Novel inhibitors of Elk-1 phosphorylation by ERK1 / 2

Abstract

The present invention describes novel synthetic peptides which are inhibitors of Elk-1 phosphorylation by ERK1 / 2 kinases. A pharmaceutical composition containing said peptides and their use as medicaments are also described. A method for identifying and optimizing novel inhibitors of Elk-1 phosphorylation by ERK1 / 2 is also described.

Description

Title of the invention: Novel inhibitors of phosphorylation of Elk-1 by ERK1 / 2 Technical field

[0001] The present invention describes novel synthetic peptides which are inhibitors of Elk-1 phosphorylation by ERK1 / 2 kinases. A pharmaceutical composition containing said peptides and their use as medicaments are also described. A method for identifying and optimizing novel inhibitors of Elk-1 phosphorylation by ERK1 / 2 is also described.

[0002] TECHNOLOGICAL BACKGROUND 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 numerous intracellular substrates, whether membrane, cytoplasmic or nuclear (Yang et al., Trends Pharmacol. Sci.2019 Nov;40(ll):897-910) and it is the amplitude and duration of the activations of the intracellular signal cascade which will direct ERK1 / 2 towards one of its substrates.

[0003] In the central nervous system, the Ras-Raf-MEKl / 2-ERKl / 2 signaling pathway is activated in neurons by neurotransmitters, neuromodulators or neurotrophic factors. By acting via receptors coupled to G proteins (monoamine receptors, or metabotropic glutamate receptors for example), to calcium inputs (NMDA glutamate receptors) or to receptors with tyrosine kinase activity (neurotrophic factors such as BDNF) the activation of this signaling cascade is involved in multiple cellular functions, during development or in the adult brain. Thus the ERK pathway controls neuronal differentiation and maturation during development. In the adult brain, it plays a key role in neuronal plasticity and learning and memory processes, notably involved in cognition, reward learning and mood.Hyperactivity of the ERK pathway is observed in many neurodevelopmental diseases, including autistic disorders and neuropathologies. fibromatosis type 1, Noonan syndrome, capillary-arteriovenous malformation syndrome, Costello syndrome, cardiofaciocutaneous syndrome, and Legius syndrome. ERK pathway deregulation is also responsible for psychiatric illnesses such as addiction, depression, post-traumatic stress, and anxiety and mood disorders.

[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 Se-lumetinib (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 a unique substrate 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 EP1988167 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 (or FXFP) and D (or DEJL) (see EP1988167).

[0008] The docking site for ERK FXFP (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 FXFP or (F / Y)X(F / Y)P type, where F is Phenylalanine (Phe), Y is Tyrosine (Tyr), P is Proline (Pro), and X is any natural amino acid (Sharrocks et al., Trends Biochem Sci. 2000 Sep;25(9):448-53). The DEF domain is specific for ERK1 / 2 substrates.

[0009] The DEJL domain (docking site for ERK and JNK LXL) 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 for MEK1 / 2, which are located upstream of ERK1 / 2 in the intracellular signaling cascade. It should be noted that this site is not specific to ERK-type kinases, but also allows the binding and phosphorylation of substrates on MAPkinases of the JNK and p38 / MAPK family.

[0010] Many teams are developing MEK1 / 2 inhibitors with the aim of providing therapeutic solutions in various pathologies, particularly in oncology, in disorders of the central nervous system and in 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 (Balagula et al., Invest. New Drugs, 2010; 29:1114-21).

[0012] Studies are also being conducted to discover molecules that may act on the activation of ERK1 / 2 in order to regulate the proliferation and survival of cells, particularly cancer cells. Over the last ten years, in silico studies have identified ligands that may interact with specific binding sites of ERK1 / 2 and selectively inhibit interactions with substrates involved in cell proliferation (Boston et al., BMC Cancer. 2011; 11: 7; Samadani et al., Biochem J. 2015 May 1; 467(3): 425-438). They have also highlighted some of the structural characteristics of the binding domains of ERK1 / 2.

[0013] Peptides containing the DEF (or FXFP) domain, and surrounding amino acids in the N- and C-terminal regions, of a specific substrate downstream of ERK inhibit the binding and subsequent phosphorylation of said substrate without impacting other ERK1 / 2 activities 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.

