Ligand controling interaction between gags with their effector molecules and use thereof

Abstract

The invention relates to new compounds that mimic Glycosaminoglycans and are able to control interaction between Glycosaminoglycans with their effector molecules. The compounds of the invention are peptides and are able to prevent or reduce the binding of at least one effector molecule with at least one glycosaminoglycan. The compounds according to the invention can be used as drug, in particular for the stimulation of the neurogenesis and more generally to treat nervous system related pathologies.

Claims

1. A ligand, or any of its pharmaceutically acceptable salts, said ligand comprising or consisting of a polypeptide of the general formula (I):
[X]n  (I) wherein n is comprised between 3 and 50, X is a peptide comprising from 4 to 6 amino acids, X comprises an amino acid selected from the group consisting of glutamic acid and aspartic acid, X comprises one or two cysteic acid (s), and X comprises at least one neutral amino acid other than cysteine, wherein said ligand is able to interact with binding of one effector molecule with at least one glycosaminoglycan (GAG).

2. The ligand according to claim 1, characterized in that n is comprised between 3 and 35.

3. The ligand according to claim 1, characterized in that X comprises two cysteic acids.

4. The ligand according to claim 1, characterized in that X comprises two homocysteic acids.

5. The ligand according to claim 1, characterized in that the at least one neutral amino acid other than cysteine is chosen from the group consisting of alanine, asparagine, glutamine, histidine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and 2-aminoisobutyric acid.

6. The ligand according to claim 1, characterized in that the neutral amino acid other than cysteine is alanine.

7. The ligand according to claim 1, characterized in that the amino acid selected from the group consisting of glutamic acid and aspartic acid is placed in the first position of peptide X starting from the C-terminus.

8. The ligand according to claim 1, characterized in that X in polypeptide [X].sub.n is selected in the group consisting of EACC, ECCA and ECAC, with C is cysteic acid, A is alanine, E is glutamic acid.

9. The ligand according to claim 1, characterized in that the ligand is able to interact with a glycosaminoglycan (GAG) binding site.

10. The ligand according to claim 9, characterized in that the said glycosaminoglycan GAG is selected from the group consisting of heparan sulfate, heparin and chondroitin sulfate.

11. The ligand according to claim 1, characterized in that the ligand is able to prevent the binding of at least one effector molecule with at least one glycosaminoglycan (GAG).

12. The ligand according to claim 11, characterized in that the said effector molecule is selected from the group consisting of transcription factors, growth factors, signaling factors, coagulation cascade proteins, an effector molecule chosen from the group consisting of Semaphorins, and homeoprotein family.

13. The method of administering a medicament to a subject, comprising administering the ligand according to claim 1 to a subject in need thereof.

14. A method of treating a disease and/or neurological conditions and/or nervous system damage, comprising administering the ligand according to claim 1 to a subject in need thereof, wherein the disease and/or neurological conditions and/or nervous system damage is selected from the group consisting of: nervous system damage following cerebrovascular accident, ischemic, hemorrhagic, neoplastic, degenerative, traumatic, and/or neurodevelopmental damage; nervous system damage following events; neurodegenerative diseases; conditions caused by nutrient deprivation or toxins; neurodevelopmental diseases; neuropsychiatric diseases; depression, epilepsy; and degenerative diseases affecting the eyes or ears.

15. A pharmaceutical composition comprising the ligand according to claim 1.

16. The ligand according to claim 1, wherein n is comprised between 3 and 15.

17. The ligand according to claim 1, wherein n is comprised between 3 and 6.

18. The ligand according to claim 11, wherein said effector molecule is selected from Semaphorins and homeoprotein family.

