Hexokinase-derived peptides and their therapeutic uses

Abstract

The inventors previously demonstrated that mitochondrial VDAC1 directly induces Schwann cell demyelination via the MAPK pathway, as well as c-jun activation after sciatic nerve injury, diabetic neuropathy, and CMT1A. They found that reducing mitochondrial calcium release by blocking VDAC1 significantly reduced the number of demyelinated Schwann cells in vivo and improved nerve conduction as well as neuromuscular activity in diabetes, Guillain-Barré syndrome, and Charcot-Marie-Tooth disease models. Herein, the inventors precisely mapped the binding region of the N-terminal HK-1 helix through ala-scanning completed by deletion studies. Furthermore, they optimized the HK-derived peptide through stabilization of the helix by replacement of non-essential amino acids with α-aminoisobutyric acid (Aib), a known helix inducer. Furthermore, they described an in-house cellular screening assay based on the ability of MJ to separate HK from VDAC, which allows for the determination of peptide potency. Overall, their data support that N-terminal HK-derived peptides acting on VDAC are promising tools for the study of demyelinating processes. The present invention therefore relates to optimized HK-derived peptides and their use in the treatment of peripheral demyelinating diseases, myocardial diseases (Refs. 10, 11), cancer (Refs. 12, 13-15), diabetes (Refs. 14, 14-16), lupus-like diseases (Refs. 17), non-alcoholic fatty liver disease (Refs. 24, 25), chemically induced neuropathy (Ref. 9), Alzheimer's disease (Refs. 18, 19), Parkinson's disease (Ref. 20), Huntington's disease (Ref. 21), ALS (Refs. 22, 23), and more generally all neurodegenerative diseases associated with protein aggregation (Ref. 28).

Description

[Technical field]

[0001] The present invention relates to hexokinase (HK-) derived peptides and their therapeutic use, in particular for treating peripheral demyelinating or neurodegenerative diseases or cancer. [Background technology]

[0002] Voltage-dependent anion channels (VDACs) present in the outer mitochondrial membrane (OMM) are essential for the exchange of ions (ref. 1) and metabolites between mitochondria and the cytosolic cellular compartment (ref. 2). VDACs are transmembrane proteins with a β-barrel structure with an N-terminal helix lying perpendicular to the pore wall that influences channel permeability providing a molecular gate function (refs. 3, 4, 5). Movement of the VDAC N-terminal helix allows it to switch between open and closed states that affect cellular homeostasis. It also controls VDAC multimerization, which triggers apoptosis by release of cytosolic cytochrome C and calcium and activates caspases (refs. 6, 7). Moreover, VDAC is a privileged docking site for up to 200 proteins (Refs. 8, 9), some of which are involved in several pathologies, including cardiomyopathy (Refs. 10, 11), cancer (Refs. 12, 13-15), diabetes (Refs. 14, 14-16), lupus-like disease (Ref. 17), Alzheimer's disease (Refs. 18, 19), Parkinson's disease (Ref. 20), Huntington's disease (Ref. 21), ALS (Refs. 22, 23), non-alcoholic fatty liver disease (Refs. 24, 25), and chemically induced neuropathy (Ref. 9). Thus, VDAC constitutes a therapeutic target, and drugs capable of modulating its permeability or disrupting / enhancing its binding to partner proteins are being scrutinized. Among the proteins known to interact with VDAC, hexokinase (HK) I and II are the main ligands. Due to the pivotal role of this protein / protein interaction in several diseases, amino acids involved in the binding of both isoforms of HK to VDAC have been identified. The binding site is located within the first 20 amino acids of the N-terminal sequence of HK, and more precisely, the first 10 amino acids are essential (References 23, 26). Strong sequence homology has been observed between the N-terminal regions of HK1 and HK2, and both HK isoforms are known to be largely localized to the outer mitochondrial membrane (OMM) in cells.The mitochondrial fraction of HK and the HK-VDAC1 complex were found to be significantly decreased in neurodegenerative diseases, and several misfolded proteins involved in neurodegenerative diseases appear to bind to VDAC (Refs. 27, 19, 28). In light of this, VDAC1 was also identified as a key player in Schwann cell (SC) demyelination (Refs. 29, 30).

[0003] Schwann cells (SCs) are responsible for myelin production in the peripheral nervous system. These cells wrap around axons and maintain protective connections to them, allowing for accurate and efficient action potential transmission (Ref. 46). Unfortunately, there are numerous inherited and acquired demyelinating diseases of the peripheral nervous system (PNS) that affect an ever-increasing number of people (Ref. 47). Acquired demyelinating diseases are more prevalent, including diabetic peripheral neuropathy (Ref. 48), drug-associated peripheral demyelinating diseases, leprosy, and peripheral demyelinating diseases of inflammatory etiology (Ref. 49). Demyelinating peripheral neuropathy is a major complication of diabetes and causes significant morbidity (Ref. 50). The chronic form of this neuropathy is characterized by demyelination of Schwann cells and loss and / or degeneration of axons, resulting in slowed nerve conduction velocities (Refs. 51, 52). Furthermore, it has been reported that at least 50% of diabetic patients will develop one or more forms of diabetic neuropathy within 25 years of diagnosis (Reference 53).

[0004] The plant hormone methyl jasmonate (MJ) can dissociate HK from VDAC1 (ref. 31) and induce spontaneous demyelination (ref. 29). On the other hand, silencing VDAC in Schwann cells or treating them with the neuroprotective drug olesoxime (refs. 32, 33), which binds to VDAC, prevents mitochondrial calcium release and blocks demyelination (ref. 29). Restoring the tight HK / VDAC association therefore represents an attractive opportunity to treat several diseases in which VDAC permeability is implicated. In this particular context of amyotrophic lateral sclerosis, it has been reported that an N-terminal HK-1-derived peptide interacts with VDAC in vitro and in cellulo and prevents the VDAC / SOD1 G93A interaction (ref. 23). Furthermore, in the hereditary demyelinating peripheral neuropathy CMT4G, mutations in the 5' non-coding sequence of HK1 promote the expression of an alternatively spliced ​​isoform that lacks the normal N-terminus of HK1 (ref. 34). In peripheral blood mononuclear cells from CMT4G patients and in HEK293 cells mimicking the disease, this leads to a lack of interaction between HK and VDAC (Fig. 1A-C). Mutant HK1 does not block VDAC-mediated mitochondrial calcium release in HEK293 cells mimicking the disease (Fig. 2). Finally, a peptide derived from the N-terminus of wild-type HK1 can prevent mitochondrial calcium release after MJ treatment (Fig. 3), whereas a peptide derived from mutant HK1 has no effect (Fig. 3). Thus, it may be possible to use a peptide containing the N-terminal region of wild-type HK1 to block calcium efflux and stop the demyelinating process in several peripheral nerve diseases, such as CMT4G. This peptide may also be a therapeutic suggestion for all diseases involving VDAC permeability.

[0005] Herein, the inventors developed optimized HK-derived peptides with improved stability and affinity for VDACs, particularly VDAC1. Summary of the Invention

[0006] The present invention relates to an HK-derived peptide comprising the amino acid sequence: QX1X2X3YYX4 (SEQ ID NO: 1), X1 is leucine (L) or tryptophan (W); X2 is leucine (L) or tryptophan (W); X3 is alanine (A), D isomer alanine (A D ) or α-aminoisobutyric acid (U), X4 is phenylalanine (F), leucine (L) or tyrosine (Y).