[0014] Patents EP1988167 and EP2337576 describe the use of synthetic TAT-DEF type peptides as specific inhibitors of substrates downstream of ERK and in particular the use of a TAT-DEF-Elk-1 type peptide for the treatment of an anxiety and mood disorder; the Elk-1 protein being a transcription factor acting downstream of the ERK signaling pathway. Summary of the invention

[0015] The present invention describes novel peptides which are inhibitors of the phosphorylation of Elk-1 by ERK1 / 2 and which are derived from those described in EP1988167. These new synthetic peptides are modified to make them more stable and more effective than those described in EP1988167.

[0016] 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 the TAT-DEF-Elk-1 peptide according to patent EP1988167 and characterize their interaction with this binding pocket. This makes it possible to define the essential and non-essential positions in the binding of the TAT-DEF-Elk-1 peptide to the ERK2 anchoring domain and to optimize potential inhibitors of the phosphorylation of Elk-1 by ERK1 / 2. This study allows the optimization of peptides containing DEF-Elk-1 domains (SEQ ID No. 1) either by stabilization and in particular by D-racemization of amino acids not essential for binding, or by deletion of amino acids not essential for binding, or by modifying the Ser residues of phosphorylation by ERK into Asp, or by combining these different modifications, and finally to consider new inhibitors.

[0017] 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.

[0018] 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

[0019] [Fig.l] [Fig.l] describes the in silico modeling of the interaction between the DEF-Elkl domain of MLK2001 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] [Fig.2] depicts the binding affinity of MLK2001 peptide (50nM) to active ERK2 by surface plasmon resonance (biaccore) technique. Recombinant ERK2 protein was immobilized by amine coupling and MLK2001 peptide (analyte) is mobile. TAT-NR2B9c peptide was used as negative control. [Fig.3] [Fig.3] describes the binding affinity of the MLK2001 peptide, of 3 peptides according to the invention (MLK2002, 2003 and 2004), and two control peptides: the TAT sequence alone, and a "Scramble" (random) peptide of the MLK2001 sequence, by the biacore technique. The resonance signal (RU) represents the affinity of the analytes (peptides) towards the immobilized active ERK2 proteins. The graph represents the profiles obtained at 50nM for each analyte. The higher the RU signal, the greater the affinity of the analyte for ERK2. It should be noted that the RU signal for MLK2002 is 1.5 times higher than for MLK2001. [Fig.4] [Fig.4] provides a graph representing the dose-dependent binding of MLK peptides 2001 to 2004 as well as the control peptides TAT ​​and Scr. [Fig.5] [Fig.5] depicts the phosphorylation of Elk-1 and ERK by the MLK2001 peptide and the modified MLK2002 peptide at low dose. [Fig.6] [Fig.6] depicts the toxicity after 24h of treatment with the MLK2001 peptide and the modified MLK2002 peptide according to the present invention. [Fig.7] [Fig.7] illustrates the result of implementing the in silico screening method for the binding of small molecules to the DEF-Elk-1 peptide binding site within the ERK2 docking domain. Three important binding sites were identified and separated. These figures illustrate the present invention and are not limiting of the claimed scope. DETAILED DESCRIPTION OF THE INVENTION

[0020] The docking experiments were carried out with the MLK2001 peptide of SEQ ID No. 1 as a ligand for the ERK2 docking domain. The in silico modeling method made it possible to precisely study the interactions between MLK2001 of SEQ ID No. 1 and the DEF-Elk-1 type ligand binding pocket. From this experiment, new compounds were designed and synthesized. These are described in Table No. 1 below. Compared to MLK2001 of SEQ ID No. 1, they are either shortened, stabilized, or both.

[0021] 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 Elk-1 including in particular the consensus sequence FQFP framed by amino acids.

[0022] The results of the in silico 3D modeling experiment from the positioning of TAT-DEF-Elk-1 of SEQ ID No. 1 in the binding pocket of the active ERK2 kinase made it possible to identify the amino acids essential for the binding of the DEF-Elk-1 and ERK2 domains and in particular the FQFP consensus sequence. Non-essential amino acids were identified N-ter of the DEF-Elk-1 sequence and within this same sequence.

[0023] Using this information, several peptides were designed and synthesized. These peptides, which illustrate each of the modifications alone or in combination, number 23 and their sequences are detailed in Table 1. Their pharmacological effects have been tested and several show interesting results.

[0024] 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.

[0025] 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.

[0026] 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 acids. 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 (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T). 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).

