Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention concerns new peptides derived from the netrin-like domain of the FRZB protein, that are useful in the prevention or treatment of Wnt/b- catenin and/or Hippo/YAP/TAZ pathway-related diseases, in particular of osteoarthritis.
• references in square brackets ([ ]) refer to the list of references at the end of the text.
• the synovial joints are specialized structures that connect the bones and support movement while at the same time restricting the range of motion between the different skeletal elements.
• the bony bearings are capped with a thin layer of hyaline or articular cartilage.
• the joint cavity in between is lined by the synovial membrane.
• these different tissues allow smooth movement with very low-friction and sufficient lubrication between joint surfaces.
• Homeostasis of the joint, in particular of the articular cartilage and subchondral bone is essential for maintenance of joint function and critically dependent on the balance between anabolic and catabolic signaling pathways (Lories and Luyten, 2011 ) [1 ].
• the homeostasis requires maintenance of the stable chondrocyte phenotype that characterizes the articular cartilage, controlled by soluble factors and extracellular matrix (ECM), under the form of either whole or cleaved proteins.
• ECM extracellular matrix
• OA osteoarthritis
• OA is a disease with a complex aetiology to which both genetic and acquired or environmental factors contribute (Valdes and Spector, 2008) [3] These factors interact and determine the onset, progression and outcome of the disease.
• the identification of genetic factors that are associated with OA also conveys important information about pathways that are important for skeletal development or for maintenance of homeostasis in the adult joint tissues and identifies therapeutic targets to maintain joint homeostasis. Polymorphisms in susceptibility genes are associated with OA without causing overt skeletal abnormalities.
• FRZB a natural Wnt modulator.
• CCDFRZB cysteine-rich domain
• NTNFRZB Netrin-like domain
• mice deficient for FRZB underwent a significant modification of their transcriptome with an increase in the activity of the canonical Wnt pathway (Lodewyckx et al., 2012) [5]
• Dickopf-related protein-1 Dkk1 , a Wnt antagonist
• Dkk1 a Wnt antagonist
• the canonical Wnt pathway, involving b-catenin activation contributes to the control of cartilage homeostasis.
• Calmodulin-Kinase II Calmodulin-Kinase II
• the size of the whole protein FRZB which has already been shown to be effective in modulating the signalling pathways involved in osteoarthritis (as well as its truncated form containing only the netrin-like domain), is 34 kDa. Because of its large size, this protein or its truncated form may be immunogenic in in vivo experiments. This is why it may be interesting to derive peptides from the protein FRZB having the same or similar properties (biological activity) than the whole protein in order to reduce the risk of an immune system reaction.
• the inventors have unexpectedly identified new peptides derived from the netrin-like domain of the FRZB protein having the ability to decrease the expression of the Wnt/p-catenin canonical pathway that is overexpressed in osteoarthritis, decrease the expression of the Hippo/YAP/TAZ pathway involved in the pathophysiology of osteoarthritis, and increase the expression of the non-canonical Cam Kll pathway having a protective role for the joint.
• the peptides were originally designed with the idea that in the sequence of the netrin-like domain of the FRZB protein, there may be elements suggesting an alpha- helix structuring, allowing the interaction of said domain with other protein partners. Therefore, two peptide sequences PEP12 (ATQKTYFRNNYN, SEQ ID NO: 4) and PEP16 (DRLGKKVKRWDMKLRH, SEQ ID NO: 2) derived from the netrin-like domain and corresponding to this criterion, have been tested. Flowever, it turned out that PEP12, although chosen by computer prediction to have a similar secondary, alpha- helix conformation, to that of PEP16, was totally ineffective in the tested systems described below, unlike PEP16. It then appeared that deriving active peptides from the netrin-like domain of the FRZB protein was not as obvious as expected.
