Novel inhibitors of Fos phosphorylation by ERK1 / 2

Abstract

The present invention describes novel synthetic peptides which are inhibitors of c-Fos phosphorylation by ERK1 / 2 kinases. A pharmaceutical composition containing said peptides and their use as medicaments are also described. A method for identifying novel inhibitors of c-Fos phosphorylation by ERK1 / 2 is also described.

Description

Title of the invention: Novel inhibitors of Fos phosphorylation by ERK1 / 2 Technical field

[0001] The present invention describes novel synthetic peptides which are inhibitors of c-Fos phosphorylation by ERK1 / 2 kinases. A pharmaceutical composition containing said peptides and their use as medicaments are also described. A method for identifying novel inhibitors of c-Fos phosphorylation by ERK1 / 2 is also described. TECHNOLOGICAL BACKGROUND

[0002] MAP kinases (for "Mitogen-Activated Protein Kinase") ERK1 and ERK2 (ERK for "Extracellular signal-Regulated Kinase") are enzymes whose function is to add phosphate groups to target proteins in order to activate them. They are ubiquitous proteins involved in many cellular functions. They play a key role in the transduction of intracellular signals. Their activation is achieved by extracellular signals that act on membrane receptors to induce the cascade activation of different intracytoplasmic actors, upstream of ERK1 / 2, in particular the Ras, Raf and MEK proteins; this is also referred to as the Ras-Raf-MEK1 / 2-ERK1 / 2 signaling pathway. ERK-type MAP kinases have many intracellular, membrane, cytoplasmic or nuclear substrates (Yang et al., Trends Pharmacol. Sci.2019 Nov; 40(11): 897-910) and it is the amplitude and duration of the activations of the intracellular signal cascade that will direct ERK1 / 2 towards one of its substrates.

[0003] In human cancers, the Ras-Raf-MEK1 / 2-ERK1 / 2 signaling pathway appears to be one of the most disrupted transduction pathways. Thus, following mutations in the Ras, Raf or MEK proteins encountered in a wide variety of cancers, aberrant ERK1 / 2 activity leads to anarchic cell proliferation and mobility. It is therefore logical to develop inhibitors of this signaling pathway in order to slow down or even stop this disruption.

[0004] Many ERK-type MAP kinase inhibitors have been developed. They generally target the Ras-Raf-MEK1 / 2-ERK1 / 2 pathway upstream of ERK, which completely blocks its activity and its binding to its various downstream substrates. Examples include PD98059 (2'-amino-3'-methoxyflavone) and U0126 (1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene), Trametinib (GSK1120212), Cobimetinib (GDC0973), Mirdametinib (PD0325901) or Selumetinib (AZD6244), which are specific MEK inhibitors.

[0005] Specific inhibitors of kinases upstream of ERK-type MAP kinases block the Ras-Raf-MEKl / 2-ERKl / 2 signaling pathway, but since their action is upstream of ERK-type MAP kinases, this results in complete inhibition of ERK-type MAP kinases, and consequently of all downstream substrates, without discrimination between them and without distinction of their cellular location.

[0006] Being able to act at the level of the interaction between the ERK-type MAP kinase and its substrate, therefore downstream of the ERK intracellular signaling pathway, makes it possible to block the ERK signaling pathway specifically while allowing its other functions, in particular on normal cellular homeostasis.

[0007] In EPI693458 the inventors have demonstrated synthetic peptides allowing the selective inhibition of the ERK-type MAP kinase pathway towards a given substrate. The different substrates of ERK1 / 2 have consensus sequences called binding or docking sites, known to those skilled in the art, which allow the binding of ERK1 / 2 to its substrates. These sequences are known under the terms, DEF and / or FXFP and D or LXL (see EP1693458).

[0008] The DEF binding domain of ERK1 / 2-specific substrates is responsible for recruiting ERK1 / 2 to immediate early genes and transcription factors (see EP1988169). This DEF binding domain can bind peptides having a consensus sequence of the type FXFP or (F / Y)X(F / Y)P in which F denotes Phenylalanine (Phe), Y denotes Tyrosine (Tyr), P denotes Proline (Pro), and X any naturally occurring amino acid (Sharrocks et al., Trends Biochem Sci. 2000 Sep; 25(9): 448-53).