[0027] 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. The D-racemization of one or more amino acids makes the peptide more stable.

[0028] 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.

[0029] 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.

[0030] [Table 1] Table 1: Synthetic peptides derived from TAT-DEF-Elk-1 SEQ. ID No. Peptide Detailed Sequence 1 MLK2001 GRKKRRORRRPPSPAKLSFOFPSSSGSAOVHI 2 MLK2002 GRKKRRORRRPPSPAKLsFOFPsSGSAOVHI 3 MLK2003 GRKKRRORRRPPSPAKLSFOFPSDGDAOVHI 2004MLK GRKKRRORRRPPSPAKLSFOFPsDGDAOVHI 5 MLK2005 GRKKRRQRRPPPSPAKLSFOFPSSSGSAOVHI 6 MLK2006 GRKKRRORRRPPSPAKLSFOFPSDGDAOVHI 7 MLK2007 GRKKRRORRRPPAKLSFOKVHIF20808 GRKKRRORRRPPAKLSFOFPSSSGSAOVHI 9 MLK2009 GRKKRRORRRPPAKLSFOFPSDGDAOVHI 10 MLK2010 GRKKRRORRRPPAKLSFOFPSDGDAOVHI 11 MLK2011 GRKKRRORRRPPAKLSFOFPSSGSAOVHI 2012 MLK GRKKRRORRRPPAKLSFOFPSDGDAOVHI 13 MLK2013 Ac-GRKKRRORRRPPSPAKLSFOFPSSGSSAOVHI 14 MLK2014 Ac-GRKKRRORRRPPSPAKLsFOFPsSGSAOVHI 15 MLK2015 Ac-GRKRRRRPPAKLSFOFPSOFPGSAOVHI 16 MLK2016 Ac-GRKKRRORRRPPSPAKLSFOFPsDGDAOVHI 17 MLK2017 Ac-GRKKRRORRRPPSPAKLSFOFPSSSGSAOVHI 18 MLK2018 Ac-GRKKRRORRRPPSPAKLSFOFPSDGDAOVHI 19 MLK2019 Ac-GRKKRRQRRPPAKLSFOFPSSSGSAOVHI 20 MLK2020 Ac-GRKKRRORRRPPAKLSFOFPSSSGSAOVHI 21 MLK2021 Ac-GRKKRRORRRPPAKLSFOFPSDGDAOVHI 22 MLK2022 Ac-GRKKRRRPQLSFOFPSDG MLK2023Ac-GRKKRRORRRPPAKLSFOFPSSGSAOVHI 24 MLK2024 Ac-GRKKRRORRRPPAKLSFOFPSDGDAOVHI

[0031] In this table 1 we see the different modifications applied according to different criteria to the TAT-DEF-Elk-1 sequence of SEQ ID N°L ​​The amino acids corresponding to the DEF-Elk-1 sequence are underlined and it is this sequence SEQ ID N°25 which is essentially modified as follows: - the N-ter region is shortened in the 2 amino acid sequence SP (see for example SEQ ID Nos. 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23 and 24); and / or - a D-type racemization (or D racemization) is applied to one or more non-essential amino acids to stabilize each of these new peptides (see for example SEQ ID No. 2, 4, 5, 6, 8, 10, 11, 12, 14, 16, 17, 18, 20, 22, 23 and 24) which is indicated by a lowercase letter in bold; and / or - the amino acids serine (S / Ser) are changed to aspartic acid (D / Asp), represented by a double highlight, to provide phospho-mimetic properties (see SEQ ID Nos. 3, 4, 6, 9, 10, 12, 15, 16, 18, 21, 22 and 24); and / or - an acetyl group (Ac) is added in N-ter to protect the molecule (see for example SEQ ID No. 13 to 24).

[0032] The present invention provides novel synthetic peptides which are ligands inhibiting phosphorylation of the Elk-1 protein by ERK1 / 2.

[0033] Described are peptides of the TAT-DEF-Elk-1 type characterized in that the 19 amino acid sequence DEF-Elk-1 (SEQ ID No. 25) located between positions 13 to 31 of SEQ ID No. 1 is modified in at least one position. In a particular embodiment, the peptides of the TAT-DEF-Elk-1 type are characterized in that the DEF-Elk-1 sequence is modified in at least two positions. Said peptides are in particular selected from the peptides of SEQ ID No. 2 to 24. 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 present invention describes a TAT-DEF-Elk-1 type peptide whose DEF-Elk-1 sequence of SEQ ID No. 25 is modified by deletion and / or racemization and / or substitution of at least one amino acid at a position located between positions 13 to 31 of SEQ ID No. 1.