• PEP16 is a peptide which sequence is predicted to have a propensity to form an alpha-helix, and which has a role in the regulation of the canonical (Wnt/p-catenin) and non-canonical (CamKII) Wnt pathways: it decreases activation of the canonical pathway which is over-expressed in osteoarthritis and increases activation of the non- canonical pathway having a protective role for the joint.
• PEP16 is capable of reducing the activation of the Flippo/YAP/TAZ pathway notably involved in the physiopathology of osteoarthritis.
• PEP16 can be vectorised with an addressing sequence specific for binding to collagen 2a1 (col2a1 ).
• the inventors derived mutated sequences from the PEP16 peptide to promote this alpha-helix conformation.
• the inventors have also included among the five sequences tested, a mutant sequence without the tryptophan residue that could be strongly linked to the activity of PEP16 (binding). This mutant sequence might then constitute a negative control of the activity, as the steric hindrance of this sequence could constitute a more selective binding via the tryptophan residue.
• the mutations of the hydrophobic amino acids into alanine made it possible to respect the predicted helicoidal shape, while favouring the turns of the helix because alanine has this structural property, therefore potentially increasing the rigidity of the helix.
• the inversions of lysine to arginine allowed to preserve the notion of electric charge of the peptide, and its total global charge, while altering its primary sequence, to elucidate a possible effect which would be solely due to the electric charge.
• the five mutant sequences are as follows (in bold the variations from PEP16): DRAGKKVKRWDMKARH (SEQ ID NO: 3)
• PEP16bis DRAGKKVKRWDMKARH, SEQ ID NO: 3
• PEP16bis DRAGKKVKRWDMKARH, SEQ ID NO: 3
• an object of the present invention concerns a peptide of 16 to 50 amino acids comprising a peptide having the following sequence :
• DRX3GKKVKRWDMKX14RH (SEQ ID NO: 1 ) wherein X3 and X14 are independently of each other any amino acid.
• X3 and Xi4 are independently of each other an alanine, a leucine, an isoleucine or a serine.
• the peptide of the present invention comprises or consists of the sequence SEQ ID NO: 2 (DRLGKKVKRWDMKLRH), the sequence SEQ ID NO: 3 (DRAGKKVKRWDMKARH), DRIGKKVKRWDMKIRH (SEQ ID NO: 9) or DRSGKKVKRWDMKSRH (SEQ ID NO: 10).
• Another object of the present invention concerns a peptide of the present invention for use as a drug.
• Another object of the present invention concerns a pharmaceutical composition
• a pharmaceutical composition comprising a peptide of the present invention, and one or more pharmaceutically acceptable excipient.
• Another object of the present invention concerns a peptide or pharmaceutical composition of the present invention for use in the prevention or treatment of a Wnt/b- catenin and/or Hippo/YAP/TAZ pathway-related disease selected from the group consisting of: osteoarthritis, osteoporosis and cancer.
• a Wnt/b- catenin and/or Hippo/YAP/TAZ pathway-related disease selected from the group consisting of: osteoarthritis, osteoporosis and cancer.
• it concerns a peptide or pharmaceutical composition of the present invention for use in the prevention or treatment of osteoarthritis.
• Figure 1 represents alterations of the articular chnodrocyte’s phenotype in the course of OA.
• the phenotype biomarkers are indicated around the cells, and the main modulators of phenotype are indicated inside the cells. + : stimulating effect, - : inhibiting effect.
• Figure 2 represents the relative expression level of (A) aggrecan, (B) col2a1, (C) ctgf, (D) mmp3 and (E) mmp13, at day 1 , 7, 14, 21 and 28 in ATDC5 micromasses, treated with the PEP16 at 30, 100, 300 ng/mL (respectively, P30, P100 and P300), compared to a non-treated control (CTL). Results were normalized with the housekeeping gene ppia. * P ⁇ 0.05. Results are representative of 3 experiments.