[0009] The D domain is essential for the activation of different series of substrates, including RSK (for 90 kDA ribosomal S6 kinase) and phosphatases (MAP kinases Phosphatases or MPK). It is also the binding site of MEK1 / 2 which are located upstream of ERK1 / 2 in the intracellular signaling cascade.

[0010] Many teams are developing MEK1 / 2 inhibitors with the aim of providing therapeutic solutions in various pathologies, particularly in oncology, central nervous system disorders and inflammation. Examples of MEK1 / 2 inhibitors that are or have been studied during clinical trials include Selumetinib, Refametinib, Cobimetinib, Trametinib (Samatar et al., Nat Rev Drug Discov . 2014 Dec; 13(12): 928-42).

[0011] These MEK1 / 2 inhibitors have a global effect on the inhibition of ERK1 / 2 and its substrates and thus have a pleiotropic, non-specific and deleterious effect. Ongoing clinical trials show in particular that the side effects are numerous depending on the doses administered and that resistance to the drug develops.

[0012] Peptides containing the DEF domain of a specific substrate downstream of ERK inhibit the binding and phosphorylation of said substrate without impacting other activities of ERK1 / 2 or the phosphorylation of other substrates. There are therefore much fewer side effects of this inhibition in the transduction of the intracellular ERK1 / 2 signal.

[0013] Patent EP1988167 describes the use of TAT-DEF type peptides, capable of penetrating into cells, as specific inhibitors of substrates downstream of ERK. Summary of the invention

[0014] The present invention describes novel peptides which are inhibitors of Fos phosphorylation by ERK 1 / 2 and which are derived from those described in EP1988167. These novel synthetic peptides are modified to make them more stable and more effective than those described in EP1988167.

[0015] The inventors used 3D in silico modeling of the binding pocket of the ERK2 anchoring domain to substrates containing the DEF domain so as to be able to position known ligands, in particular TAT-DEF-Fos peptides according to patent EP1988169 and characterize their interaction with this binding pocket. This makes it possible to define the essential and non-essential positions in the binding of known peptides, in particular TAT-DEF-Fos type peptides to the DEF anchoring domain of ERK2 and to optimize potential inhibitors of Fos phosphorylation by ERK1 / 2. This study makes it possible to optimize peptides containing TAT-DEF-Fos domains (SEQ ID No. 1) either by stabilization and in particular by D-racemization of the amino acids not essential for binding, or by deletion of the amino acids not essential for binding, or to combine these different modifications, and finally to envisage new inhibitors.

[0016] In the present description, reference is made to the intracellular signaling pathway Ras-Raf-MEKl / 2-ERKl / 2 or Ras-Raf-MEK-ERK or ERK or ERK1 / 2. These terms are equivalent and refer to the same signaling pathway or cascade.

[0017] The term "docking method" refers to the method that calculates the preferred orientation of one molecule toward a second when they are bound to form a stable complex. Determining this preferred orientation is used to predict the strength of the bond between two molecules and possibly its effectiveness. Docking, fixation, anchoring or docking studies are therefore used to determine the affinity between the molecules studied and to quantify the consequences of the binding of a ligand to a receptor. The terms fixation method, anchoring method or docking method can be used to refer to the docking method. DESCRIPTION OF DRAWINGS

[0018] [Fig.l] describes the in silico modeling of the interaction between the DEF-Fos domain of MLK1001 and the binding pocket of the ERK2-DEF domain of the active kinase. This modeling allows the visualization of the amino acids essential for said interaction. The amino acids represented start at the first amino acid. [Fig.2] describes the antiproliferative properties of TAT-DEF-Fos inhibition by MLK1001 on different human tumor cell lines. Cell proliferation is measured by BrdU incorporation in the presence of bovine serum. [Fig.3] represents the antiproliferative properties of the new peptides according to the invention on HPAF-II pancreatic carcinoma cells. [Fig.4] depicts cell cycle progression by Ki-67 immunostaining on HPAF-II cells after addition of serum (10%) in the presence of Selumetinib (MEK inhibitor, reference product), MLK-1001 peptide and some of the novel peptides according to the invention. [Fig.5] describes the surface plasma resonance (SPR) method by Biacore, to determine the interaction levels and affinities of certain peptides (MLK1001 and MLK1008) according to the invention. [Fig.6A] describes the kinetics of association and dissociation of the MLK1001 peptide according to the invention at 1.25; 2; 5; 10 and 20 nM on the ERK2 protein by the Biacore method. [Fig.6B] describes the kinetics of association and dissociation of the MLK1008 peptide according to the invention at 1.25; 2; 5; 10 and 20 nM on the ERK2 protein by the Biacore method. These figures illustrate the present invention and are not limiting of the claimed scope. DETAILED DESCRIPTION OF THE INVENTION