[0034] In a particular embodiment, the peptides according to the invention are characterized in that the DEF-Elk-1 sequence of SEQ ID No. 25 is shortened in the N-ter region of the DEF-Elk-1 sequence by 1 to 2 amino acids, preferably by 2 amino acids. In the case of the modification of the N-ter region of the DEF-Elk-1 sequence, the peptides are in particular selected from the peptides of SEQ ID No. 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23 and 24.

[0035] In another particular embodiment, in the peptides according to the invention at least one serine amino acid (S / Ser) of the consensus sequence SSGS located in positions 23 to 26 of SEQ ID No. 1 is replaced by an aspartic acid amino acid (D / Asp). In this embodiment the peptides according to the invention are in particular selected from the peptides of SEQ ID No. 3, 4, 6, 9, 10, 12, 15, 16, 18, 21, 22 and 24. In a preferred embodiment, two S amino acids (Ser) of the SSGS consensus sequence are replaced by two D amino acids (Asp), said residues being preferentially located in positions 24 and 26 of SEQ ID No. 1.

[0036] In another particular embodiment, a D-type racemization is applied to at least one amino acid located at any one of positions 18, 20, 23 and 28 of SEQ ID No. 1. In a preferred embodiment, a D-type racemization is applied to the amino acids located at positions 18, 23, and 28 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. 2, 4, 14 and 16. In another preferred embodiment, a D-type racemization is applied to the amino acids located at positions 18, 20, 23 and 28 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. 5, 6, 17 and 18.When the peptides according to the invention have a shortened sequence in N-ter of the DEF-Elk-1 sequence, then the numbering of the amino acids changes; the D-type racemization is applied to the same amino acids as those cited above with reference to SEQ ID No. 1. In this context, the peptides according to the invention can be selected from the peptides of SEQ ID No. 2, 4, 5, 6, 8, 10, 11, 12, 14, 16, 17, 18, 20, 22, 23 and 24.

[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 No. 13 to 24.

[0038] In a preferred embodiment, the peptides according to the invention are selected from the peptides of SEQ ID No. 2 to 24, preferentially from the peptides of SEQ ID No. 2, 4, 5, 6, 8, 10, 11, 12, 14, 16, 17, 18, 20, 22, 23 and 24. In another embodiment, the peptides according to the invention correspond to SEQ ID No. 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23 and 24. In another embodiment, the peptides according to the invention correspond to SEQ ID No. 3, 4, 6, 9, 10, 12, 15, 16, 18, 21, 22 and 24.

[0039] Advantageously, the invention describes a method for identifying new inhibitors of phosphorylation of Elk-1 by ERK1 / 2. This identification method comprises the following steps:

[0040] a. develop a 3D model of the ERK2 anchoring domain towards substrates containing a DEF site, based on the crystal structure of the ERK2 complex AMP-PNP (pdb code 4S32) as a receiver; 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 ERK2 anchoring pocket to substrates containing a DEF site; f. optimize the peptide modeled in b. then design and synthesize new peptides taking into account all the information obtained in c., d. and e.; g. test biochemical affinity by protein / protein interaction studies h. test the biological activity and / or properties of the synthesized peptide(s) in f.

[0041] With this method it is possible to identify other phosphorylation inhibitory peptides, presenting good binding affinities on ERK2 and likely to have interesting effects in mood and anxiety disorders, as well as their comorbidity (addiction, anxiety) or PTSD.

[0042] Once the receptor model has been developed, this method of identifying molecules capable of binding to the DEF domain of ERK2 towards Elk-1 in a similar manner to the peptides detailed above, comprises the following steps:

[0043] a. use the 3D model of the DEF domain of ERK2 towards Elk-1 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 Elk-1; 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.

[0044] Advantageously, the present invention also describes a method for identifying small peptidomimetic molecules of the new peptides according to the invention, therefore capable of binding very precisely in the binding pocket of DEF-Elk-1 type ligands towards ERK2 and thus of inhibiting the phosphorylation of Elk-1 by ERK1 / 2.