[0007] In particular, the invention is defined by the claims. [Brief description of the drawings]

[0008] [Figure 1A-C]Amount of VDAC1 co-immunoprecipitating with HK in peripheral blood mononuclear cells (PBMCs) from patient blood and in HEK293 cells expressing wild-type HK or CMT4G mutant HK (or nothing - control). A. PBMCs from peripheral blood of CMT4G patients or controls were collected by centrifugation, washed, and lysed in detergent solution to extract proteins. HK was precipitated with a specific monoclonal antibody using sepharose beads coupled to G protein. After washing, the co-immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting using a polyclonal antibody against VDAC1. The amount of co-immunoprecipitated VDAC1 was normalized to the amount of protein in the cell lysate. B. Sequences of the major and alternatively spliced ​​AlT2 isoforms of human HK1 showing the contribution of exons 1 and 2, and alternative exons T3 and T4, to the N-terminal sequence of each isoform. See Hantke, J. et al. 2009. C. HEK293 cells were transfected with plasmids expressing wild-type (wt) Flag-tagged human HK1 or CMT4G mutant Flag-tagged human HK1. After 48 h, cells were washed and lysed with detergent solution to extract proteins. HK was precipitated with monoclonal anti-Flag antibody using Sepharose beads coupled to G protein. After washing, co-immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting using a polyclonal antibody against VDAC1. The amount of co-immunoprecipitated VDAC1 was normalized to the amount of protein in the cell lysate. [Diagram 2]Fluorescence intensity of a mitochondrial calcium probe in HEK293 cells overexpressing wild-type HK or CMT4G mutant HK (or nothing - control). HEK293 cells were transfected with a plasmid expressing mito-GCaMP2, a fluorescent probe that detects calcium in the mitochondrial matrix, alone (control) or together with a plasmid expressing wild-type Flag-tagged human HK1 or CMT4G mutant Flag-tagged human HK1. After 48 h, cells were washed, fixed with paraformaldehyde, and treated with DAPI to detect nuclei. GFP fluorescence was recorded using an LSM700 Zeiss confocal microscope and normalized to background values ​​for each picture. [Diagram 3] Time-lapse recording of the fluorescence intensity of a mitochondrial calcium probe in HEK293 cells treated with methyl jasmonate (MJ, 6 mM) and the N-terminal peptide of wild-type HK1 (HK1-Nt peptide) or the N-terminal peptide of mutant HK1 (HKmut-Nt peptide). HEK293 cells were transfected with a plasmid expressing mito-GCaMP2. After 48 h, cells were imaged using a Zeiss Axio-observer designed for live imaging and treated with MJ (6 mM) and / or 5 mM peptide. Peptide sequences: wild-type HK1 peptide Ac-MIAAQLLAYYFTELKGRKKRRQRRRPPQ-NH2 (SEQ ID NO: 90), CMT4G mutant HK1 peptide Ac-MGQICQRESATAAEKGRKKRRQRRRPPQ-NH2 (SEQ ID NO: 91) and control peptide Ac-GRKKRRQRRRPPQ-NH2 (SEQ ID NO: 92). [Figure 4] Peptide libraries 1-6 designed to optimize binding to VDAC. 1a and 2a represent the initial sequences of peptides that were subjected to ala scanning, deletion, optimization and stabilization assays. nL represents norleucine, a non-oxidative substitute for methionine. tat sequences are highlighted in blue, and NHK1 recognition sequences are shown in red and preceded by the sequence number. [Diagram 5]Quantification of fluorescence levels over time of mitoGCaMP2 (A) and GCaMP2 (B) in mitochondria and in the cytosol, respectively. Quantification of fluorescence levels in HEK-293 cells transfected with mitoGCaMP2 (A) and GCaMP2 (B) probes. Controls (circles) represent mitochondrial fluorescence levels upon treatment of cells with MJ and the diluents 0.1 DMSO and 5% EtOH used for solubilization of the compounds. MJ was tested at 6 mM and compound 1a at 33 μM. Statistical analysis using two-way ANOVA followed by Tukey's multiple comparison test (N=3 independent experiments). Results are expressed as mean ± SEM. **p<0.01, ****p<0.0001, ns indicates no significant difference, AU is arbitrary unit. [Figure 6A-D] Figure 6 shows the effect of ala scan studies (A, B) and deletion studies (C, D) on compounds 1a and 2a. All compounds were tested by screening assay at 10 μM (N=5 independent experiments). ala scan substitutions are displayed in bold. Statistical analysis showing one-way ANOVA followed by Dunnett's multiple comparison test between compounds 1a (A and C dark grey plots) or 2a (B and D dark grey plots) and other compounds. Blue plots represent compounds where ala scan studies revealed key amino acids involved in the interaction with VDAC or deletion studies caused significant loss of activity. *p<0.05, **p<0.01, ***p<0.001. Statistical tests were not significant (white plots) unless otherwise stated. Results are expressed as mean ± SD. AU is arbitrary unit. [Figure 7A-B]Effect of combinations of isomeric substitutions on amino acids involved in VDAC interaction in compounds 3c (A) and 4d (B). Substitutions are displayed in bold. All compounds were tested at 3 μM (N=3 independent experiments) except compounds 3c and 4d, which were tested at 10 μM (dark grey plots) and 3 μM (light grey plots). Statistical analysis showing one-way ANOVA between compounds 3c or 4d at 3 μM and other compounds followed by Dunnett's multiple comparison test. Red plots represent compounds where the combination of isomeric substitutions led to the most significant increase in activity. *p<0.05, **p<0.01. Statistical tests were not significant unless otherwise stated. Results are expressed as mean ± SD. AU is arbitrary unit. [Figure 8A-C] A) Structure of the N-terminal modification introduced in 7f. Effect of the introduction of the helicogenic Aib(U) in the 3c or 5x sequence. The modified amino acids are indicated in bold. All compounds were tested at 10 μM (A) and 3 μM (B). The control condition (dotted line) represents the mitochondrial fluorescence level when cells were treated with MJ and the diluents 0.1 DMSO and 5% EtOH used for solubilization of the compounds. MJ (line) represents the mitochondrial fluorescence level when cells were treated with only 6 mM MJ. Statistical analysis using one-way ANOVA followed by Dunnett's multiple comparison test (N=3 independent experiments) between 3c or 5x and the other compounds (10 μM shown in dark grey, 3 μM in light grey). The red plots correspond to peptides showing a significant increase in activity. Results are expressed as mean ± SD. *p<0.05, ***p<0.001. ns indicates no significant difference, AU is arbitrary unit. [Figure 9] Effect of SAR optimization studies on mitochondrial Ca2+ efflux via VDAC showing improved activity. The graph shows representative dose-response curves for compounds 1a, 5x, and 7g in screening assays. IC50 is shown for each compound (N=3 independent experiments). Results are expressed as mean ± SD. AU is arbitrary unit. [Figure 10]Stability study of NHKI-derived sequences (3c', 7a', 7d', 7f'-g') against rat serum (N=3 independent experiments). All peptides were tested at a concentration of 66.6 μmol / L in the presence of 25% (v / v) rat serum and water after 24 h incubation at 37 °C. Error bars indicate standard deviation. [Figure 11] Effects of NHKI-derived peptides 3c, 5x, 7d and 7g on sciatic nerve explants cultured in medium supplemented with serum. (A) Representative CARS images showing myelin (green) of an intact sciatic nerve that was harvested and immediately fixed in 4% PFA, and a sciatic nerve explant cultured in medium supplemented with FBS, referred to as a negative control. (B) Representative CARS images showing myelin (green) of a sciatic nerve explant cultured for 24 h in medium containing 3 μM of NHKI-derived peptides and supplemented with FBS. All nerves are represented in longitudinal section. Healthy myelin sheaths (white arrows), nodes of Ranvier (white stars), and myelin globules (orange arrows) are shown. Scale bar: 20 μm. (C) Graphs showing the percentage of damaged fibers in the intact nerve (white plots), the negative control (light gray plots), 3c and its analog 7d (blue plots), and 5x and its analog 7g (red plots). (N=3 independent experiments). Results are expressed as mean ± SD. Statistical analysis using one-way ANOVA followed by Dunnett's multiple comparison test. *p<0.05, **p<0.01. ns indicates no significant difference. [Figure 12] AAV9 represents an efficient way to sustain the expression of anti-demyelinating peptides in target cells. HEK293 cells were infected with control AAV9 or AAV9-HK peptide or were left uninfected. After 2 days, cells were incubated with the fluorescent dye Rhod-2, which fluoresces with calcium in mitochondria. After 15 min, infected cells were incubated with methyl jasmonate (6 mM) and non-infected cells were incubated with methyl jasmonate (6 mM) + 5z peptide (5 μM) for 40 min. Pictures imaging the Rhod-2 dye were taken every 5 min. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The inventors precisely mapped the binding region of the N-terminal HK-1 helix through an ala scan completed by deletion studies. Furthermore, they optimized the HK-derived peptide through stabilizing the helix by replacement of non-essential amino acids with α-aminoisobutyric acid (Aib), a known helix inducer. Furthermore, they described an in-house cellular screening assay based on the ability of MJ to separate HK from VDAC, allowing the potency of the peptide to be determined. Overall, their data confirm that N-terminal HK-derived peptides acting on VDAC are promising tools for the study of demyelination processes.