19. The ligand according to claim 11, wherein said effector molecule is Otx2 or Semaphorin-3A.

20. The method according to claim 14, wherein: the nervous system damage follows a stroke or an injury; the neurodegenerative disease is selected from the group consisting of multiple sclerosis, amyotrophic lateral sclerosis, subacute sclerosing panencephalitis, Parkinson's disease, Huntington's disease, muscular dystrophy, Alzheimer's disease, idiopathic dystonia, Spinal muscular atrophy or Wilson's disease; the neurodevelopmental disease is an autism or a dyslexia; the neuropsychiatric disease is a schizophrenia or a bipolar disorder, and the degenerative disease affecting the eyes or ears is a glaucoma or an amblyopia.

Description

(1) In addition to the above arrangements, the invention also comprises other arrangements, which will emerge from the description, which refers to exemplary embodiments, with reference to the Figures in which:

(2) FIG. 1. Ligands interact with Otx2. (a) Example dot blot (DB) of biotinylated sulfopeptides incubated or not with Otx2 protein. (b) Quantification of DB chemiluminescence with at least 3 duplicate experiments per data point.

(3) FIG. 2. Ligands interact with the GAG-binding site of Otx2. (a) Electrophoretic mobility shift assay (EMSA) with biotinylated IRBP1 DNA probe and Otx2 protein shows loss of shift when incubated with hexaCSE and (EC′AC′).sub.5. (b) Dot blot (DB) of (EC′AC′).sub.6 incubated with GAG motif peptide (RKpep) or control peptides (AApep, SCpep). (c) Quantification of DBs. All values: N=3; mean±SEM; one-way ANOVA with Bonferonni posthoc test; **P<0.01.

(4) FIG. 3. Ligand pull-down experiments with lysates of adult mouse visual cortex. (a) Comparison of GAG-binding motifs. (b) Western blot (WB) for Sema-3A after pull-down with ligands retained on streptavidin beads (Control is beads alone). (c) Quantification of WBs. (d) WB for Sema-3A after pull-down with (EC′AC′).sub.6 incubated with GAG motif peptide (RKpep) or control peptides (AApep, SCpep). (e) Quantification of WBs. All values: N=3; mean±SEM; one-way ANOVA with Bonferonni posthoc test; *P<0.05, **P<0.01, ***P<0.001.

(5) FIG. 4. Ligands have in vivo activity. (a) Representative images of staining for Wisteria floribunda agglutinin (WFA), which labels PNNs, and parvalbumin (PV) in adult mouse primary visual cortex layer IV infused or not with 40 fmol (EC′AC′).sub.6. (b) Quantification of WFA+ cell numbers. (c) Quantification of PV cell numbers. Scale bar=100 μm. All values: N=3-6; mean±SEM; t-test; *P<0.05, **P<0.01, ***P<0.001.

(6) FIG. 5. GAG mimics peptide scaffold of the invention: EC′C′A and EC′AC′, where C′ represents cysteic acid.

EXAMPLE

Materials and Methods

Compounds

(7) The sequences (ECCA)n and (ECAC)n (n=3-6) were synthesized by Fmoc strategy on a Rink amide MBHA resin. A 6-aminohexanoic acid (Ahx) spacer was introduced at the N-terminus and the peptidyl resins were either acetylated (Ac) or acylated with a biotin sulfone (Biot(SO2)). The peptides were cleaved from the resin by TFA and precipitated in diethyl ether to give either Ac-Ahx-(ECAC)n-NH2, Ac-Ahx-(ECCA)n-NH2, Biot(S02)-Ahx-(ECAC)n-NH2, or Biot(S02)-Ahx-(ECCA)n-NH2. These precursors were oxidized with performic acid to give cysteic acid (C′) peptides, then neutralized with aqueous ammonia resulting in GAG mimics (EC′AC′)n and (EC′C′A)n, with either an acetyl or a biotin sulfone at the N-terminus and an amide at the C-terminus. Crude sulfopeptides were desalted on Sephadex G25, purified by reverse phase HPLC and characterized by mass spectroscopy. These GAG mimic peptides can be stored for months at −20° C. as sodium salts.