[0045] Peptidomimetic molecules or peptidomimetic ligands are understood to mean small molecules, which mimic the activity of active peptides. In research on Peptidomimetic molecules The objective is to 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 the DEF-Elk-1 peptide and capable of inhibiting the phosphorylation of Elk-1 by ERK1 / 2.

[0046] This method for identifying small peptidomimetic molecules comprises the following steps:

[0047] a. use the 3D model of the DEF domain of ERK2 towards Elk-1 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 Elk-1; c. synthesize said molecules with good binding affinity in silico; d. test 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) selected in d. and synthesized in c.

[0048] This method makes it possible to highlight small molecules of a non-peptide nature and capable of regulating a biological process. Thus, the present invention describes a method for identifying peptides that inhibit the binding of Elk-1 to the anchoring domain of ERK2 and its phosphorylation, but also a method for identifying small molecules of a non-peptide nature that also exhibit an inhibitory activity on the binding of Elk-1 to the anchoring domain of ERK1 / 2. These molecules or peptidomimetics are inhibitors of the phosphorylation of Elk-1 by ERK1 / 2.

[0049] In one embodiment the present invention describes a method for identifying small molecules strongly interacting with the DEF-Elk-1 peptide binding site towards ERK2, as well as small molecules strongly interacting with the DEF-Elk-1 peptide binding site towards ERK2 which are selected from beta-lactam, 2-piperacetazine, levocabastine, prostaglandin E1 type molecules as well as their analogues.

[0050] The invention describes a novel peptide inhibiting phosphorylation of Elk-1 by ERK1 / 2 for medical use. The invention also relates to a pharmaceutical composition containing a therapeutically active dose of at least one peptide according to the present invention.

[0051] In one embodiment, the new peptides according to the invention interact with the DEF-Elk-1 peptide binding site towards ERK2 are used for the treatment of cancer, anxiety and mood disorders and in particular resistant depression, addictions, Down syndrome, Alzheimer's disease, lupus erythematosus and proximal spinal muscular atrophy.

[0052] In another embodiment, the small molecules strongly interacting with the DEF-Elk-1 peptide binding site towards ERK2 are used for the treatment of cancer, anxiety and mood disorders and in particular resistant depressions, addictions, Down syndrome, Alzheimer's disease, lupus erythematosus and proximal spinal muscular atrophy.

[0053] The pathologies and / or conditions that can be treated by the novel peptides of the present invention are cancers (breast, bladder, prostate, ovarian, glioblastoma, leukemia, etc.), anxiety and mood disorders, addictions, Down syndrome, Alzheimer's disease, lupus erythematosus and proximal spinal muscular atrophy. Many studies highlight the link between these pathologies and / or conditions and the phosphorylation of Elk-1 by ERK1 / 2.

[0054] By "mood disorder" is meant a disturbance in the emotional state and well-being of an individual over an extended period of his or her life. Mood disorders include, but are not limited to, major depression (unipolar disorder), typical or melancholic and atypical depression, pre- and postpartum depression, manias, dysphorias, bipolar disorders, dysthymias, cyclothymias, psychotic depression, depressive personality disorder, seasonal affective disorder and many others as defined by the "Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSMV).

[0055] Anxiety disorder means an unpleasant emotional state, which includes psychophysiological responses in anticipation of unreal or imagined danger and the ostensibility resulting from unrecognized intrapsychic conflict. Concomitant physiological effects include increased heart rate, altered breathing, sweating, trembling, weakness and fatigue. Psychological concomitants include a sense of imminent danger, a sense of helplessness, apprehension and tension. Anxiety disorders include, but are not limited to, panic disorder, obsessive compulsive disorder, post-traumatic stress disorder (PTSD), social phobias, social anxiety disorder, specific phobias, generalized anxiety disorder.

[0056] By "addictions" we mean brain pathologies defined by a dependence on a substance or an activity, with harmful consequences. The most frequent addictions are linked to the repeated consumption of psychoactive substances such as tobacco, alcohol, medications, psychotropic drugs, etc. or to the excessive practice of a behavior (games, social networks, sport, etc.) which leads to a loss of control over the level of consumption and / or practice, a change in emotional balance, medical disorders and disruptions to personal, professional and social life.

[0057] In a particular embodiment, the invention describes a novel peptide inhibiting phosphorylation of Elk-1 by ERK1 / 2 for the treatment of anxiety and mood disorders and in particular resistant depression.