• Figure 3 represents the relative expression level of (A) aggrecan, (B) col2a1, (C) mmp3, (D) mmp13, and (E) ankrd at day 1 , 21 and 28 in ATDC5 micromasses treated with the PEP 16 and PEP16bis at 300 ng/mL, compared to a non-treated control (CTL). Results were normalized with the housekeeping gene ppia. * P ⁇ 0.05. Results are representative of 2 experiments (3 experiments in (E)).
• Figure 4 represents glycosaminoglycans quantification in ATDC5 micromasses treated with the PEP16 and the PEP16bis at 300 ng/mL and compared to a non-treated control (CTL). Alcian blue staining has been performed at day 28. Results are expressed in absorbance (650nm) after a biochemical quantification. * P ⁇ 0.05. Results are representative of 2 experiments.
• Figure 5 represents transcriptionnal activity of promoters of interest evaluated using constructs containing the firefly luciferase gene under the control of (A) 7X TCF/LEF response elements (Super 8x TOPFIash reporter) in ATDC5 micromasses treated one day after transfection with the PEP16 or the PEP16bis at 300 ng/mL, and simultaneously the Wnt3a at 100 ng/mL, for 24h, (B) 8X YAP reponse elements (8x GTIIC reporter) or (C) 7X TCF/LEF response elements (Super 8x TOPFIash reporter) in ATDC5 micromasses treated one day after transfection with the PEP16 at 3, 30, 100, 300 ng/mL (respectively P3, P30, P100 and P300), and simultaneously with the Wnt3a at 100 ng/mL, for 24 h.
• TK-RL tyrosine kinase promoter
• Figure 6 represents glycosaminoglycans quantification (A) with computer program (blue pixels quantification) (B) with biochemical quantification (solubilisation of the staining in Guanidine HCI 6M), in ATDC5 micromasses treated with the PEP16 at 30, 100 and 300 ng/mL (respectively, P30, P100 and P300) and cultured during 28 days in comparison of a non-treated control (CTL). Alcian blue staining was performed at day 28. * P ⁇ 0.05. Results are representative of 3 experiments.
• Figure 7 represents western blot exploring (A) the Wnt/p-catenin pathway activation (B) the Camkll pathway activation, in ATDC5 micromasses at day 1 , 7, 14, and 21 after treatment with the PEP16 at 100 and 300 ng/mL in comparison to a non- treated control (CT or CTL). Results are expressed in active protein form over total protein form. Results are representative of 3 experiments.
• Figure 8 represents western blot exploring (A) the Wnt/p-catenin pathway activation (B) the CamKII pathway activation, in ATDC5 micromasses at day 21 and 28 after treatment with the PEP16 or the PEP16bis at 300 ng/mL in comparison to a non-treated control (CT or CTL). Results are expressed in active protein form (i.e. P- CamKII for (B)) over total protein form. Results are representative of 3 experiments.
• Figure 9 represents MMP13 enzymatic activity in culture supernatants of mouse articular chondrocytes treated during 24h with PEP16 at 300 ng/mL and/or with IL1 b at 100 pg/mL and compared to a non-treated control (CTL).
• CTL non-treated control
• IL1 b was used as a positive control for MMP13 activity. * P ⁇ 0.05.
• Figure 10 represents the relative expression level of (A) aggrecan, (B) col2a1, at 24 and 48h in mouse articular chondrocytes treated with the PEP16 at 300 ng/mL and/or wnt3a at 100 ng/mL, compared to a non-treated control (CTL). Results were normalized with the housekeeping gene ppia. Results are representative of 3 experiments.
• Figure 11 represents the relative expression level of (A, B) mmp3 and (C) ankrd, in mouse articular chondrocytes treated with the PEP16 or PEP16bis at 300 ng/mL, and/or wnt3a at 100, 50, 30 ng/mL, compared to a non-treated control (CTL). Results were normalized with the housekeeping gene ppia. * P ⁇ 0.05. Mmp3 expression was monitored after 24h of stimulation, while ankrd expression was monitored after 12h of stimulation. Results are representative of 3 experiments.
• Figure 13 represents the influence of PEP16 on osteogenic differentiation.