[0019] The docking experiments were carried out with the peptide MLK1001 of SEQ ID No. 1 as a ligand for the ERK2-DEF domain. The in silico modeling method made it possible to precisely study the interactions between MLK1001 of SEQ ID No. 1 and the DEF-Fos type ligand binding pocket. From this experiment, new compounds were designed and synthesized. These are described in Table No. 1 below. Compared to MLK1001 of SEQ ID No. 1, they are either shortened, stabilized, or both.

[0020] In patent EP1988167 the inhibitory peptides were designed as follows: they contain a sequence rich in basic amino acids of the HIV-TAT protein followed by two prolines (PP) and then by the DEF binding domain of Fos flanked by amino acids.

[0021] The results of the in silico 3D modeling experiment from the positioning of TAT-DEF-Fos of SEQ ID No. 1 in the DEF binding pocket of the ERK2 kinase active have identified the essential amino acids for binding of the DEF-Fos and ERK2 domains. These essential amino acids form a beta sheet on ERK2 and exclude an amino acid (T) from the FVFT sequence (consensus FXFP binding site sequence) of c-Fos (see [Fig.l]). Non-essential amino acids have been identified N-ter of the DEF-Fos sequence and within this same sequence.

[0022] Using this information, several peptides were designed and synthesized. These peptides, which illustrate each of the modifications alone or in combination, number 12 and their sequences are detailed in Table 1. Their pharmacological effects have been tested and several show interesting results.

[0023] The terms amino acid, amino acid or amino acid are equivalent and refer to the structural units of proteins and peptides. These are molecules organized around the same basic structure, a carbon atom, which carries the acid function -COOH, the basic amine function -NH2, a hydrogen atom -H and a radical -R of variable structure which distinguishes and characterizes the twenty amino acids found naturally in proteins. The twenty natural standard amino acid residues are called A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V in the one-letter classification or Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val in the three-letter classification.

[0024] The term "deletion" of an amino acid is understood in its primary sense, namely the act of deleting an amino acid from the peptide sequence of interest. In general, an amino acid considered non-essential to the functionality of the peptide is deleted, which makes it possible to reduce the size of the peptide sequence and possibly to optimize the functionality of the peptide.

[0025] The term "substitution" as used herein refers to the replacement of one amino acid residue with another selected from the 20 naturally occurring standard amino acid residues, rare naturally occurring amino acid residues, and unnaturally occurring amino acid residues. Preferably, the term "substitution" refers to the replacement of one amino acid residue with another selected from the 20 naturally occurring standard amino acid residues (A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V). The substitution(s) may be conservative or non-conservative substitutions. The term "conservative substitution" as used herein refers to a substitution of one amino acid residue with another that has similar chemical or physical properties (size, charge, or polarity).

[0026] The term "racemization" refers to a physical property which is the transformation of an optically active compound into a racemic form which is optically inactive; for amino acids it is the passage from one enantiomer or stereoisomer (optical isomers with opposite specific rotational power) to another. This transformation has an impact on the structure and functionality of the peptides. In the present invention, it is a D-type racemization (for dextrorotatory) as opposed to L (for levorotatory) because the amino acids in their natural state are of the L type with the exception of glycine. D-racemization of one or more amino acids makes the peptide more stable.

[0027] As used herein, the term "comprising" or "comprises" is used with reference to substances, compounds, methods or processes which are essential to the invention, but which are open to the inclusion of non-specific elements, whether essential or not.

[0028] The term "and / or" as used herein should be considered a specific description of each of the two specified features or components, with or without the other. For example, "A and / or B" should be considered a specific disclosure of each of the following: (i) A, (ii) B, and (iii) A and B, as if each were presented individually.