[0058] In another embodiment the invention describes a method of administering a therapeutically active dose of at least one peptide according to the invention.

[0059] As used herein, "therapeutically active dose" defines the minimum amount of the peptide according to the invention to produce a therapeutic benefit to a patient in need thereof. It is an amount of the active ingredient that induces, ameliorates, or causes an improvement in pathological symptoms, disease progression, or physical conditions associated with the disease affecting the patient.

[0060] The present invention describes more particularly the use of a novel peptide according to the invention for the treatment and / or prevention of mood and anxiety disorders.

[0061] As used herein, the term "treatment" of mood and anxiety disorders refers to reversing, alleviating, or inhibiting one or more symptoms associated with such disorders. In the present application, the term "prevention" of mood and anxiety disorders refers to preventing one or more symptoms associated with such disorders.

[0062] The present invention also describes a method for treating depressions resistant to conventional antidepressant treatments 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 Elk-1 by ERK1 / 2, alone or in combination with conventional antidepressants.

[0063] "Major depressive disorder" or "unipolar disorder" refers to a mood disorder involving, on a daily basis and for at least two weeks, at least 5 of the following symptoms: persistent sadness, anhedonia (loss of the ability to experience pleasure), anxiety, feelings of worthlessness or guilt, thoughts of planning or attempting suicide, fatigue or loss of energy, sleep disturbances, appetite disturbances, difficulty thinking, concentrating or indecisiveness, psychomotor retardation or agitation. These symptoms of major depression, as described in the DSMV, therefore include emotional, neurovegetative, and neurocognitive disorders.

[0064] By "conventional antidepressants" is meant selective serotonin reuptake inhibitors (SSRIs), such as, but not limited to, fluoxetine (PROZAC ®), ci-talopram (Cipramil ®), daxopetine (Priligy ®), Tescitalopram (Cipralex ®), flu-voxamine (Faverin ®), paroxetine (Seroxat ®), sertraline (Lustral ®), vor- tioxetine (Brintellix ®); monoamine oxidase inhibitors (MAOs), norepinephrine reuptake inhibitors (NERIs), combined serotonin-norepinephrine reuptake inhibitors (SNRIs), phosphodiesterase-4 (PDE4) inhibitors, and other compounds. These antidepressants have an onset of action of several weeks, and many patients respond only partially or not at all to these treatments. In addition, most of these agents produce significant side effects, such as sexual dysfunction, gastrointestinal disturbances, with manifestations of nausea and vomiting, insomnia, weight gain, the development of diabetes, and cardiac problems. These side effects often discourage patients from continuing their antidepressant treatment.A new class of antidepressants includes ketamine and its derivatives, which, although possessing faster onsets of action, produce dissociative and addictive effects.

[0065] In the present application the term "resistant depression" refers to the persistence of the depressive episode despite at least two successive well-conducted antidepressant treatments or to an absence of favorable progress despite these treatments. Resistant depression would concern 15 to 30% of major depressive episodes. The present invention will be better understood by reading the examples and figures detailed below.

[0066] EXAMPLES The advantages of the peptides according to the invention are detailed in the following non-limiting examples.

[0067] Example 1: 3D modeling to position MLK2001 in the binding pocket to the DEF domain of the active ERK2 kinase. The crystal structure of the ERK2 AMP-PNP complex (pdb code 4S32) was used as a receptor to perform the 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 spaces 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 binding is performed using Libdock methodology and software. Libdock uses protein site characteristics such 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 docking mode. The protein / protein docking experiment is performed using ZDOCK software.This software is a protein / protein rigid structure-based docking algorithm based on the FFT (Fast Fourier Transform) correlation technique that 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 specific residue selection on ERK or peptides. The obtained poses (2000 per cycle) are refined using the rDOCK module (DS Modeling 2.5). rDOCK is an energy minimization procedure to refine and rank the docking poses using the 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. The result of this modeling is presented in [Fig. 1].

[0068] Example 2: The level of interaction and affinity of the MLK2001 peptide towards the active ERK2 protein was tested by the surface plasmon resonance technique (Biacore). The recombinant ERK2 protein was immobilized by amine coupling on a chip, and the peptides were used as mobile analytes. A TAT-NR2B9c peptide, which does not have an anchoring domain on the DEF domain of ERK2, nor an ERK phosphorylation site, was used as an inactive reference control. The results are presented in [Fig.2] where it is observed that the resonance signal (RU) is proportional to the binding of the electrolyte to its ligand (ERK2).