• MC3T3-E1 cells were stimulated with PEP16 at different concentrations for 21 days, and compared to non-treated controls. Results were normalized with the housekeeping gene S29. * P ⁇ 0.05.
• the relative expression level of (A) osteocalcin and osterix was studied, as well as (B) western blot exploring the Wnt/p-catenin pathway and the CamKII pathway activation, and (C) the mineralization level after 21 days of differentiation culture. Results are representative of 3 experiments.
• Figure 14 represents the results of example 3: a) the biochemical quantitation of the extracellular matrix mineralization (absorbance : 405 nm) for CTL, PEP12 300 ng/ml and PEP12 1000 ng/ml, and b) the specific alkaline phosphatase activity (pmol of para-nitrophenol/min/mg of protein) for PEP16 and PEP12, at a concentration of 300 ng/ml, 1000 ng/ml or without any peptide stimulation (CTL), at day 1 (D1 ) and day 21 (D21 ).
• CTL specific alkaline phosphatase activity
• Figure 15 represents qPCR analyses (normalization against PPIA housekeeping gene) performed at D1 and D14 for type II collagen and aggrecan, CTGF and MMP3, on ATDC5 cells cultured as explained in Example 4, with a concentration of 300 ng/ml of peptides PEP16, PEP I or PEP s or without any peptide stimulation (CTL). * indicates a p value ⁇ to 0.05 in comparison to D14 control (CTL), and £ indicates a p value ⁇ to 0.05 in comparison to D1 CTL (ANOVA).
• Figure 16 represents Biochemical Alcian Blue quantitation (Absorbance read at 650 nm after Guanidine HCI 6M dissolution) for PEP16, PEP I and PEP s, as explained in Example 4. * indicates a p value ⁇ to 0.05 in comparison to D14 control (CTL).
• Figure 17 represents qPCR analyses (normalization against PPIA housekeeping gene) performed at D1 and D14 for type II collagen and aggrecan, CTGF and MMP3, on ATDC5 cells cultured as explained in Example 5, with a concentration of 300 ng/ml of PEP16 or PEP50, or 1000 ng/ml (PEP50 molarity condition) or without any peptide stimulation (CTL).
• * indicates a p value ⁇ to 0.05 in comparison to D14 control (CTL), and £ indicates a p value ⁇ to 0.05 in comparison to D1 CTL (ANOVA).
• Figure 18 represents Biochemical Alcian Blue quantitation (Absorbance read at
• Figure 19 represents Western blot membrane and SDS PAGE of FRZB tentative production as explained in Example 6.
• Figure 20 represents Western blot membrane and SDS PAGE of NTN tentative production as explained in Example 6.
• Figure 21 represents qPCR analyses performed at D1 and D14 for ANKRD (YAP/TAZ target gene, indicator of the pathway activity), on MC3T3-E1 cells treated with a concentration of 300 ng/ml of PEP16 or MC3T3-E1 cells stably overexpressing the NTN domain.
• Treated cells and NTN overexpressing cells were compared to control cells (CTL).
• * indicates a p value ⁇ to 0.05 in comparison to D14 control (CTL), and £ indicates a p value ⁇ to 0.05 in comparison to D1 CTL (ANOVA).
• Mouse chondrogenic ATDC5 cells (cartilage-specific chondrogenic strain) were cultured until confluent in maintenance medium containing 1 :1 Dulbecco’s modified Eagle’s medium (DMEM): Ham’s F-12 mix (Gibco) supplemented with 1% antibiotic (AB, Penicillin-Streptomycin, Gibco), 5% fetal bovine serum (FBS) (Gibco) and enriched with 10 pg/ml human transferrin (Sigma) and 30 nM sodium selenite (Sigma).
• DMEM Dulbecco’s modified Eagle’s medium
• Ham Ham’s F-12 mix (Gibco) supplemented with 1% antibiotic (AB, Penicillin-Streptomycin, Gibco), 5% fetal bovine serum (FBS) (Gibco) and enriched with 10 pg/ml human transferrin (Sigma) and 30 nM sodium selenite (Sigma).