[0010] <Peptides of the Invention> The present invention relates to an HK-derived peptide comprising the amino acid sequence: Alanine (A)-Glutamine (Q)-X1-X2-X3-Tyrosine (Y)-Tyrosine (Y)-X4 (SEQ ID NO: 1), X1 is leucine (L) or tryptophan (W); X2 is leucine (L) or tryptophan (W); X3 is alanine (D)-isomer alanine (A D ) or α-aminoisobutyric acid (U), X4 is phenylalanine (F), leucine (L) or tyrosine (Y).

[0011] The term "hexokinase" (HK) as used herein has its general meaning in the art and refers to an enzyme that phosphorylates hexoses to form hexose phosphates. Hexokinase I and II are two isoforms of hexokinase and are the primary ligands for VDAC.

[0012] As used herein, the term "VDAC" has its common meaning in the art and refers to voltage-dependent anion-selective channel protein 1. VDAC is a major component of the outer mitochondrial membrane, facilitating the exchange of metabolites and ions across the outer mitochondrial membrane, and may regulate mitochondrial function as well as cell physiology and differentiation. The protein also forms multimeric channels in the cell membrane and may be involved in apoptosis and transmembrane electron transport. Alternative splicing results in multiple transcript mutations. VDAC has numerous binding partners that control its permeability, especially hexokinase (HK). Binding of HK to VDAC reduces the permeability of the pore, especially to calcium. VDAC includes three VDAC isoforms: VDAC1, VDAC2 and VDAC3.

[0013] The term "peptide" as used herein corresponds to a chemical entity belonging to the protein family. Peptides are composed of a mixture of several amino acids. Depending on the number of amino acids involved, peptides are classified as dipeptides, which are composed of two amino acids, tripeptides, which are composed of three amino acids, etc. Peptides composed of 10 or more amino acids are called polypeptides. Thus, the peptides of the present invention can be considered as polypeptides.

[0014] The peptides according to the invention can be produced by conventional automated peptide synthesis methods or by recombinant expression. The general principles for designing and making proteins are well known to those of skill in the art.

[0015] The peptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols described in Stewart and Young; Tam et al., 1983, Merrifield, 1986, and Barany and Merrifield, Gross and Meienhofer, 1979. The peptides of the present invention can also be synthesized by solid-phase techniques using exemplary peptide synthesizers. The purity of any protein produced by automated peptide synthesis or recombinant methods can be measured using reverse-phase HPLC analysis. The chemical authenticity of each peptide can be established by any method known to those skilled in the art. As an alternative to automated peptide synthesis, recombinant DNA techniques can be used in which a nucleotide sequence encoding a selected protein is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression as described herein below. Recombinant methods are particularly preferred for producing longer polypeptides. A variety of expression vector / host systems can be utilized to contain and express the peptide or protein coding sequence. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors (Giga-Hama et al., 1999), insect cell systems infected with viral expression vectors (e.g., baculovirus, see Ghosh et al., 2002), plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids; see, e.g., Babe et al., 2000), or animal cell systems. Those skilled in the art are aware of various techniques for optimizing mammalian protein expression, as exemplified by Kaufman, 2000; Colosimo et al., 2000.Mammalian cells useful for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Exemplary protocols for recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are known to those of skill in the art and are briefly described below. U.S. Patent Nos. 6,569,645; 6,043,344; 6,074,849; and 6,579,520 provide specific examples of recombinant production of peptides, and these patents are expressly incorporated herein by reference for their teachings. Mammalian host systems for expressing recombinant proteins are also well known to those of skill in the art. Host cell lines may be selected based on their particular ability to process the expressed protein or to generate particular post-translational modifications useful for providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cuts off the "prepro" form of a protein, may also be important for correct insertion, folding and / or function. Various host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular and characteristic mechanisms for such post-translational activity, and can be selected to ensure the correct modification and processing of the introduced foreign protein.

[0016] As used herein, the term "amino acid" refers to natural or unnatural amino acids that are D and L stereoisomers of chiral amino acids. It is understood to refer to both amino acids and the corresponding amino acid residues, for example, as present in peptidyl structures. Natural and unnatural amino acids are well known in the art. Common naturally occurring amino acids include, but are not limited to, alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Uncommon and unnatural amino acids include, but are not limited to, α-aminoisobutyric acid (Aib, U), allylglycine (AllylGly), norleucine (Nle), norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphthylalanine (2-Nal), ornithine (Orn), cyclohexylalanine (Cha, Fx), and pentafluorophenylalanine.

[0017] In some embodiments, the HK-derived peptides of the invention comprise 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.

[0018] In some embodiments, the HK-derived peptides of the invention do not consist of the amino acid sequence: alanine (A)-glutamine (Q)-leucine (L)-leucine (L)-alanine (A)-tyrosine (Y)-tyrosine (Y)-phenylalanine (F) (SEQ ID NO: 95).

[0019] In some embodiments, the HK-derived peptides of the invention do not consist of or contain the sequence alanine(A)-alanine(A)-glutamine(Q)-leucine(L)-leucine(L)-alanine(A)-tyrosine(Y)-tyrosine(Y)-phenylalanine(F)-threonine(T)-glutamic acid(E)-leucine(L)-lysine(K) (SEQ ID NO:96).

[0020] In some embodiments, the HK-derived peptide comprises the amino acid sequence: Alanine (A)-Glutamine (Q)-X1-X2-X3-Tyrosine (Y)-Tyrosine (Y)-X4-Threonine (T)-Glutamic acid (E)-X5-Lysine (K) (SEQ ID NO: 2); X1 is leucine (L) or tryptophan (W); X2 is leucine (L) or tryptophan (W); X3 is alanine (A), D isomer alanine (A D ), or α-aminoisobutyric acid (U), X4 is phenylalanine (F), leucine (L) or tyrosine (Y); X5 is leucine (L) or tryptophan (W).

[0021] In some embodiments, X3 is α-aminoisobutyric acid (U).

[0022] In some embodiments, the HK-derived peptide comprises or consists of an amino acid sequence in Table 1.

[0023] [Table 1-1]

[0024] [Table 1-2]

[0025] In some embodiments, the HK-derived peptide comprises or consists of the amino acid sequence of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:29.

[0026] Furthermore, we demonstrated the importance of the AUAU patch or 3-CF3-Ph[Tz]Aib fused to the N-terminus to enhance the stability of the HK-derived peptide (see Figure 7). We also demonstrated that the replacement of the second alanine with α-aminoisobutyric acid is important to enhance the stability of the HK-derived peptide.

[0027] In our previous work, we showed that the N-terminal capping 3-CF3Ph[Tz]U dipeptide promoted peptide insertion into membranes (see Figure 4A and Das et al., Chemistry, December 24, 2017).

[0028] As used herein, the term 3-CF3Ph[TZ]U dipeptide has its general meaning in the art and refers to 2-methyl-2-{4-[(3-trifluoromethyl)phenyl]-1H-1,2,3-triazol-1yl}propanoic acid, which has the formula C 16 H 16 It is also known as 1,4-disubstituted-1,2,3-triazole combined with α-aminoisobutyric acid, represented as F3N3O.

[0029] [ka]

[0030] In some embodiments, the sequence AUAU (SEQ ID NO:54) or AU (SEQ ID NO:55) is attached to the HK-derived peptide.

[0031] In some embodiments, the sequence AUAU (SEQ ID NO:54) or AU (SEQ ID NO:55) is attached to the N-terminus of the HK-derived peptide.

[0032] In some embodiments, the sequence AUAU (SEQ ID NO:54) or AU (SEQ ID NO:55) is attached to the C-terminus of the HK-derived peptide.