[0029] [Table 1] Table 1: Synthetic peptides derived from TAT-DEF-Fos SEQ. NO Peptide Detailed sequence 1 MLK1001 GRKKRRQRRRPPCTTYTSSFVFTYPEADSFPS 2 MLK1002 GRKKRRQRRRPPTSSFVFTYPEADSFPS 3 MLK1003 Ac-GRKKRRORRRPPTSSFVFTYPEADSFPS 4 MLK1004 GRKKRRQRRRPPTySFVFTYPEAySFPy 5 MLK1005 Ac-GRKKRRORRRPPTsSFVFTYPEAsSFPs 6 MLK1006 GRKKRRORRRPPTsSFVFDYPEAsSFPs 7 MLK1007 Ac-GRKKRRORRRPPTsSFVFDYPEAsSFPs 8 MLK1008 GRKKRRORRRPPCTTY TSSFVFTY 9 MLK1009 Ac-GRKKRRQRRRPPCTTY TSSFVFTY 10 MLK1010 GRKKRRORRRPPCTTY TSSFVFTY 11 MLK1011 Ac-GRKKRRQRRRPPCTTY Ts SFVFTY 12 MLK1012 GRKKRRORRRPPCTTY TySFVFDY 13 MLK1013 Ac-GRKKRRQRRRPPCTTY TsSFVFDY

[0030] In this table 1 we see the different modifications applied according to different criteria to the TAT-DEF-Fos sequence of SEQ ID N°L ​​The amino acids corresponding to the DEF-Fos sequence are underlined and it is this sequence of 20 amino acids which is essentially modified as follows: - the N-ter region is shortened in the 4 amino acid sequence CTTY (see for example SEQ ID Nos. 2, 3, 4, 5, 6 and 7); and / or - the C-ter region is shortened so as to allow perfect positioning of the beta sheet (see for example SEQ ID Nos. 8, 9, 10, 11, 12 and 13); and / or - a D-type racemization (or D racemization) is applied to the non-essential amino acids to stabilize each of these new peptides (see for example SEQ ID Nos. 4, 5, 6, 7, 10, 11, 12 and 13) which is indicated by a lowercase letter in bold; and / or - in the FVFT consensus sequence the threonine residue (T / Thr) is substituted by an aspartic acid residue (D / Asp), represented by a double highlight, to provide phospho-mimetic properties (see SEQ ID Nos. 6, 7, 12 and 13); and / or - an acetyl residue (Ac) is added in N-ter to protect the molecule (see for example SEQID N°3, 5,7,9, 11 and 13).

[0031] The present invention provides novel synthetic peptides which are inhibitory ligands for phosphorylation of the Fos protein by ERK1 / 2.

[0032] Described are peptides of the TAT-DEF-Fos type characterized in that the 20 amino acid sequence DEF-Fos of SEQ ID No. 14 located in positions 13 to 32 of SEQ ID No. 1 is modified in at least one position. In a particular embodiment, the peptides of the TAT-DEF-Fos type are characterized in that the DEF-Fos sequence is modified in at least two positions. Said peptides are in particular selected from the peptides of SEQ ID No. 2 to 13. By modification is meant the deletion or substitution of one or more amino acids and / or the D racemization of one or more amino acids and / or the mutation of a phosphorylation site Ser into a constitutively active residue Asp, and / or the acetylation of the N-terminal region.The invention describes a peptide of the TAT-DEF-Fos type characterized in that the DEF-Fos sequence of SEQ ID No. 14 is modified by deletion and / or substitution and / or racemization of at least one amino acid in a position located between positions 13 to 32 of SEQ ID No. 1.

[0033] In a particular embodiment, the peptides according to the invention are characterized in that the DEF-Fos sequence of SEQ ID No. 14 is shortened in the N-ter region by 1 to 4 amino acids, preferably by 4 amino acids. In the case of the modification of the N-ter region of the DEF-Fos sequence, the peptides are in particular selected from the peptides of SEQ ID No. 2, 3, 4, 5, 6 and 7.