• Cells were trypsinized, washed and resuspended at 2.10 7 cells/mL in a chondrogenic medium made of maintenance medium enriched by 1X of ITS premix (resulting in 10 pg/mL insulin, 10 pg/mL human transferrin and 30 nM sodium selenite) (Life technologies).
• a chondrogenic medium made of maintenance medium enriched by 1X of ITS premix (resulting in 10 pg/mL insulin, 10 pg/mL human transferrin and 30 nM sodium selenite) (Life technologies).
• ITS premix resulting in 10 pg/mL insulin, 10 pg/mL human transferrin and 30 nM sodium selenite
• One droplet of cell suspension (10 pL) was placed at the center of each well of a 24-well plate. Cells were allowed to adhere for 2 h at 37°C, followed by addition of 500 pL chondrogenic medium supplemented.
• the cells were cultured in the absence or in the presence of PEP16 (SEQ ID NO: 2) and/or PEP16bis (SEQ ID NO: 3) at different concentrations (30 ng/mL, 100 ng/mL and/or 300 ng/mL).
• PEP16 SEQ ID NO: 2
• PEP16bis SEQ ID NO: 3
• concentrations 30 ng/mL, 100 ng/mL and/or 300 ng/mL.
• induction of hypertrophic differentiation and mineralization was induced by the mineralization medium made of a-MEM medium (Gibco) containing 1 % AB, 5% FBS, 5 pg/ml human transferrin, 1X of ITS premix, 50 pg/ml ascorbic acid- 2-phosphate (AA) (Sigma) and 10 mM b-glycerophosphate (BGP) (Sigma).
• Mouse osteogenic MC3T3-E1 cells (bone-specific osteogenic strain) were cultured until confluent in maintenance medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 1 % AB (Gibco), 10% FBS (Gibco) and enriched with 1 % sodium pyruvate (Gibco). Cells were trypsinized, washed and then seeded at 2600 cells/cm 2 in 6-well plates.
• DMEM Dulbecco’s modified Eagle’s medium
• MC3T3-E1 differentiation was initiated the day after (D1) by culturing cells for 21 days in a-Minimal Essential medium (a-MEM, Gibco), supplemented with 10% FBS, 1% AB, 1 % sodium pyruvate, 10 mM BGP and 50 pg/ml AA.
• a-MEM a-Minimal Essential medium
• the cells were cultured in the absence or in the presence of PEP16 (SEQ ID NO: 2) at different concentrations (100 ng/mL, 300 ng/mL or 1000 ng/ml). Cells were maintained in a humidified atmosphere of 5% C02 at 37°C. Cells were collected at time points 1 , 7, 14 21 days. Each time point was processed in three technical replicates. The medium was changed every two days. Readouts included mineral content, signaling pathways activation status and genes of interest evaluation. The genes of interest were osteognic differentiation markers (osteocalcin, osterix).
• MACs Mouse articular chondrocyte
• Explants of human articular cartilage were obtained from total knee replacement surgeries, from the orthopedic department from the CFIRU of Nancy (Pr. Didier Mainard). Samples were washed three times in 1 % AB/PBS and incubated for 24 h in growth medium (DMEM-F12, containing 1% AB (Gibco), 10% FBS (Gibco) and 5 % L- glutamine (Thermoscientific). Explants were then stimulated with PEP16 at the concentration of 300 ng/ml during 7 days. The culture supernatants were then harvested in order to perform MMP13 enzymatic activity assay.