[0033] In some embodiments, the dipeptide 3-CF3Ph[Tz]U is attached to the N-terminus of the HK-derived peptide.

[0034] In some embodiments, a cell membrane penetrating sequence is attached to the HK derived peptide.

[0035] The term "cell membrane penetrating sequence" as used herein has its general meaning in the art and refers to a short sequence that facilitates cellular intake and uptake of the peptides of the present invention. Based on the origin of the peptide, CPPs are classified as chimeric, protein-derived and synthetic. Cell membrane penetrating sequences include, but are not limited to, penetratin, octaarginine (R8), tat, transportan and xentry. Penetratin is a first generation cell membrane penetrating peptide derived from the Drosophila antennapedia homeodomain. Penetratin overcomes the barrier of the cell membrane of mammalian cells through the macropinocytosis pathway and efficiently delivers molecular cargo in a biologically active form. The tat peptide is derived from the transcriptional activator (tat) of human immunodeficiency virus. TAT is an arginine-rich peptide that directly penetrates cell membranes and stabilizes DNA. Transportan is a chimeric CPP derived from galanin and mastoparan. Zentry is a short peptide derived from the N-terminal region of the X protein of the Hepatitis B virus. Zentry penetrates adherent cells using syndecan-4 as a portal of entry. Horton peptide is a synthetic cell membrane penetrating peptide that can enter mitochondria. The sequence of MPP was designed to exhibit two properties known to be important for crossing both the cellular and mitochondrial membranes, namely positive charge and lipophilicity as described by Horton et al, Chem Biol. 2008 (ref. 58).

[0036] In some embodiments, the cell membrane permeable sequence is attached to the N-terminus or C-terminus of the HK-derived peptide.

[0037] In some embodiments, the cell membrane permeable sequence consists of a sequence in Table 2:

[0038] [Table 2]

[0039] In some embodiments, the cell membrane permeable sequence is tat (SEQ ID NO:58).

[0040] In some embodiments, the sequence AUAU (SEQ ID NO:54) or AU (SEQ ID NO:55) is attached to the N-terminus of the HK-derived peptide, and the cell membrane permeable sequence is attached to the C-terminus of the HK-derived peptide.

[0041] In some embodiments, the sequence AUAU (SEQ ID NO:54) or AU (SEQ ID NO:55) is attached to the C-terminus of the HK-derived peptide and the cell membrane penetrating sequence is attached to the N-terminus of the HK-derived peptide.

[0042] In some embodiments, the dipeptide 3-CF3Ph[Tz]U is attached to the N-terminus of the HK-derived peptide, and the cell membrane penetrating sequence is attached to the C-terminus of the HK-derived peptide.

[0043] In some embodiments, the HK-derived peptide comprises or consists of the amino acid sequence of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:29, and the dipeptide 3-CF3Ph[Tz]U is attached to the N-terminus of the HK-derived peptide.

[0044] In some embodiments, the HK-derived peptide comprises or consists of the amino acid sequence of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:29, and the sequence AUAU (SEQ ID NO:54) or the sequence AU (SEQ ID NO:55) is linked to the N-terminus of the HK-derived peptide.

[0045] In some embodiments, the HK-derived peptide comprises or consists of the amino acid sequence of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:29, wherein the dipeptide 3-CF3Ph[Tz]U is attached to the N-terminus of the HK-derived peptide and the cell membrane permeable sequence is attached to the C-terminus of the HK-derived peptide.

[0046] In some embodiments, the HK-derived peptide comprises or consists of the amino acid sequence of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:29, wherein the sequence AUAU (SEQ ID NO:54) or the sequence AU (SEQ ID NO:55) is attached to the N-terminus of the HK-derived peptide, and the cell membrane permeable sequence is attached to the C-terminus of the HK-derived peptide.

[0047] In some embodiments, the HK-derived peptide comprises or consists of the amino acid sequence of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:29, wherein the sequence AUAU (SEQ ID NO:54) or the sequence AU (SEQ ID NO:55) is attached to the C-terminus of the HK-derived peptide, and the cell membrane permeable sequence is attached to the N-terminus of the HK-derived peptide.

[0048] In some embodiments, the cell membrane permeable sequence is tat (SEQ ID NO:58).

[0049] In a second aspect, the present invention relates to a vector comprising the HK-derived peptide of the present invention.

[0050] Typically, the peptide can be delivered in combination with a vector. The HK-derived peptide of the present invention is contained in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector. Thus, a further object of the present invention relates to a vector comprising the peptide of the present invention. Typically, the vector is a viral vector, which is an adeno-associated virus (AAV), a retrovirus, a bovine papilloma virus, an adenovirus vector, a lentivirus vector, a vaccinia virus, a polyoma virus, or an infectious virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO and variants thereof. The AAV vector can have one or more AAV wild-type genes, preferably with the rep gene and / or the cap gene deleted in whole or in part, but with functional adjacent ITR sequences. Retroviruses are potential gene delivery vectors of choice because they can transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines to integrate their genes into the host genome. To construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in place of a specific viral sequence, generating a replication-defective virus. To produce virions, a packaging cell line is constructed that contains the gag, pol and / or env genes but does not contain the LTR and / or packaging components. When a recombinant plasmid containing cDNA is introduced into this cell line along with the retroviral LTR and packaging sequences (e.g., by calcium phosphate precipitation), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles that are then secreted into the medium. The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a wide variety of cell types.Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. The increased complexity allows the virus to regulate its life cycle, such as during latent infection. Examples of lentiviruses include human immunodeficiency viruses (HIV1, HIV2) and simian immunodeficiency viruses (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV pathogenicity genes, e.g., genes env, vif, vpr, vpu, nef have been deleted, making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, vectors are plasmid-based or virus-based and are constructed to carry sequences essential for the integration, selection, and introduction of foreign nucleic acid into a host cell. The gag, pol, and env genes of a vector of interest are also known in the art. Thus, the relevant genes are cloned into a selected vector and used to transform the target cell of interest. In recombinant lentiviruses capable of infecting non-dividing cells, suitable host cells are transfected with two or more vectors carrying packaging functions, i.e., gag, pol and env, as well as rev and tat, and this lentivirus is described in U.S. Pat. No. 5,994,136, which is incorporated herein by reference. This describes a first vector capable of providing nucleic acid encoding the viral gag and pol genes, and another vector capable of providing nucleic acid encoding the viral env that produces packaging cells. The vector providing the heterologous gene is introduced into the packaging cells, resulting in producer cells that release infectious viral particles carrying the foreign gene of interest. env is preferably an amphotropic envelope protein that allows transduction of cells of human and other species.Typically, the nucleic acid molecule or vector of the invention comprises a "control sequence," which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively are responsible for the replication, transcription, and translation of the coding sequence in the recipient cell. Not all of these control sequences need always be present, so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell. Another nucleic acid sequence is a "promoter" sequence, which, as used herein in the usual sense, refers to a nucleotide region that contains a DNA regulatory sequence, the regulatory sequence being derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0051] In some embodiments, the vector is an adeno-associated virus (AAV).

[0052] In some embodiments, the vector is AAV9 or AAVrhlO.

[0053] <Treatment method> The formation of myelin sheaths around peripheral nerve axons by Schwann cells is essential for the rapid propagation of action potentials. Several peripheral neuropathies have the process of demyelination as a pathological physiology. We previously demonstrated that mitochondrial VDAC1 directly induces Schwann cell demyelination via the MAPK pathway, as well as c-jun activation after sciatic nerve injury, diabetic neuropathy, and CMT1A. They found that reducing mitochondrial calcium release by blocking VDAC1 significantly reduced the number of demyelinated Schwann cells in vivo and improved nerve conduction as well as neuromuscular activity in diabetes, Guillain-Barré syndrome, and Charcot-Marie-Tooth disease models.

[0054] Therefore, restoring the close HK / VDAC relationship represents an attractive opportunity for different peripheral demyelinating diseases and all other diseases in which VDAC permeability is involved.

[0055] That is, the present invention relates to an HK-derived peptide or vector of the present invention for use as a pharmaceutical.

[0056] In other words, the present invention relates to an HK-derived peptide or vector of the present invention for use in therapy.