[0069] Example 3: The affinity of the peptides optimized according to the invention (MLK2002 to MLK2004) (50nM) on binding to active ERK2 is determined by the Biacore technique, compared to the peptide MLK2001 (at 50nM). Peptides: TAT sequence alone, or a random sequence (Scramble: Scr) of the MLK2001 sequence were used. The results are detailed in [Fig.3]. It is noted that the peptide MLK2002 has a 1.5 times greater affinity towards active ERK2, as indicated by the resonance signal (RU). On the other hand, the peptides MLK2003 and MLK2004 have a lower affinity towards active ERK2.

[0070] Example 4: Graph showing the affinities of the peptides MLK2001, Scr, TAT and MLK2002 to MLK2004 according to the concept, towards active ERK2, at three different concentrations: 10, 20 and 50 nM. The results are presented in [Fig.4]. There is an increasing, dose-dependent affinity for the peptides MLK2001 to MLK2004, with, for each dose, a greater affinity for the peptide MLK2002 compared to the peptide MLK2001.

[0071] Example 5: Phosphorylation of Elk-1 by ERK by the peptides according to the invention The efficacy of the peptide MLK2001 and the modified peptide MLK2002 at low dose (5 pM) was tested on primary cultures of striatal neurons. The striatal extracts are made from mouse embryos (E14). After dissociation of the tissues, the cells are placed in culture wells in a complete Neurobasal medium supplemented with B27 (Invitrogen, Cergy Pontoise, France), 500 nM L-glutamine, 60 pg / ml pe-nicillin G and 25 pM [3-mercaptoethanol (Sigma, L'Isle d'Abeau, Chasnes, France) at 37°C in a humid atmosphere (95% air and 5% CO2). The treatments are carried out after 7 days of culturing, when the majority of the cells are of a neuronal phenotype. The peptides MLK2001 and MLK2002 were added to the culture medium at a dose of 5pM one hour before BDNF treatment (Brain Derived Neurotrophic Factor, Peprotech France; 50nM).The MEK inhibitor U0126 is used at a dose of 100M and added 30 minutes before treatment. 20 minutes after BDNF treatment, cells are fixed using paraformaldehyde and immunolabeled as described in Lavaur et al. (J. Neuroscience, 2007) and Besnard et al. (J. Neuroscience 2011) using antibodies specific for the active, phosphorylated forms of ERK and Elk-1-1 (rabbit phospho-ERK 1 / 500, supplier to be specified; and mouse phospho-Elk-1-1 1 / 200, Santa Cruz). Separate wells are used for each immunolabeling. After revelation using secondary antibodies (anti-rabbit Alexa Fluor 488 (1:400), anti-mouse Alexa Fluor 488 (1:400)), counterlabeling of the nucleus is performed with Hoechst. The wells thus treated are mounted under a coverslip in vec-tashield (ThermoFischer). Image acquisition is done with a 40x objective on a Leica DM400 microscope assisted by a CCD camera. The results are shown in [Fig.5] where on the top panel we can observe a stronger fluorescence corresponding to the phospho-Elk-1-1 labeling that is observed in BDNF-treated neurons; while the peptides MLK2001, MLK2002 and U0126 produce an inhibition of this fluorescence. On the bottom panel we see that BDNF increases the phosho-ERK fluorescence while U0126 totally inhibits this phospho-ERK fluorescence, the peptides MLK2001 and 2002, as expected, do not affect this fluorescence. All these results indicate that the low dose (5pM) MLK2002 peptide produces an inhibition of Elk-1 phosphorylation, in the same way, or even in a different way. more effective than the MLK2001 peptide at the same dose.