• DMEM-F12 growth medium
• PEP16 5 % L- glutamine
• ATDC5 cells were transfected with either the YAP/TAZ firefly luciferase reporter plasmid (8x GTIIC, Plasmid #34615, Addgene), the b-catenin payhway reporter plasmid (Super 8x TOPFIash, Plasmid #12456, Addgene) or the inactive b-catenin control reporter plasmid (Super 8x TOPFIash, #12457, Addgene). Results were normalized by-cotransfecting each of the above plasmid with the tyrosine kinase-renilla luciferase plasmid (Promega). Transfections were carried out using the Dharmafect kb transfection reagent (Fisher Scientific).
• reporter plasmids and transfection reagent were diluted in serum-free medium, and then the diluted Dharmafect kb was added to the plasmids for an incubation time of 10 minutes at room temperature. During that time, cells were rinsed with PBS and were placed in fresh maintenance medium. When incubation was over, the mixture of transfection reagent-plasmids was dispensed in each well. Stimulations (with Wnt3a and/or PEP16 and/or PEP16bis) were carried out the next day.
• RNA extraction cDNA synthesis and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) (to see expression of healthy and hypertrophic chondrocyte markers)
• Results were expressed using the comparative threshold method and were normalized to housekeeping gene PPIA (Peptidylprolyl Isomerase A) mRNA level for ATDC5 cells and MACs, or normalized to housekeeping gene S29 (ribosomal protein S29) mRNA level for MC3T3-E1 cells.
• PPIA Protidylprolyl Isomerase A
• S29 ribosomal protein S29
• Micromasses were washed with PBS and fixed with 95% ice-cold ethanol for 30 min at 4°C for staining. After washing with distilled water, micromasses were stained with Alcian Blue (0.1 % Alcian Blue 8GX, (Sigma)), washed three times with distilled water to remove unbound staining and air-dried. Quantification of the staining was performed by blue pixel quantitation, using the ImageJ software (NIH Image, National Institutes of Health, Bethesda, MD, USA), and then by dissolving the micromasses with 6M Guanidine-HCI (Sigma) and by measuring the absorbance at 650 nm with a spectrophotometer (Varioskan Flash, Thermo Fisher Scientific).
• Alcian Blue 0.1 % Alcian Blue 8GX, (Sigma)
• tubes were centriguated at 13000 rpm for 15 minutes, the liquid phase was harvested and mixed with 100 mI of 10% ammonium hydroxide. Absorbance was measured at 405 nm with a spectrophotometer (Varioskan Flash, Thermo Fisher Scientific).
• Proteins were isolated from the micromasses using 1x Laemmli Buffer (2%SDS, 10% glycerol, 5% 2-betamercaptoethanol, 0.002% bromophenol blue, 125 mM Tris HCI (pH 6.8)). Samples were denatured for 5 min at 95°C, chilled on ice and separated on a Tris-Glycine extended gradient gel 4-20% (Biorad) by electrophoresis using a migration buffer, containing 192 mM glycine, 20 mM Tris Base, 0,1 % SDS. (Invitrogen).
• PVDF polyvinylidine difluoride
• BioRad Trans-blot turbo system
• the MMP13 enzymatic activity was assessed using the MMP13 fluorogenic substrate (Merck Millipore). Briefly, culture supernatants harvested at the end of the different experiments were mixed with 1.5 mM of 4-aminophenylmercuric acetate, and incubated for 1 h at 37°C, in order to turn every pro-MMPs into active MMPs. Then, the fluorogenic substrate was added, and the mixture was incubated for 3 h at 37°C. Fluorescence was evaluated using the following parameters, excitation 325 nm and emission 393 nm, on the Varioskan Flash device (Thermo Fisher Scientific). Recombinant human MMP13 was used as a positive control (Merck Millipore). Negative control consisted in growth medium.
• PEP16 significantly enhanced the induction of the expression of markers of the healthy joint cartilage phenotype during ATDC5 chondrogenesis (aggrecan and type 2 collagen). Moreover, PEP16 efficiently reduced the expression of osteoarthritis-related MMPs, i.e. MMP3 and MMP13, as well as the expression of Ctgf, a target genes of the Flippo/YAP/TAZ pathway. In addition, PEP16 significantly increased the accumulation of glycosaminoglycans in the extracellular matrix during chondrogenesis. Finally, PEP16 increased the activation of the CamKII pathway in the pre-mineralization phase of chondrogenesis (D14), while it reduced in the activation of the Wnt/p-catenin pathway during chondrogenesis.