[0057] More specifically, the present invention relates to an HK-derived peptide or vector of the present invention for use in the treatment of peripheral demyelinating diseases.

[0058] In other words, the present invention relates to a method for treating a peripheral demyelinating disease in a subject in need of treatment, comprising administering a therapeutically effective amount of the HK-derived peptide of the present invention or the vector of the present invention to the subject.

[0059] As used herein, the term "subject" refers to a human or another mammal (e.g., a mouse, rat, rabbit, hamster, dog, cat, cow, pig, sheep, horse, or primate). In some embodiments, the subject is a human. Typically, the subject suffers from or may suffer from a disease affecting the peripheral nervous system. Typically, the subject suffers from or may suffer from a peripheral demyelinating disease.

[0060] As used herein, the term "treatment" or "treating" refers to both curative or disease-modifying treatment and prophylactic or preventive treatment, including treatment of subjects at risk of or suspected of having a disease, and subjects diagnosed as suffering from a disease or medical condition, as well as suppression of clinical recurrence. Treatment may be administered to a subject with a medical disorder or a subject who may eventually develop a disorder to prevent, cure, delay the onset, reduce the severity, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. "Treatment regimen" refers to a pattern of treatment of a disease, e.g., a dosing pattern used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the subject during the initial period of the treatment regimen. Induction regimen may adopt (partially or entirely) "loading regimen", which may include administering a larger amount of drug than the doctor adopts during maintenance regimen, administering drug more frequently than the doctor administers drug during maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a part of a treatment regimen) used to maintain a subject during disease treatment, for example, to keep the subject in remission for an extended period of time (months or years). Maintenance regimen may adopt continuous therapy (e.g., administering drug at regular intervals such as weekly, monthly, yearly, etc.), or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment when certain predetermined criteria are achieved [e.g., disease manifestation, etc.]).

[0061] As used herein, a "therapeutically effective amount" refers to the minimum amount of active agent (i.e., the peptide of the present invention) required to provide a therapeutic effect to a patient. For example, a "therapeutically effective amount of an active agent" for a patient is an amount of active agent that induces, promotes, or causes an improvement in pathological symptoms, disease progression, or physical condition associated with a disease affecting the patient. It will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for a particular patient will vary depending on a variety of factors, including the patient's age, weight, general health, sex, diet; administration time, route of administration, excretion rate of the particular compound used; duration of treatment; drugs used in combination with or simultaneously with the particular polypeptide used; and similar factors well known in the medical field.

[0062] As used herein, the term "peripheral demyelinating disease" has its general meaning in the art and refers to a series of diseases involving substantial damage to axons and glial cells, particularly Schwann cells (SCs) in the peripheral nervous system (PNS). The wide variety of forms exhibited by peripheral demyelinating disease may each be uniquely attributed to an equally wide variety of causes. For example, peripheral demyelinating disease may be genetically generated ("genetic peripheral demyelinating disease"), may be caused by systemic disease, or may be induced by toxic or infectious agents ("acquired peripheral demyelinating disease").

[0063] The methods of the present invention are broadly applicable to the treatment or prevention of peripheral demyelinating diseases that affect the regulation of peripheral nerves, including peripheral ganglion neurons, sympathetic neurons, sensory neurons, and myelinated motor and sensory neurons.

[0064] In particular, the methods of the invention are useful for treatments designed to rescue, for example, the ocular nerves, the inner ear and auditory nerves, and myelinated motor and sensory neurons. In particular, the methods of the invention are particularly suited to preventing demyelination of peripheral nerves.

[0065] The peptides of the present invention are suitable for the treatment of inherited peripheral demyelinating diseases.

[0066] Inherited peripheral demyelinating diseases are caused by genetic abnormalities that are passed from generation to generation. For some of these, the genetic defect is known and tests are available for diagnosis and prenatal counseling. In particular, the diagnosis of inherited peripheral demyelinating diseases is usually suggested by early onset of neuropathic symptoms, especially if the family history is positive. Prior to recent genetic advances, the diagnosis was supported by typical findings of markedly decreased nerve conduction on electromyography and nerve biopsy. Typical findings on nerve biopsy include the presence of so-called onion-like structures, which indicate repeated demyelination and remyelination of nerve fibers. There are several inherited neuropathies that are directly or indirectly associated with peripheral nerve demyelination. Examples include, but are not limited to, Lifsum disease, abetalipoproteinemia, Tangier disease, Krabbe disease, metachromatic leukodystrophy, Charcot-Marie-Tooth (CMT) disease, Fabry disease, hereditary neuropathy with pressure palsies (HNPP), familial amyloid neuropathy, hereditary sensory neuropathy type II (HSN II), hereditary porphyrias, muscular dystrophies such as congenital muscular dystrophy 1A, and Dejerine-Sottas syndrome.

[0067] In some embodiments, the inherited demyelinating disease is Charcot-Marie-Tooth (CMT) disease.

[0068] CMT disease is the most common inherited neurological disorder. It is characterized by muscle weakness and atrophy due to partial demyelination of peripheral nerves and associated degeneration of axons and anterior horn cells. In the past 15 years, knowledge of the genetic basis of Charcot-Marie-Tooth disease (CMT) has increased significantly, with more than 60 genes now known. A regularly updated list can be found at http: / / www.molgen.ua.ac.be / CMTMutations / Home / IPN.cfm. Autosomal dominant inheritance is common, and associated central nervous system degenerative disorders, such as Friedreich's ataxia, are also common. In some embodiments, the peptides of the invention can be used to treat Charcot-Marie-Tooth disease types 4G and 1A.

[0069] The peptides of the invention are also suitable for the treatment of acquired peripheral demyelinating diseases.

[0070] Acquired peripheral demyelinating diseases have their general meaning in the art and include, but are not limited to, diabetic neuropathy, immune-mediated neuropathies; acute and chronic motor neuropathies; acute and chronic sensory neuropathies; acute and chronic autonomic neuropathies; and Miller-Fisher syndrome, which presents with gaze palsies, incoordination and unsteady gait.

[0071] In some embodiments, the peptides of the present invention are used to treat diabetic neuropathy. Diabetes is the most common known cause of neuropathy. Approximately 50% of diabetic patients are symptomatic. In most cases, neuropathy is primarily sensory, with pain and loss of sensation in the hands and feet. However, some diabetic patients suffer from chronic demyelinating neuropathy, mononeuritis or mononeuritis multiplex, which causes weakness of one or more nerves, or lumbosacral plexopathy or muscle atrophy, which causes weakness, inflammation, necrosis and abscesses in the legs.

[0072] In some embodiments, the peptides of the invention are used to treat immune-mediated neuropathies. The main function of the immune system is to protect the body from infectious microorganisms that invade from the outside. In some cases, however, the immune system may rebel against the body, resulting in autoimmune diseases. The immune system is composed of several types of white blood cells, including T lymphocytes (which also control the immune response); and B lymphocytes or plasma cells, which secrete special proteins called "antibodies." Sometimes, for unknown reasons, the immune system may mistakenly attack parts of the body, such as peripheral nerves. "This is an 'autoimmune' peripheral neuropathy. There are several different types, depending on the part of the peripheral nerve that is attacked and the type of immune response. For example, the method of the invention is suitable for treating Guillain-Barre syndrome (GBS). It is an acute neurological disorder that develops suddenly or rapidly. Guillain-Barre syndrome may progress to paralysis and respiratory failure within days or weeks of onset. Neuropathy is caused by the immune system destroying the myelin sheath of motor and sensory nerves. It is often preceded by an infection, vaccination, or trauma that is thought to trigger an autoimmune reaction. The disease is self-limited, with spontaneous recovery within 6 to 8 weeks. However, recovery is often incomplete.

[0073] Another acquired peripheral demyelinating disease that may be treated by the peptides of the present invention is chronic inflammatory demyelinating polyneuropathy (CIDP). CIDP is considered to be a chronic, more indolent form of Guillain-Barre syndrome. The disease may have repeated attacks, called relapses, or progress in a stepwise or steady manner. As with GBS, there appears to be destruction of the myelin sheath by antibodies and T lymphocytes. However, there is no specific test for CIDP, so the diagnosis is based on clinical and laboratory features.