Dot Blots

(8) For competition assays, 400 pmol of biotinylated ligand or hexaCSE were incubated 30 min at 37° C. in 100 mM ammonium acetate with 1 μg of Otx2 protein (in-house) and with 3 μg of RK-, AA- or SC-peptide. Each solution was then spotted on a nitrocellulose membrane and biotin was detected by 30-min incubation with streptavidin-HRP (ThermoFisher Scientific) followed by chemiluminescence (#34580, ThermoFisher Scientific) reaction. Membranes were digitized with an LAS-4000 (Fujifilm) and quantified by densitometry with ImageJ.

Gel Shift

(9) Otx2 protein (0.1 μg) was incubated at room temperature for 30 min with 40 fmol of biotinylated IRBP1 oligonucleotide and 4 pmol of (EC′AC′).sub.5 or hexaCSE in 50 ng/μl dldC, PBS. Samples were separated on 6% native polyacrylamide gels at 100 V in TBE then transferred onto a nylon membrane at 380 mA for 45 min, crosslinked with UV (120,000 μJ/cm.sup.2, Amersham). The LightShift Chemiluminescent EMSA Kit (#89880, ThermoFisher Scientific) was used for detection and membranes were digitized with an LAS-4000 (Fujifilm) and quantified by densitometry with ImageJ.

Immunoprecipitation

(10) Visual cortex of adult mice were dissected and lysed in homogenization buffer (0.32 M sucrose, 5 mM HEPES, 10 mM MgCl.sub.2 and protease inhibitors). Samples were centrifuged (8 min, 1700 g) at 4° C. and the supernatant was incubated 2 hours at 37° C. with 5 nmol of GAG mimics. For the competition assay, ligands were pre-incubated 30 min with 50 nmol of RK-, AA- or SC-peptide at 37° C. prior to incubation overnight at 4° C. with streptavidin-coupled Dynabeads (Life technologies). The loaded beads were washed with homogenization buffer and heated 10 min at 95° C. in Laemmli buffer (with DTT) to detach proteins for western blot analysis.

Western Blot

(11) Immunoprecipitated proteins were separated on NuPAGE 4-12% Bis-Tris pre-cast gels (Invitrogen) for 1 h at 200 V and transferred onto a methanol-activated PVDF membrane at 400 mA for 1 h. Membranes were blocked with 5% non-fat dry milk for 1 h before incubation with primary antibody anti-Sema3A (rabbit, 1/1000, Millipore) overnight at 4° C. Membranes were washed and incubated 1 h with secondary antibody anti-rabbit HRP-linked (Cell Signaling). Membranes were digitized with an LAS-4000 (Fujifilm) and quantified by densitometry with ImageJ.

Brain Infusions and Immunohistochemistry

(12) Three-month-old C57BL/6J mice (Janvier) were infused for 7 days into V1 (lambda: x=1.7 mm, y=0 mm, z=0.5 mm) with various concentrations of ligands (4 pM, 400 pM or 4 μM), using Alzet micro-osmotic pumps (0.5 μL/h). Animals were then perfused with PBS and 4% paraformaldehyde. Cryostat sections (20 μm) were incubated overnight with primary antibody anti-parvalbumin (rabbit, 1/500, Swant) and WFA-FITC (1/100, Vector), followed by secondary antibody anti-rabbit Alexa Fluor-546 (1/2000, Molecular Probes) for 1 h. Images were acquired with a Leica SP5 confocal microscope and quantified by analysis with ImageJ.

Statistical Analysis

(13) Analysis was performed with Prism 6 (GraphPad). Single comparisons were made by t-test, whereas multiple group analyses were made by ANOVA followed by Bonferonni's posthoc test.

Results

(14) To evaluate affinity of the biotinylated (EC′C′A).sub.n and (EC′AC′).sub.n libraries to Otx2 protein, we performed dot blots with nitrocellulose membranes for which sulfopeptide retention requires interaction with protein (FIG. 1a). While Otx2 binding did not favor one motif over the other, there was a clear increase in affinity as a function of n repeats (FIG. 1b). For comparison, dot blots were performed with hexaCSE, which was previously shown to bind Otx2 and interfere with its in vivo activity in the mouse brain. Sequences with n=4 or 5 repeats were found to bind to Otx2 equally as well as hexaCSE, while those with n=6 where found to bind 2- to 3-fold better.

