Polypeptides capable of inhibiting the binding between leptin and Neuropilin-1

Abstract

The present invention relates to agents capable of inhibiting the binding between Leptin and Neuropilin-1 (NRP1) and uses thereof in the therapeutic field.

Claims

1. An isolated, a synthetic or a recombinant polypeptide capable of inhibiting the binding between leptin and NRP-1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:3 (ENLRDLLHVLAFSKSCHLPWASGLETL) or SEQ ID NO:4 (EGNKPVLFOGNTNPTDVYVAVFPK), and wherein the polypeptide is fused to at least one heterologous polypeptide.

2. A nucleic acid encoding the polypeptide fused to the at least one heterologous polypeptide of claim 1.

3. The nucleic acid of claim 2 which is included in a suitable vector.

4. An isolated host cell comprising the nucleic acid of claim 2.

5. A pharmaceutical composition comprising the polypeptide fused to the at least one heterologous polypeptide of claim 1 or a nucleic acid encoding the polypeptide fused to the at least one heterologous polypeptide.

6. The nucleic acid of claim 3 wherein the vector is a plasmid, a cosmid, an episome, an artificial chromosome, a phage or a viral vector.

Description

FIGURES

(1) FIG. 1 shows the OB:NRP-1 interaction with VEGF

(2) FIG. 2 shows the OB:VEGF165:NRP1 docking

(3) FIG. 3 shows the NRP1:OB peptides interaction

(4) FIG. 4 shows the increase of NRP-1/OBR complex formation and MDAMD231 cell migration following Avastin treatment

EXAMPLE

(5) Material & Methods

(6) Bio-Layer Interferometry

(7) Bio-layer interferometry (BLI) is a label-free technique that is sensitive to an increase of mass bound to the biosensor enabling protein-protein interaction characterization.

(8) Ligands preparation: proteins were incubated in a PBS buffer with a 1:3 ratio molar ratio of biotin (biotin-PEG4-NHS from Pierce EZ kit, prepared following the manufacturer's instructions). Free biotin was removed using a desalting column (Pierce). The biotinylated protein (called ligand) was immobilized onto streptavidin biosensor tips and dipped into wells containing the buffer with the analyte of interest (association) or without (dissociation).

(9) Experimental conditions were as follow: total volume in each well: 200 l; shake speed: 1,000 rpm. For simple protein:protein interactions an association phase was followed by a dissociation phase. For competition experiments, the association phase was followed by another association phase with a second analyte instead of a dissociation phase.

(10) Sensorgrams were background corrected, smoothed with the Savitzky-Golay algorithm and analyzed using OctetRED instrument software (ForteBio Data Analysis version 7.1). Experimental sensorgrams were first fit to a 1:1 model. The 1:1 model was accepted if the Chi.sup.2 test was below 3 and the R.sup.2 was above 0.9. When the 1:1 model was rejected, the model with the lowest Chi.sup.2 and the highest R.sup.2 was then selected.

(11) Molecular Docking Experiment

(12) Preparation of the Protein Structures

(13) The structure of VEGF (PDB ID: 4DEQ) and Leptin (PDB ID: 1AX8) were extracted from the protein databank (ref Berman). Since the leptin structure was mutated in the original PDB (W100E), we reversed the mutation to the wild type leptin with PyMol (ref Delano). Hydrogens and partial charges were added using the dockprep routine from Chimera (ref Pettersen).

(14) Blind Docking Experiment.

(15) We used a hierarchical blind docking protocol comprising PatchDock web server (ref Schneidman-Duchovny) for the first step with default parameters. The top 1000 solutions from PatchDock were refined and reranked using Firedock server (ref Schneidman-duchovny 2). The top 10 reranked solutions were optimized with RosettaDock as implemented in ROSIE (ref Lyskov) with the no-refine parameter. Consensus binding mode, illustrated in FIG. 2 was extracted from the top solutions.

(16) NRP-1 and OBR Complex Detection in MBA-MB231 Breast Cancer Cell Line by Immunocytochemistry Using a PLA Technology

(17) The detection of NRP-1/OBR complex in human MDA-MB231 breast cancer cell line was assessed by proximity ligation assay (PLA) or duolink technology (www.olink.com). The detection of the NRP-1/OBR complex was assessed on MDA-MB231 cell line cultivated in normal human serum (human male AB plasma, USA origin, MDL number MFCD00165829 H4522 Sigma) and treated or not with Avastin 40 g/ml final concentration for 48 h. The goal by using human serum was to mimic a physiologic condition during therapy with Avastin. The immunostained samples were analysed by the acquisition of the Z stacks through confocal microscopy on Zeiss LSM 700, Inverted confocal microscope. The acquired images were analyzed using Image J software for the quantification of NRP-1/OBR complex expressed by cells.

(18) Results

(19) By using a BioLayer Interferometry technology (BLI, http://www.fortebio.com), we were able to demonstrate a direct interaction between recombinant proteins leptin and NRP-1 (FIG. 1A). VEGF165 has been used as positive control of the experiment. Surprisingly, in contrast to other NRP-1 ligands, VEGF, and Sema3A known as competitors, leptin binds directly to NRP-1 but do not compete with VEGF binding and similarly VEGF does not prevent leptin binding to NRP-1. These observations suggest that leptin and VEGF should have a distinct binding domain.

(20) Since BLI technology have shown that leptin and VEGF could interact with NRP-1 in non-competitive way and since we could demonstrate that leptin and VEGF form a complex in obese people tissue and by BLI technology using recombinant protein we assessed a docking of NRP-1 and leptin complexed with VEGF165. From the best consensus mode, peptide sequences SEQ ID NO:3 for leptin and SEQ ID NO:4 for NRP-1 have been identified (FIG. 2). The Leptin (OB) binding domain (SEQ ID:3) to NRP-1 (SEQ ID:2) was validated by BLI using a synthetized peptide (SEQ ID:3) and the extracellular domain of the recombinant protein NRP-1 from RnD systems (FIG. 3).

(21) Since we have demonstrated that VEGF play a negative feed-back regulatory role for leptin signaling and since we demonstrated that Avastin increased MDA-MB231 cell migration, this raises the question of whether of not the Avastin effect occurs or not through the increase of NRP-1/OBR complex formation? Interestingly, compared to MDA-MB231 cell line cultivated in human serum, cells cultivated in the same condition and treated with Avastin40 mg/ml presented a high number of NRP-1/OBR complex which my explain the increase of the migration of the cells.

REFERENCES

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