[0034] In another particular embodiment, the peptides according to the invention are characterized in that the DEF-Fos sequence of SEQ ID No. 14 is shortened in the C-ter region by 1 to 8 amino acids, preferably by 8 amino acids. In the case of the modification of the C-ter region of the DEF-Fos sequence, the peptides are in particular selected from the peptides SEQ ID No. 8, 9, 10, 11, 12 and 13.

[0035] In a particular embodiment, in the peptides according to the invention the amino acid threonine (T / Thr) of the consensus sequence FVFT is replaced by an amino acid aspartic acid (D / Asp). In this particular embodiment the peptides according to the invention are in particular selected from the peptides of SEQ ID No. 6, 7, 12 and 13

[0036] In another particular embodiment, a D-type racemization is applied to one or more non-essential amino acids located in positions 18, 28 and 32 of SEQ ID No. 1. In this embodiment, the peptides according to the invention are in particular selected from the peptides of SEQ ID No. 4, 5, 6, 7, 10, 11, 12 and 13.

[0037] In a particular embodiment, the N-ter region is additionally acetylated. In this embodiment, the peptides according to the invention are in particular selected from the peptides of SEQ ID Nos. 3, 5, 7, 9, 11 and 13.

[0038] In a particular embodiment, the peptides according to the invention are selected from the peptides of SEQ ID No. 2 to 13, preferentially from the peptides of SEQ ID No. 8 and SEQ ID No. 12. In a preferred embodiment, the peptides according to the invention correspond to SEQ ID No. 8. In another preferred embodiment, the peptides according to the invention correspond to SEQ ID No. 12.

[0039] Advantageously, the invention describes a method for identifying new inhibitors of Fos phosphorylation by ERK1 / 2. This identification method comprises the following steps: a. develop a 3D model of the ERK2 anchoring domain towards substrates containing a DEF site, based on the crystal structure of the ERK2 AMP-PNP complex (pdb code 4S32) as a receptor; b. 3D model the binding of a peptide known as a ligand of this domain to bind to the binding site of the active ERK2 kinase; c. perform a protein-ligand docking study using the LibDock algorithm; d. perform a protein-protein docking study using the ZDOCK algorithm and refine the results using RDOCK; e. identify the essential and non-essential amino acids for the binding of the peptide modeled in b. in the DEF-type binding pocket of ERK2; f. optimize the peptide prepared in b. then design and synthesize new peptides taking into account all the information obtained in c., d. and e.; g. test the biological activity and / or properties of the peptide(s) synthesized in f.

[0040] With this method it is possible to identify other peptides inhibiting Fos phosphorylation, exhibiting good binding affinities on ERK2 and likely to have interesting effects on different cell lines, in particular tumor cell lines.

[0041] Once the receptor model has been developed, this method of identifying molecules capable of binding to the DEF domain of ERK2 towards c-Fos in a similar manner to the peptides detailed above, comprises the following steps: a. use the 3D model of the DEF domain of ERK2 towards c-Fos based on the crystal structure of the ERK2 AMP-PNP complex as a receptor; b. test in silico the fixation or “docking” of different molecules on the binding pocket of the DEF domain of ERK2 towards c-Fos; c. synthesize said molecules having good binding affinity; d. study the affinity of each molecule for the binding pocket of the DEF domain of ERK2 and select the best binding affinities; e. test the biological activity and / or properties of the molecule(s) selected in d. and synthesized in c.

[0042] Advantageously, the present invention also describes a method for identifying peptide-like molecules of the new peptides according to the invention, therefore capable of binding very precisely in the binding pocket of DEF-Fos type ligands towards ERK2 and thus of inhibiting the phosphorylation of c-Fos by ERK1 / 2.

[0043] Peptidomimetic molecules or peptidomimetic ligands are understood to mean small molecules, which are designed to mimic the activity of active peptides. In research on peptidomimetic molecules the sole objective is to design and synthesize, or identify, small molecules that have a very specific effect on the identified target with minimal or no side effects. In the present invention the peptidomimetic molecules are ligands of the binding pocket of the DEF domain of ERK2 towards c-Fos and capable of inhibiting the phosphorylation of c-Fos by ERK1 / 2.