[0074] Chronic polyneuropathy with antibodies against peripheral nerves is another acquired peripheral demyelinating disease in which the peptides of the invention can be used. In several types of chronic neuropathies, antibodies against specific components of the nerves have been identified. These include demyelinating peripheral diseases associated with antibodies against myelin-associated glycoprotein (MAG), motor neuropathies associated with antibodies against gangliosides GM1 or GDla, and sensory neuropathies associated with anti-sulfatide or GD1b ganglioside antibodies. The antibodies in these cases bind to oligosaccharides or sugar-like molecules that are attached to proteins (glycoproteins) or lipids (glycolipids or gangliosides) in the nerves.

[0075] The peptides of the present invention can also be used to treat peripheral demyelinating diseases associated with vasculitis or inflammation of blood vessels in peripheral nerves. Peripheral demyelinating diseases can be caused by vasculitis, which is inflammation of blood vessels in peripheral nerves. It can cause small "strokes" along the peripheral nerve pathways and can be limited to the nerve or can be systemic, including skin rashes and affecting other organs. Some rheumatic diseases, such as rheumatoid arthritis, lupus, periarteritis nodosa or Sjogren's syndrome, are associated with systemic vasculitis and can affect peripheral nerves. Vasculitis can cause polyneuritis, mononeuritis or mononeuritis multiplex, depending on the distribution and severity of the lesions.

[0076] In some embodiments, the method of the invention is suitable for the treatment of peripheral demyelinating diseases associated with monoclonal gammopathy, in which a single clone of B cells or plasma cells in the bone marrow or lymphoid organs proliferates to form benign or malignant tumors and secretes antibodies. "Monoclonal" refers to the presence of a single clone of antibodies. And "Gammopathy" corresponds to immunoglobulin, another name for antibodies. In some cases, the antibodies react with neural components, and in other cases, fragments of the antibodies form amyloid deposits.

[0077] In some embodiments, the methods of the invention are suitable for the treatment of peripheral demyelinating disease associated with a tumor or neoplasm. Neuropathy may be due to direct invasion of the nerve by tumor cells or indirect effects of the tumor. The latter is called paraneoplastic neuropathy. Several types have been described. For example, the methods of the invention can be used to manage sensory neuropathy associated with lung cancer. Similarly, the methods of the invention can be used to treat peripheral demyelinating disease associated with multiple myeloma. In some embodiments, the methods of the invention are suitable for the treatment of peripheral demyelinating disease associated with Waldenstrom's macrocytosis, chronic lymphocytic leukemia or B-cell lymphoma. In some embodiments, the methods of the invention are used as part of a treatment protocol for the treatment of patients with cancer with peripheral demyelinating disease that is the result of local irradiation or caused by chemotherapeutic agents. Chemotherapeutic agents known to cause sensory and / or motor neuropathy include vincristine, an antineoplastic drug used in the treatment of hematological malignancies and sarcomas, along with cisplatin, taxol, etc. Neurotoxicity is dose-related and manifests as decreased intestinal motility and peripheral neuropathy, especially in the distal muscles of the limbs, orthostatic hypotension, as well as bladder atony. Similar problems have been reported with taxol and cisplatin (MoUman, JE, 1990, New Eng Jour Med. 322:126-127), but cisplatin-associated neurotoxicity can be reduced with nerve growth factor (NGF) (Apfel, SC et al. 1992, Annals of Neurology 31:76-80). Neurotoxicity may be reversible after removal of the neurotoxic agent, but recovery can be a very slow process (Legha, S., 1986, Medical Toxicology 1:421-427; Olesen, et al, 1991, Drug Safety 6:302- 314).

[0078] In some embodiments, the methods of the present invention are suitable for the treatment of peripheral demyelinating diseases caused by drugs such as chloroquine, FK506 (tacrolimus), perhexiline, procainamide, and zimeldine.

[0079] In some embodiments, the method of the present invention is suitable for treating peripheral demyelinating diseases caused by infection. Peripheral demyelinating diseases can be caused by infection of peripheral nerves. Viruses that cause peripheral demyelinating diseases include the AIDS virus or HIV-I, which causes a slowly progressive sensory neuropathy, the cytomegalovirus, which causes a rapidly progressive paralytic neuropathy, the varicella zoster virus, which causes shingles, and the poliovirus, which causes a motor neuropathy. Hepatitis B or C infections can be accompanied by vasculitic neuropathy. Bacterial infections that cause neuropathy include leprosy, which causes a patchy sensory neuropathy, and diphtheria, which causes a rapidly progressive paralytic neuropathy. Other infections that cause neuropathy include Lyme disease, which is caused by a spirochete, and trypanosomiasis, which is caused by a parasitic worm. Both are generally accompanied by multifocal neuropathy.

[0080] In some embodiments, the peptide of the present invention is suitable for treating peripheral demyelinating diseases caused by nutritional imbalance.For example, deficiency of vitamin B12, vitamin B1 (thiamine), vitamin B6 (pyridoxine) or vitamin E can cause polyneuropathy with degeneration of peripheral nerve axons.This can be caused by poor diet or reduced ability to absorb nutrients from stomach or intestine.In addition, high intake of vitamin B6 can cause peripheral demyelinating diseases, and the peptide of the present invention can be used as part of detoxification program in such cases.

[0081] In some embodiments, the peptides of the present invention are suitable for the treatment of peripheral demyelinating diseases occurring in renal disease. Chronic renal failure can lead to peripheral neuropathy, mainly sensory, involving degeneration of peripheral nerve axons.

[0082] In some embodiments, the peptides of the present invention are suitable for treating hypothyroidism neuropathy.Hypothyroidism may be associated with sensory polyneuropathy, which involves axonal degeneration and causes pain.Mononeuropathies or multiple mononeuropathy may also occur due to compression of peripheral nerves by swollen tissue.

[0083] In some embodiments, the peptides of the present invention are suitable for the treatment of peripheral demyelinating diseases caused by alcohol and toxins. Certain toxins can cause peripheral neuropathy. Lead toxicity is associated with motor neuropathy; arsenic and mercury cause sensory neuropathy; thallium can cause sensory and autonomic neuropathy and autonomic neuropathy, and some organic solvents and pesticides can cause polyneuropathy as well. Alcohol is directly toxic to the nerves, and alcohol abuse is a major cause of neuropathy. The peptides of the present invention can be used in some embodiments as part of a more extensive detoxification program. In yet another embodiment, the peptides of the present invention can be used to treat peripheral demyelinating diseases caused by drugs. Several drugs are known to cause neuropathy. These include, among others, nitrofurantoin used in pyelonephritis, amiodarone in arrhythmias, disulfiram in alcoholism, ddC and ddl in AIDS, and dapsone used to treat leprosy. As mentioned above, the peptides of the present invention can be used in some embodiments as part of a more extensive detoxification program.

[0084] In some embodiments, the peptide of the present invention is suitable for treating peripheral demyelinating diseases caused by trauma or compression.Localized neuropathy can be caused by external pressure or by compression of nerves by covering tendons and other tissues.The most well-known of these is carpal tunnel syndrome caused by wrist compression, and cervical or lumbar radiculopathy (sciatica) caused by compression of nerve roots as they exit the spine.Other common nerve compression sites include elbows, armpits and backs of knees.

[0085] The peptides of the present invention are also useful for treating various idiopathic peripheral demyelinating diseases. The term "idiopathic" is used when no cause of the peripheral demyelinating disease can be found. In these cases, the peripheral demyelinating disease is classified according to its symptoms, i.e., sensory, motor or sensorimotor idiopathic polyneuropathy.

[0086] The VDAC pore is a special docking site for proteins involved in many diseases, making it a therapeutic target for drugs able to disrupt or enhance binding with partner proteins. Blocking this channel upstream of the signaling pathways it activates is important in the fight against cardiomyopathy (10, 11), cancer (12, 13-15), diabetes (14, 14-16), lupus-like diseases (17), non-alcoholic fatty liver disease (24, 25), chemically induced neuropathy (9), Alzheimer's disease (18, 19), Parkinson's disease (20), Huntington's disease (21), ALS (22, 23), and more generally all neurodegenerative diseases associated with protein aggregation (28).