(15) To confirm the biotinylated ligands interact with Otx2 through its previously identified GAG-binding site, we performed DNA chase and peptide binding experiments (FIG. 2). In Otx2, a GAG-binding motif is located in the first helix of its DNA-binding domain, thus specific binding of GAG molecules may interfere with DNA binding. Assays show the (EC′AC′).sub.5 mimic is able to chase the IRBP1 DNA probe from Otx2 to the same extent as biotinylated hexaCSE positive control (FIG. 2a). The GAG-binding site in Otx2 (RKQRRER) contains an arginine-lysine doublet (RK) which when mutated to an alanine doublet (AA), no longer binds ECM and causes critical period defects in the Otx2.sup.+/AA mouse model. Dot blot assays with peptides (15-mer) based on this motif were used to assess whether (EC′AC′).sub.6 binds specifically (FIG. 2b). While the wild type peptide (RKpep: RKQRRERTTFTRAQL) retained the ligand, the mutated peptide (AApep: AAQRRERTTFTRAQL) did not; neither did a scrambled peptide (SCpep: RTQTRFRTRARLEQK) containing the same residues as the RKpep but in random order (FIG. 2c). These assays confirm the interaction is not simply electrostatic but requires a specific residue sequence.

(16) To measure the in vivo activity and specificity of biotinylated GAG mimics, we performed biochemical and immunohistochemical analyses. Otx2 and Semaphorin-3A (Sema-3A) are both keys actors of visual cortex plasticity and share a similar motif for binding CS-E (FIG. 3a). We first focused on Sema-3A, as cortical Otx2 levels are too low to be reliably detected biochemically. By using ligands in pull-down experiments with lysates of adult mouse visual cortex, we found that (EC′AC′).sub.5 and (EC′AC′).sub.6 but not (EC′C′A).sub.6 interact with Sema-3A (FIG. 3b-c). The RKpep specifically disrupts Sema-3A interaction with (EC′AC′).sub.6 in these lysates (FIG. 3d-e), suggesting involvement of Sema-3A GAG-binding motif.

(17) After 7-day infusion of either (EC′AC′).sub.5 or (EC′AC′).sub.6 in adult mouse visual cortex, 40 fmol caused a reduction in PNN assembly (FIG. 4a-b). Infusion with up to 10.sup.5-fold more of either mimic provided the same reduction. A feedback loop exists between Otx2 accumulation in PV cells and assembly of surrounding PNNs; PNNs attract extracellular Otx2 while Otx2 activity in PV cells increases PNN expression. These results suggest that these ligands interfere with Otx2 signaling enough to break this feedback loop and diminish PNN assembly. Furthermore, interfering with Otx2 signaling in the adult visual cortex has been shown to reverse PV cell maturation state and induce plasticity. Here, only infusion of (EC′AC′).sub.6 (>40 fmol) resulted in significantly reduced PV expression (FIG. 4c). This modest reduction (>25%) has been previously shown to be sufficient for re-opening brain plasticity.

(18) These pull-down and infusion experiments confirm in vitro findings that the longer ligands have higher affinity for GAG-binding proteins; only when n=6 for the (EC′AC′).sub.n mimic do we observe full effects on PV cell maturation (FIG. 4c). They also suggest that (EC′AC′).sub.n and (EC′C′A).sub.n mimics can provide specificity and selectivity. While Otx2 shows no preference for either motif in vitro (FIG. 1b), Sema-3A interacts with the (EC′AC′).sub.n motif but not the (EC′C′A).sub.n motif in brain lysates (FIG. 3c). Thus, these GAG mimics contain valid electrostatic patterns to recapitulate specific sulfation patterns present in natural GAGs.