[0044] This method for identifying peptidomimetic molecules comprises the following steps: a. use the 3D model of the DEF domain of ERK2 based on the crystal structure of the ERK2 AMP-PNP complex (pdb code 4S32) described above, as a receptor; b. perform in silico a high-throughput screen of different small molecules capable of binding to the binding pocket of the DEF domain of ERK2 towards c-Fos; c. synthesize said molecules with good binding affinity; d. study the affinity of the molecules selected in the screening for their binding affinity towards ERK2; e. test the biological activity and / or properties of the molecule(s) synthesized in c and selected in d.

[0045] This method makes it possible to highlight peptides but also small molecules of non-peptide nature and capable of regulating a biological process. Thus, the present invention describes a method for identifying peptides inhibiting the binding of c-Fos to the DEF domain of ERK1 / 2, but also a method for identifying small molecules of non-peptide nature also exhibiting an inhibitory activity on the binding of c-Fos to the DEF domain of ERK1 / 2. These peptides or peptidomimetics are inhibitors of the phosphorylation of c-Fos by ERK1 / 2.

[0046] The invention describes a novel peptide inhibiting c-Fos phosphorylation by ERK1 / 2 for medical use. The invention describes a pharmaceutical composition containing a therapeutically active dose of at least one peptide according to the present invention.

[0047] The present invention also describes the use of a novel peptide according to the invention for the treatment of inflammatory diseases, restenosis or cancer. Cancer is understood to mean, in particular, colon cancer, pancreatic cancer, melanoma, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, bladder cancer, leukemia, gliomas, neurofibromatosis, and osteosarcomas. Inflammatory diseases are understood to mean, in particular, chronic inflammatory bowel diseases with ulcerative colitis and Crohn's disease, as well as rheumatoid arthritis. Restenosis is understood to mean the inflammatory reactions of smooth vascular cells after the placement of a stent (endoprosthesis) in cardiovascular diseases.

[0048] Finally, the present invention describes a method of treating cancer comprising the administration of a pharmaceutical composition containing a sufficient dose of a peptide according to the invention, to allow the inhibition of the phosphorylation of Fos by ERK1 / 2.

[0049] In one embodiment the invention describes a method for inhibiting and / or preventing the proliferation of cell lines selected from the group comprising vascular smooth muscle cells, fibroblasts and cells nerve cells, said method involving the administration of a therapeutically active dose of at least one peptide according to the invention.

[0050] The advantages of the peptides according to the invention will be better understood upon reading the following non-limiting examples. EXAMPLES

[0051] Example 1: 3D modeling to position MLK1001 in the DEF domain binding pocket of active ERK2 kinase. The crystal structure of the ERK2 AMP-PNP complex (pdb code 4S32) was used as a receptor for docking experiments. The protein was cleaned and prepared for docking (e.g., separation of structures into separate molecules, addition of missing atoms, correction of connectivity, correction of names, insertion of missing loops, etc.). The resulting peptide is considered as a small compound or as a small protein. Putative docking sites were selected based on cavities in the receptor structure when peptides are considered as small compounds or by using the ZDOCK software (a scheme for exhaustive search of all rotational and translational gaps between the ligand protein and the receptor protein, which is fixed in its starting orientation when the peptide is considered as a small protein). Ligand protein docking is performed using Libdock methodology and software. Libdock uses protein site characteristics as “HotSpots” which are classified into two categories: polar and apolar HotSpots. The receptor HotSpot map was established before the docking procedure. Ligand conformations were randomly generated from the initial ligand structure using high temperature molecular dynamics in a “Catalyst” algorithm before docking. Rigid ligand poses were positioned in the receptor active site and HotSpots were matched by triplets. The poses were trimmed and an optimization step is performed before the pose is classified. Hydrogen ligands that were removed during the docking phase are added back to the ligand poses and optimized by minimization.The poses with the highest LibDock scores are identified and then grouped according to their fixation mode.

[0052] The protein / protein docking experiment is performed using the ZDOCK software. This software is a docking algorithm based on a rigid protein / protein structure based on the FFT (Fast Fourier Transform) correlation technique which is used to explore the rotational and translational spaces of the protein / protein system (Chen et al. 2003). Docking is also performed without constraints or selection of specific residues on ERK or on the peptides. The obtained poses (2000 per cycle) are refined using the rDOCK module (DS Modeling 2.5). rDOCK is an energy minimization procedure for refining and ranking fixation poses using energy ranking functions (Li et al., 2003). All poses are classified into different groups (based on energy / number / position criteria) and the most significant groups are selected for interaction studies.