[0087] Thus, the present invention also relates to an HK-derived peptide of the present invention or a vector of the present invention for use in the treatment of neurodegenerative diseases, such as myocardial disease, cancer, diabetes, lupus-like diseases, non-alcoholic fatty liver disease, chemically induced neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease (Reference 1) or ALS.

[0088] As used herein, the term "lupus-like disease" has its general meaning in the art and refers to a disorder that has clinical, histological and immunological characteristics similar to idiopathic systemic lupus erythematosus.

[0089] As used herein, the term "non-alcoholic fatty liver disease (NAFLD)" has its general meaning in the art and refers to the condition caused by the accumulation of fat in the liver.The main stages of NAFLD are simple fatty liver (steatosis); non-alcoholic steatohepatitis (NASH), in which the liver becomes inflamed; fibrosis, in which persistent inflammation causes scar tissue around the liver and nearby blood vessels; and cirrhosis, which is the most severe stage that occurs after many years of inflammation, in which the liver shrinks, scars, and becomes lumpy.

[0090] The term "neurodegenerative disease" as used herein has its general meaning in the art and refers to a disease involving neurodegeneration, which is a progressive loss of neuronal structure or function, including neuronal death. Many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease, occur as a result of neurodegenerative processes. Such diseases are incurable and result in progressive degeneration and / or death of neuronal cells. As research progresses, many similarities are revealed that link these diseases to each other at the subcellular level. The discovery of these similarities brings hope for the advancement of treatments that can improve many diseases simultaneously. There are many similarities between various neurodegenerative diseases, including aberrant protein aggregation and induced cell death (Rubinsztein DC (2006). Nature. 443 (7113): 780-6 and Bredesen DE, et al (2006). Nature. 443 (7113): 796-802). In some embodiments, the neurodegenerative disease is a disease associated with protein aggregation (Reference 28).

[0091] Transdegenerative diseases include, but are not limited to, Alzheimer's disease, especially chemically induced neuropathy, Alzheimer's disease (References 18, 19), dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis with frontotemporal dementia (ALS), inclusion body myopathy dementia with bone and / or frontotemporal Paget's disease (IBMPFD), frontotemporal lobar degeneration, synucleopathies, Huntington's disease and Parkinson's disease, amyloidosis including amyloid angiopathy, tauopathies including frontotemporal dementia with Parkinson's disease associated with chromosome 17, neuromuscular diseases with protein inclusions, and developmental diseases including Down's syndrome.

[0092] In some embodiments, the peptides of the invention can be used to treat or at least reduce the severity of chemically induced neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease or ALS.

[0093] The term "diabetes" as used herein has its common meaning in the art and refers to a common metabolic disorder characterized by chronic hyperglycemia. Diabetes is associated with increased risk of heart disease, stroke, peripheral neuropathy, kidney disease, blindness, and amputation. There are three main types of diabetes: type 1 diabetes, type 2 diabetes, and gestational diabetes. Previous studies have demonstrated that VDAC1 inhibition restores beta cell function and prevents hyperglycemia in diabetic mice.

[0094] The term "cancer" as used herein has its general meaning in the art and refers to abnormal cell proliferation that may invade or spread to other parts of the body. Cancer cells share several characteristics that distinguish them from normal cells, such as avoiding apoptosis. Defects in apoptotic regulation, or even avoidance of apoptosis, are hallmarks of cancer. VDAC1 serves as a key regulator of energy, metabolism, and apoptosis, making it a unique target for anti-cancer therapy (Reference 8). Voltage-dependent anion channel 1 is highly expressed in many types of cancer compared to levels in normal cells (Reference 8). The peptides of the present invention are suitable for the treatment of cancer by disrupting the binding of anti-apoptotic proteins, such as hexokinase, to VDAC, thereby allowing the induction of apoptosis.

[0095] According to the present invention, the cancer may be selected from the group consisting of adrenal cortical carcinoma, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman's disease, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, vaginal cancer, vulvar cancer, uterine cancer.

[0096] <Pharmaceutical Composition> The peptides of the present invention may be used or prepared in pharmaceutical compositions.

[0097] In another aspect, the present invention relates to a pharmaceutical composition comprising a peptide of the present invention.

[0098] The present invention relates to a pharmaceutical composition comprising a peptide of the present invention or a vector of the present invention for use in the treatment of neurodegenerative diseases such as peripheral myelination diseases, myocardial diseases, cancer, diabetes, lupus-like diseases, non-alcoholic fatty liver disease, or chemically induced neuropathy (Reference 9), Alzheimer's disease, Parkinson's disease, Huntington's disease, and ALS.

[0099] Typically, the peptides of the present invention can be combined with pharma- ceutically acceptable excipients, and optionally sustained release matrices, such as biodegradable polymers, to form a therapeutic composition.

[0100] As used herein, the terms "pharmaceutical" or "pharmaceutical acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when properly administered to a mammal, particularly a human. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0101] In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient can be administered to animals or humans alone or in combination with another active ingredient in unit dosage forms, mixed with conventional pharmaceutical carriers. Suitable unit dosage forms include oral route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms. Preferably, the pharmaceutical composition contains pharma- ceutically acceptable excipients for injectable preparations. These may be in particular isotonic, sterile, saline (such as monosodium or disodium phosphate, chlorides of sodium, potassium, calcium or magnesium, or mixtures of these salts) or dry, in particular lyophilized compositions, which allow the constitution of a solution for injection by adding, optionally, sterile water or saline. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions containing the inhibitors of the present invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The inhibitors of the present invention can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), formed with inorganic acids, such as hydrochloric or phosphoric acid, or organic acids, such as acetic, oxalic, tartaric, mandelic, and the like.Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents that delay absorption, such as aluminum monostearate and gelatin.

[0102] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in a suitable solvent with some of the other ingredients listed above, and then filter sterilization is performed as necessary. In general, dispersions are prepared by incorporating various sterilized active ingredients into sterile vehicles containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. When formulated, the solution is administered in a therapeutically effective amount in a manner compatible with the administration formulation. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions mentioned above, although drug-release capsules and the like can also be used. For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in view of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0103] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the invention in any way.

[0104] [Example 1] Materials and Methods Peptides 1a–6r used in the SAR studies (truncations and Ala-scan) were purchased from Proteomic Solutions (Saint-Marcel, France). Peptides 7a–g, 3c', 5x', and 7a'–g' were synthesized on an automated microwave peptide synthesizer CEM Liberty One (CEM Corporation). Amino acids and Rink Amide MBHA resin were purchased from Iris Biotech (Germany), and Rink Amide MBHA LL resin was purchased from Sigma-Aldrich / Novabiochem (St. Louis, MO, USA). Oxyma pure and DIC were obtained from Iris Biotech (Marktredwitz, Germany). HOBt, DIEA, and TIS were obtained from Sigma-Aldrich (St. Louis, MO, USA), and dichloromethane and acetonitrile were obtained from VWR Chemicals (Radnor, PA, USA). DMF was obtained from Carlo Erba Reagents (Val-de-Reuil, France), piperidine was obtained from Acros Organics (Illkirch, France), and acetic anhydride was obtained from Prolabo (Paris, France). Rat serum and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Elastase (from porcine pancreas, EC 3.4.21.36) was purchased from Promega (Madison, WI, USA).

