaining an image of the labeled peptides.

Description

[0001] related application [0002] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 60 / 647,852, filed January 27, 2005, which is hereby incorporated by reference in its entirety. [0003] government interest [0004] This invention was made with government support under National Institutes of Health grant numbers CA82713 and HD25938 and Department of Defense grant number DAMD17-01-1-0168. The US Government has certain rights in this invention. technical field [0005] The present application relates to antagonistic peptides that selectively bind to class B Eph receptors including EphB1, EphB2 and EphB4. Also included are methods of identifying such peptides. Background technique [0006] Eph receptors are a large family of receptor tyrosine kinases that regulate many biological processes in developing and adult tissues by binding to a class of ligands known as Ephrins (Ephrins) (Murai, K.K. and Pasquale , E.B. 2003 J Cell ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/52A61K38/19A61K51/08
CPCA61K49/0056C07K2319/60A61K47/48246B82Y5/00A61K38/00G01N33/574C07K14/52A61K51/088A61K47/64A61P25/00A61P35/00A61P43/00A61P9/00C07K14/00
Inventor 埃琳娜·B·帕斯夸里米切尔·库尔普
Owner THE BURNHAM INST
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