[0053] Example 2: Pharmacological tests Antiproliferative effect on tumor cell lines The inventors have already shown an antiproliferative effect of the compound MKL1001 of SEQ ID No. 1 on cell proliferation and this effect is described in EP3164413. The antiproliferative properties were studied by a BrdU assay on different human tumor cell lines: colon, bladder, prostate and pancreatic carcinoma. The compound of SEQ ID No. 1 was tested in increasing doses (from 2 to 100 pM) and, in parallel, two controls were carried out with prior art MEK inhibitors: AZD6244 and PD 184352 at 1 pM. Tumor cells were cultured in a humidified 5% CO2 chamber at 37°C in the presence of complete medium (RPMI 1640 or DMEM / F12 depending on the cell type) supplemented with 10% fetal bovine serum, 50 units / ml penicillin and 50 pg / ml streptomycin (GIBCO). Then: - the compound MKL1001 of SEQ ID No. 1 at different concentrations and the reference products (MEK inhibitors) are incubated with the cells for 24 hours at 37°C. - BrDu (final concentration 100M) is then added and the cells incubated for a further 20 hours at 37°C. - BrDu incorporation into DNA is revealed by an ELISA test using a BrdU kit according to the supplier's recommendation (Roche®, 11647229001). The results are detailed in [Fig.2] which shows cell proliferation measured by BrdU incorporation after addition of serum on tumor cell lines of human origin. Left panels: dose response of the MLK1001 peptide compared to reference products (MEK inhibitors: selumetinib and PD184352). The MLK1001 peptide produces proliferation inhibition from 20pM on pancreatic carcinoma, 60pM on bladder adenocarcinoma and prostate carcinoma lines, and 6qM on colon adenocarcinoma, with a rebound effect on the latter line. The inhibitory effects are comparable to the reference product Selumetinib (dotted line). Left panels: Inhibitory effects of MLK1001 peptide on melanoma and neurofibromatosis type I cell lines.

[0054] Study of the new peptides according to the invention Based on the results obtained above, the inventors tested all the peptides described in Table 1 for their antiproliferative properties on a human pancreatic adenocarcinoma cell line (HPAF-II) after addition of serum. Cell proliferation following serum stimulation was determined by colorimetry using the BrDU kit (Roche, Cat. No. 11 647 229 001). Prior art MEK inhibitors are used as positive controls: selumetinib and PD 184352. Cells are first synchronized for 48h with 0.1% FBS (no serum) followed by 48h with 10% FBS (10% serum). When indicated, the MEK inhibitors Selumetinib or PD 184352 (1 µM each) or the peptides according to the invention are applied during FBS treatment. BrdU is added to the medium 8h after serum addition, in the presence of peptides or not, and left for 36h. After treatment, cell proliferation is determined by immunostaining of BrdU, and quantified by comparison with total nuclei (as labeled with DAPI). Results are twice as high for BrdU / DAPI positive cells in the presence of 10% serum (without effect of DMSO addition). BrdU staining shows that MEK inhibitors cause a significant decrease in proliferation with FBS treatment (10%) ([Fig. 3]). MLK1001 does not exhibit antiproliferative properties at the tested doses of 0.1 to 30 pM ([Fig. 3]).Similarly, the new peptides MLK1002 to MLK1007 do not exhibit antiproliferative properties at these same doses on the HPAF-II line (data not shown). However, a significant effect is observed from 30 pM for MLK1008, 1009, 1010, 1011 and 1012 and at 10 pM for MLK1011 and 1012 (see [Fig.3]). Peptides MLK1008 to MLK1010 produce an inhibition of cell proliferation from 30 pM similar to the reference product PD 183452 (grey dotted line in [Fig.3]) and peptides MLK1011 and MLK1012 from 10 pM. Peptide MLK1013 does not produce inhibition of cell proliferation on the HPAF-II line.