• PEP16bis also favoured the induction of the expression of markers of the healthy joint cartilage phenotype during ATDC5 chondrogenesis (aggrecan and type 2 collagen). Moreover, PEP16bis efficiently reduced the expression of osteoarthritis-related MMPs, i.e. MMP3 and MMP13, as well as the expression of Ctgf, a target gene of the Hippo/YAP/TAZ pathway. In addition, PEP16bis significantly increased the accumulation of glycosaminoglycans in the extracellular matrix during chondrogenesis.
• PEP16 and PEP16bis at 300 ng/ml were able to significantly reduce the activity of the canonical pathway in the Super TOPFIash luciferase reporter system (ATDC5 cells transfected with the reporter plasmid, and stimulated with 10 or 100 ng/mL of recombinant Wnt3a protein which activates the system), while the other four sequences are unable to do so.
• PEP16 at 30 ng/ml significantly reduced the activity of the Hippo/YAP/TAZ pathway in a luciferase-type reporter system (ATDC5 cells transfected with the reporter plasmid of the YAP pathway, which activates spontaneously on contact with the plastic).
• PEP16bis increased the activation of the CamKII pathway, while it reduced in the activation of the Wnt/p-catenin pathway during chondrogenesis.
• the results obtained show a protective role of the peptides under test.
• PEP16 significantly reduced the ILi p-induced MMP13 activity measured in the culture supernatant of mouse articular chondrocytes.
• PEP16 had a tendancy to counteract the deleterious effects of b-catenin activation by Wnt3a, on the decrease of markers of the healthy joint cartilage phenotype (aggrecan and type 2 collagen), and on the increase of NGF (pain, data not shown).
• the peptides under test are also able to counteract the deleterious effects induced by the use of the recombinant Wnt3a, in particular, PEP16 counteract the inductive effects of Wnt3a on the expression of MMP3.
• PEP16 and PEP16bis are capable of reducing the expression of Ankrd, a target gene of the Hippo/YAP/TAZ pathway in this primary culture system. A similar trend is observed for Cyr61 and Ctgf, two other target genes of the Hippo/YAP/TAZ pathway.
• MC3T3-E1 cells were differentiated during 21 days as mentioned previously. A concentration of 300 ng/ml of different peptides (PEP12 or PEP16) was used and introduced at each culture medium change (every two days). Treated cells were compared to cells without any peptide stimulation (CTL).
• CTL peptide stimulation
• the mineralization level was evaluated by a biochemical assay as previously described after 21 days of differentiation culture. Results are representative of 3 experiments.
• Alkaline phosphatase activity was measured by placing the harvested supernatants in para-nitrophenyl phosphate (pNPP) solution. The alkaline phosphatase will cleave this substrate and generate para-nitrophenol (pNP).
• pNPP para-nitrophenyl phosphate
• a solution of 4 mg/mL of pNPP is prepared in buffer A (0.1 M glycine pH 10.5, 0.11 % (m/v) Zinc acetate and 0.011 % magnesium chloride (m/v)).
• buffer A 0.1 M glycine pH 10.5, 0.11 % (m/v) Zinc acetate and 0.011 % magnesium chloride (m/v)
• buffer B 0.24% (m/v) cobalt chloride prepared in buffer A.
• Standard curve of pNP was prepared using a commercial preparation of 10 mM pNP diluted 1/20 (0.5 mM pNP) in buffer A : 0 ; 0.01 ; 0.02 ; 0.04 ; 0.05 ; 0.075 ; 0.1 et 0.2 mM.
• Results are representative of 1 experiment for PEP12 and 2 experiments for PEP16.
