Metod of modulation of interaction between receptor and ligand

Abstract

The present invention relates to a method for modulating the interaction between at least two proteins, wherein at least one of the two proteins is a functional cell-surface receptor and the other protein is the receptor ligand. The invention features a binding site of said functional cell-surface receptor on the receptor ligand and discloses a series of amino acid sequences, which are part of the structure of said binding site and/or involved in the interaction between the receptor and the ligand. Moreover, the present invention features methods for molecular design and screening of a candidate compound capable of modulating the interaction between the functional cell-surface receptor and receptor ligand through the described binding site, and provides a screening assay for identification of such a compound. The invention further describes an antibody capable of binding to the above binding site and/or to an epitope comprising an amino acid sequence essential for executing the receptor ligand interaction through said binding site. The invention also concerns a variety of uses of the disclosed methods, peptide sequences and antibodies. The invention in preferred embodiments concerns the binding site of the fibroblast growth factor receptor (FGFR) on FGFR ligands, compounds capable of modulating the receptor ligand interaction through said binding site, and antibody capable of recognition of said binding site.

Description

FIELD OF INVENTION [0001] The present invention relates to a method for modulating the interaction between at least two proteins, wherein at least one of the two proteins is a functional cell-surface receptor and the other protein is the receptor ligand. The invention preferably concerns interaction of the fibroblast growth factor receptor (FGFR) and FGFR ligands. The invention further relates to a series of amino acid sequences involved in forming a binding site for FGFR in a FGFR ligand. Moreover, the present invention features the methods for molecular design and screening of a candidate compound capable of modulating the interaction between FGFR and a protein having the above binding site, and provides a screening assay for identification of such a compound. BACKGROUND OF INVENTION [0002] Neural cell adhesion molecules (CAMs) of the immunoglobulin superfamily nucleate and maintain groups of cells at key sites during early development and in the adult. In addition to their adhesi...

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[0050]FIG. 5 shows the effect of the NCAM F3 module 2 and the FG loop peptide on phosphorylation of FGFR and demonstrates the immunoprecipitation of NCAM by FGFR. A) TREX-293 cells, stably transfected with FGFR containing a C-terminal Strepll-tag, were stimulated for 20 min with either 10 ng/ml FGF2, 5 μM F3 module 2 or 2.5 μM dendrimeric FG loop peptide. After stimulation, FGFR was immunopurified using anti-phosphotyrosine antibodies and then analyzed by immunoblotting using antibodies against the Strepll-tag. B) HEK293 cells, transiently transfected with a His-tagged version of FGFR1, were stimulated for 20 min with either 5 μM F3 module 2 or 25 μM FG loop peptide. The total amount of FGFR1 and the amount of FGFR phosphorylation was estimated by immunoblotting using anti-pentahis (anti-His) and anti-phosphotyrosine (anti-pY) antibodies, respectively. Quantification of FGFR phosphorylation was performed by densitometric analysis of the band intensity. Phosphorylation was estimated relative to the control (untreated cells), which has been normalized to 1.0. Error bar represents one standard error of the mean. P<0.05 by paired t-test when comparing treated cells with controls (the t-test was performed on six independent sets of non-normalized data). Ctl stands for the control, F3-F3 module 2, FGL-FG loop peptide. C) TREX-293 cells, stably transfected with FGFR containing a C-terminal Strepll-tag, were transiently transfected with a control vector or NCAM. FGFR was purified from the cell lysate via the Strepll-tag and analyzed by immunoblotting using antibodies against NCAM.

[0051]FIG. 6 shows the effect of the NCAM F3 module 2 and its FGFR binding part (the FG loop peptide (SEQ ID NO: 1)) on neurite outgrowth from hippocampal neurons. a) Phase contrast micrograph of control (untreated) neurons. b) Phase contrast micrograph of neurons treated with 5 μM F3 module. c) Neurite length versus the concentration of the F3 module, the FG-loop peptide and a truncated version of the peptide. d) Effect of an anti-FGFR antibody on neurite outgrowth induced by 5 μM F3

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However, the role extracellular ATP, which is one of the most abundant neurotransmitters

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