[0055] Cell cycle The cell cycle is determined by Ki67 immunostaining after treatment of HPAF-II cells with 10% SBF serum for 48 hours, in the presence or absence of the peptides according to the invention. MEK inhibitors are used as a positive control. Top left shows the effect of the reference product (Selumetinib) on the prolongation of the G0 / G1 phase. Stabilization of tumor cells in a quiescent state is observed. Top right: MLK1001 peptide produces a decrease in G2 / S phase and an increase in G0 / G1 phase from OpM as an increase in quiescent cells is observed. The bottom six panels show the comparative effects of peptides MLK1008 to MLK1013 on the cell cycle measured by Ki67 immunostaining. It is noteworthy that peptides MLK1008 and MLK1012 were effective from 3qM. Peptides MLK1008 to MLK1012 exhibit an increase in GO / Gi phases relative to G2 / S, indicating a cell cycle block. This GO / Gi phase block is observed at lower doses for peptides MLK1008 and MLK1012, down to 3 pM ([Fig. 4] bottom panels) compared to MLK1001 ([Fig. 4], top right panel).

[0056] Conclusion Among all the peptides according to the invention which were tested on the human HPAF-II cell line, two of them exhibit improved antiproliferative properties compared to MLK1001 on human pancreatic adenocarcinoma cell lines (HPAF-II) by decreasing cell cycle activity.

[0057] Example 3: Biochemical test by Biacore™ test The affinities of the MLK1001 and MLK1008 peptides towards ERK2 proteins were tested by the surface plasma resonance (SPR) technique using a Biacore 3000 device ([Fig.5]). The recombinant ERK2 protein was immobilized by an amine coupling on the chip composed of a layer of dextran placed on a gold foil, itself placed on a glass plate. The MLK1001 and MLK1008 peptides were used as analytes in the microfluidic cell; these peptides are mobile. The analyses carried out are a quantification of the resonance signal (RU) therefore an analysis on a single bond and an analysis on kinetic cycles with calculations of affinity constants. The analysis of the ERK2 interaction with the peptides was studied via the SPR signal. Negative controls included a Scramble peptide (TAT+random AA sequence: GRKKRRQRRRPPQSKPSGSQHPIFSLAFVAS, cf Lavaur and Caboche, J.Neurosci, 2007 for validation) and a TAT sequence comprising an interfering sequence between the NMDA2B receptor and PSD 95 (YGRKKRRQRRRKLSSIESDV). Single-binding analyses show the level of interaction with the target, and kinetic cycle analyses determine the affinity with the target.

[0058] [Table 2] Table 2: Affinity constants of MLK1001 and MLK1008 peptides defined by the Biocore technique s® Ks ■ -y- ■ :ld: w <1 1 f- ..... ISl rM km «S rssi-îî& : •: ; :tfxs --4-- SM «is there?? s&Knm-

[0059] The analyses made it possible to highlight an affinity (equilibrium dissociation constant KD) twice as high for the MLK1008 peptide towards ERK2 (KD: 4.54nM) compared to the MLK1001 peptide (10.5nM) (see Table 2 above and [Fig.6A] and 6B).

Claims

Claims

1. DEF-Fos peptide characterized in that the DEF-Fos sequence (of SEQ ID No. 14) of SEQ ID No. 1 is modified by deletion and / or substitution and / or racemization of at least one amino acid of SEQ ID No. 14 and, the DEF-Fos sequence of SEQ ID No. 14 being shortened in the C-ter region by 8 amino acids.

2. Peptide according to claim 1 characterized in that the amino acid T (Thr) of the consensus sequence FVFT is replaced by the amino acid D (Asp).

3. Peptide according to any one of claims 1 to 2 characterized in that a D-type racemization is applied to a non-essential amino acid located in position 18 of SEQ ID No.

1.

4. Peptide according to any one of claims 1 to 3, characterized in that the N-ter region of SEQ ID No. 1 is acetylated.

5. Peptide according to any one of the preceding claims, characterized in that it has a sequence selected from SEQ ID Nos. 8 to 13.

6. Peptide according to any one of claims 1 to 5, characterized in that it is selected from the peptides of SEQ ID No. 8 and SEQIDN°12.

7. Peptide according to any one of claims 1 to 6 for medical use.

8. A peptide according to claim 7 for use in the treatment of inflammatory diseases, restenosis or cancer.

9. Pharmaceutical composition containing a therapeutically active dose of at least one peptide according to one of claims 1 to 6.

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