• PEP12 was not able to increase extracellular matrix mineralization during osteogenesis, and was not able to increase the alkaline phosphatase activity.
• PEP16 tended to increase the alkaline phosphatase activity (see Figure 14).
• PEP16 with two substitution i.e. PEP i, where L in position 3 and 14 were changed to an Isoleucine (I) (corresponding to SEQ ID NO: 9), and PEP s, where L in position 3 and 14 were changed to a Serine (S) (corresponding to SEQ ID NO: 10), where tested.
• I Isoleucine
• S Serine
• ATDC5 cells were cultured as micromasses and differentiated during 14 days using ITS as mentioned previously. A concentration of 300 ng/ml of different peptides (PEP16, PEP I or PEP s) was used and introduced at each culture medium change (every day). Treated cells were compared to cells without any peptide stimulation (CTL). RNA was isolated and qPCR were performed for different target genes, i.e. type II collagen and aggrecan (articular chondrocyte extracellular matrix), CTGF (YAP/TAZ target genes, indicators of the pathway activity) and MMP3 (cartilage degrading enzyme increased during osteoarthritis) (Figure 15). Alcian blue staining was performed on the micromasses cultures, and biochemical quantitation was performed as previously described ( Figure 16).
• PEP50 50 amino acid long peptide
• ATDC5 cells were cultured as micromasses and differentiated during 14 days using ITS as mentioned previously. A concentration of 300 ng/ml of PEP 16 or PEP50, or 1000 ng/ml (PEP50 molarity condition) was used and introduced at each culture medium change (every day). Treated cells were compared to cells without any peptide stimulation (CTL). RNA was isolated and qPCR were performed for different target genes, i.e. type II collagen and aggrecan (articular chondrocyte extracellular matrix), CTGF (YAP/TAZ target gene, indicator of the pathway activity) and MMP3 (cartilage degrading enzyme increased during osteoarthritis) (Figure 17). Alcian blue staining was performed on the micromasses cultures, and biochemical quantitation was performed as previously described (Figure 18).
• Results are described below. * indicates a p value ⁇ to 0.05 in comparison to D14 control (CTL), and £ indicates a p value ⁇ to 0.05 in comparison to D1 CTL (ANOVA).
• the PEP 50 condition yielded very similar results compared to those obtained using PEP16, i.e., an increase in articular chondrocyte extracellular matrix genes, and a significant decrease in YAP/TAZ target genes and in MMP3.
• the PEP50 molarity condition demonstrated that, when used at the same molar concentration than PEP16, PEP50 significantly reduced articular chondrocyte extracellular matrix genes expression. Flowever, it kept its ability to significantly reduce MMP3 and CTGF expression. Each peptide was able to increase the proteoglycans deposition in the extracellular matrix.
• EXAMPLE 7 NTN DOMAIN OVEREXPRESSION IS NOT ABLE TO MIMIC PEP16 IN REGULATING THE YAP/TAZ PATHWAY
• MC3T3-E1 cells were differentiated during 14 days as mentioned previously. A concentration of 300 ng/ml of PEP16 was used and introduced at each culture medium change (every two days). In the same experiment, we also used MC3T3-E1 cells stably overexpressing the NTN domain. Treated cells and NTN overexpressing cells were compared to control cells (CTL).
• CTL control cells
• Results are described below. * indicates a p value ⁇ to 0.05 in comparison to D14 control (CTL), and £ indicates a p value ⁇ to 0.05 in comparison to D1 CTL (ANOVA).

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  • 2020-05-18 EP EP20315243.4A patent/EP3912991A1/en not_active Withdrawn
• 2021
  • 2021-05-18 CA CA3178537A patent/CA3178537A1/en active Pending
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  • 2021-05-18 EP EP21725546.2A patent/EP4153611A1/en active Pending
  • 2021-05-18 JP JP2022571252A patent/JP2023541087A/ja active Pending
  • 2021-05-18 CN CN202180035631.5A patent/CN115996944A/zh active Pending

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