[0072] Example 6: Toxicity of the peptides according to the invention The toxicity of the MLK2001 peptide and the modified MLK2002 peptide was tested at 5 and 10 qM on primary cultures of striatal neurons. The striatal neurons were prepared as indicated [Fig.5]. After 7 days of culture, the peptides (5 and 10 qM) were added directly into the medium; the MLK2001 and MLK2002 peptides were added to the culture medium. Twenty-four (24) hours later, the neurons were fixed with paraformaldehyde. MAP2 immunostaining (bottom panel) was performed to visualize the integrity of the neurites. Labeling of the nucleus with Hoechst allowed visualization of nuclear integrity. The wells thus treated were mounted under coverslips in vectashield (ThermoFischer). Image acquisition was done with a 40x objective on a Leica DM400 microscope assisted by a CCD camera. The results are presented in [Fig.6]. In the top panel, nuclear retraction is observed with the MLK2001 peptide at 5 and 10 qM. Better nuclear integrity is observed with the MLK2002 peptide at both doses. In the bottom panel: MAP2 labeling is strongly altered by the MLK2001 peptide at 5 and 10 qM. This labeling is intact, compared to the control with the MLK2002 peptide at 5 and 10 qM. All these results indicate that the MLK2002 peptide is less toxic at two doses (5 and 10 qM) on striatal neurons in culture.

[0073] Example 7: Identification of ERK2 binding sites The in silico screening method of small molecule binding to the DEF-Elk-1 peptide binding site within the ERK2 anchoring domain identified 3 binding sites that were separated.

[0074] Example 8: Small molecule screening method or virtual repositioning screening The identification method applied to the identification and optimization of peptides has been seen in detail above. The method according to the invention can also be applied to the screening of small molecules. Collections of known small molecules are found in academic chemical libraries and / or commercial chemical libraries. Prestwick Chemical offers reference drug libraries, available in both physical and electronic forms, which are particularly well suited for virtual repositioning screening. Implementation of the identification method described in the present invention with the Prestwick Chemical library allowed the identification of small molecules strongly interacting with the central binding site of the DEF-Elk-1 peptide towards ERK2. These molecules are detailed below and grouped into four series. These are molecules with antibacterial properties of the beta-lactam type (1st series), molecules with antipsychotic properties of the 2-piperacetazine type (2nd series), molecules with anti-H1R properties of the levocabastine type (3rd series) and molecules of the prostaglandin El type (4th series).

[0075] 1st series: Beta-lactam molecule with antibacterial effect

[0076] [Chem.l] 1st series: Beta-lactam molecule with antibacterial effect

[0077] [Chem.2] 2nd series: 2-Piperacetazine type molecule (Quide) with antipsychotic effect

[0078] [Chem.3] 3rd series: Levocabastine with anti-H1R effect

[0079] [Chem.4] 4th series: Prostaglandin El, EDEX

[0080] The first series describes a broad class of antibiotics which include penicillin derivatives, cephalosporins, monobactams, carbapenems, etc. which are known to those skilled in the art for their antibacterial properties. The second series describes a prodrug with antipsychotic effect, often used in the treatment of schizophrenia, as well as its structural analogues. The third series describes a selective H1 antihistamine called levocabastine and its structural analogues. The fourth series describes prostaglandin El (PGEi) which is used as a drug for its vasodilatory properties, as well as its structural analogues. All these molecules have a strong affinity with the DEF-Elk-1 peptide binding site towards ERK2. As detailed above, these beta-lactam molecules, 2-piperacetazine, levocabastine, prostaglandin El and their analogues can be used for the treatment of cancer, anxiety and mood disorders and in particular resistant depression, addictions, Down syndrome, Alzheimer's disease, lupus erythematosus and muscular atrophy. proximal spinal.

Claims

Claims

1. DEF-Elk-1 peptide characterized in that the DEF-Elk-1 sequence of SEQ ID No. 25 is modified by deletion in the N-ter region of 1 to 2 amino acids and / or racemization and / or substitution of at least one serine amino acid (S) of the SSGS sequence located in positions 23 to 26 of SEQ ID No. 1 by an aspartic acid amino acid (D).

2. Peptide according to claim 1, characterized in that two serine residues (S) of the SSGS sequence located in positions 23 to 26 of SEQ ID No. 1 are replaced by two aspartic acid residues (D), said residues being preferentially located in positions 24 and 26 of SEQ ID No.

1.

3. Peptide according to any one of claims 1 or 2 characterized in that a D-type racemization is applied to one or more amino acids located in positions 18, 20, 23 and 28 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. 2 to 24, preferentially from SEQ ID Nos. 2, 8 and 12, and even more preferentially that it is from SEQ ID No.

2.

6. Peptide according to any one of claims 1 to 5 for medical use.

7. Peptide according to any one of claims 1 to 6 for use in the treatment of cancer, anxiety and mood disorders and in particular resistant depressions, addictions, Down syndrome, Alzheimer's disease, lupus erythematosus and proximal spinal muscular atrophy.

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