[0105] [Solid-phase peptide synthesis] All peptides were prepared by standard solid-phase peptide synthesis using the Fmoc method on a CEM Liberty One microwave-assisted peptide synthesizer. The resins used were Rink Amide MBHA (100–200 mesh, loading 0.67 mmol / g) for the synthesis of 12–16 peptide residues (compounds 3c', 5x', 7a'–f') on a 0.1 mmol scale, and Rink Amide MBHA LL (100–200 mesh, loading 0.36 mmol / g) for the synthesis of 25–29 peptide residues (compounds 5x, 7a–f) on a 0.033 mmol or 0.055 mmol scale. DIC / Oxyma (0.5 M / 2 M in DMF) was used as coupling reagent with a 5-fold excess of each protected amino acid. In the case of Fmoc-Arg(Pbf)-OH coupling, double couplings were performed. A solution of 20% piperidine in DMF was used for deprotection of the Fmoc group. The resin was swollen in DMF overnight in the reaction vessel, then the elongation process was carried out under microwave irradiation (1 mL DIC + 0.5 mL Oxyma pure, 70 °C (25 W) for 10 min). Deprotection cycles were performed with a 20% piperidine solution in DMF (7 mL at 75 °C for 30 s, then 7 mL at 70 °C for 3 min). If further modifications / additional amino acids were required at the N-terminal moiety (compounds 7a, 7b, 7d, 7e, 7f), the resin was split into two or three parts. After completion of the synthesis, the peptide-bound resin was washed twice with 15 mL DMF and twice with 15 mL DCM. Finally, the side chain deprotection and cleavage of the peptide from the resin were performed by treating with TFA / water / triisopropylsilane (95 / 2.5 / 2.5) for 2-3 h. The trifluoroacetic acid solution was evaporated under reduced pressure, followed by diethyl ether precipitation and washing with diethyl ether to give the crude peptide as a white powder. The analogues were purified by RP-HPLC on a C18 column and the identity of the products was confirmed by LCMS. The purity of the peptides was found to be 95% or higher for all peptides.

[0106] [Analytical HPLC] Peptides were analyzed on a Thermo Fisher Scientific LC-MS device, Accela HPLC and 3D ion trap analyzer connected to an LCQ fleet equipped with an electrospray ionization source (cone voltage 30 V). The column used was a Phenomenex BioZen™ 2.6 μm peptide XB-C18 (LC column 50 × 2.1 mm) eluted with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), with an elution gradient: 0-2 min, 20% B; 2-5 min, 20-90% B; 5-6 min, 90% B; 7-10 min, 20% B, flow rate 0.5 mL / min, 10 μL injection.

[0107] [HPLC purification] Peptides were purified by semi-preparative HPLC using a Waters 1525 chromatography system operated by Breeze software and equipped with a Waters 2487 variable absorbance detector set at 214 nm and 254 nm. A GRACE Vydac C-18 column (250 x 10 mm, 5 μm) was used with a flow rate of 3 mL / min. Two purification gradients were run depending on the polarity of the peptide.

[0108] Method A. Crude peptides were eluted with an isocratic gradient of 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B) from A / B (90:10) to A / B (50:50) for 30 min, then A / B (90:10) for 5 min, followed by A / B (90:10) for 2 min.

[0109] Method B. Crude peptides were eluted with an isocratic gradient of 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B) from A / B (80:20) to A / B (30:70) for 30 min, then A / B (90:10) for 5 min, followed by A / B (90:10) for 2 min.

[0110] [CD Spectroscopy] Circular dichroism (CD) experiments were recorded on a Jasco J815 spectropolarimeter. Spectra were acquired over the wavelength range of 190-260 nm at 20 °C using 1 mm path length CD cuvettes in MeOH or DPBS pH 7. Continuous scan mode was used with a response of 1.0 s, with 0.2 nm steps and 2 nm bandwidth. The signal-to-noise ratio was improved by obtaining spectra from the average of each triplicate scan. The baseline was corrected by subtracting the background from the sample spectra. The alpha-helical content was determined using the following equation: % helicity = ([θ]obs x 100) / (-39500 x (1-2.57) / N), where [θ]obs is the average residual ellipticity at 220 nm and N is the number of peptide bonds.

[0111] [NMR 3D structure analysis] NMR samples were prepared by dissolving NHKI analogs (3c', 7c', 7d', and 7g') in PBS (10% DO) at pH 6.8 to a final concentration of 2 mM. If necessary, the pH was adjusted using traces of 0.1 M NaOH or HCl solution. Up to 10% DMSO was added in cases of solubility issues. Compounds 3c', 7d', and 7f' were studied in the presence of 40% TFE (PBS, 10% DO, pH 6.8). Chemical shifts were referenced to trimethylsilylpropanoic acid (TSP).

[0112] All spectra were recorded on a Bruker Avance 600 AVANCE III spectrometer equipped with a 5 mm triple resonance cryoprobe (1H, 13C, 15N) at the "Laboratoire de Mesures Physiques (LMP)" of the University of Montpellier (UM). Homonuclear 2D spectra DQF-COSY, TOCSY (DIPSI2), ROESY, and NOESY were typically recorded in phase-sensitive mode using the States-TPPI method as a data matrix of 256–400 real (t1) × 2048 (t2) complex data points; 8–48 scans with recovery delays of 1.0–1.5 s per t1 increment, and a spectral width of 6009 Hz in both dimensions were used. Mixing times were 80 ms for TOCSY experiments and 150 ms for ROESY / NOESY experiments. Spectra were processed with Topspin (Bruker Biospin) and visualized with Topspin on a Linux station or NMRview 64. After shifted sine squared multiplication in the F1 domain and apodization by linear prediction, the matrix was zero-filled to 1024 (t1) × 2048 (t2) points.

[0113] Proteolytic stability assay A stock solution of 1 mg / mL elastase was prepared in Tris.HCl buffer (50 mM, pH 8, containing 0.5 mM CaCl2). The stock solution was diluted to 0.94 mg / mL with 658 μL of stock solution in 42 μL of Tris.HCl buffer. All peptides were dissolved in DMSO to prepare a stock solution of 6.66 mmol / L. A further diluted peptide solution (0.666 mmol / L) was prepared with 70 μL of stock solution in 630 μL of Tris.HCl buffer pH 8. In a 1.5 mL eppendorf, 890 μL of Tris.HCl pH 8 was introduced, followed by 100 μL of peptide solution (0.666 mmol / L) and incubated at 37 °C for 15 min before digestion. Then 10 μL of elastase solution (0.94 mg / mL) was added. The reaction mixture was incubated at 37 °C for up to 4 h with shaking at 1000 rpm. Aliquots (50 μL) were taken at various time points, quenched with 450 μL MeOH and centrifuged for 20 min (14000 rpm) at 4 °C. The supernatant was transferred to injection vials and analyzed by LC-MS using an elution program of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (see analytical data section). The relative concentrations of the remaining peptides and cleavage products were calculated by integration of the corresponding peaks in the HPLC chromatogram / MS trace. Control peptide solutions were prepared without enzyme. Hydrolysis of the control peptide solutions was found to be stable after 4 h at 37 °C in Tris buffer, except for compound 5x'. All proteolysis experiments were performed in triplicate.

[0114] [Structural calculation] 1H chemical shifts were assigned following classical procedures. NOE cross peaks were integrated and assigned within NMRView software. The volume of the methylene-proton NOE peak was used as a reference of 1.8 Å. The lower limit of all constraints was fixed at 1.8 Å, and the upper limit was fixed at 2.7, 3.3, and 5.0 Å for strong, medium, and weak correlations, respectively. Upper pseudoatom corrections were applied to the unresolved aromatic, methylene, and methyl proton signals as previously described. Structure calculations using AMBER 16 were performed in two stages: cooking and simulated annealing using a generalized Born implicit solvent model. The cooking stage was performed at 1000 K to generate 100 initial random structures. The simulated annealing calculations were performed for 20 ps (20000 steps, 1 fs length). First, the temperature was rapidly increased and maintained at 1000 K for the first 5000 steps, then the system was gradually cooled from 1000 K to 100 K from steps 5001 to 18000, and finally the temperature was reduced to 0 K for the remaining 2000 steps. For the first 3000 steps, the force constant of the distance restraints was gradually increased from 2.0 to 20 kcal mol-1 Å. For the remainder of the simulation (steps 3001–20000), the force constant was maintained at 20 kcal mol-1 Å. The 20 lowest energy structures with no violations greater than 0.3 Å were considered to be representative of the peptide structure. Representation and quantitative analysis were performed using MOLMOL and PyMOL.

[0115] [In-vitro metabolic stability in rat serum] Before digestion, the protein content of rat serum was measured by Bradford assay and found to be 108 mg / mL. For each peptide, a stock solution in DMSO was prepared at a concentration of 6.66 mmol / L. 70 μL of the solution was removed and added to 630 μL of MilliQ water to prepare an aqueous peptide solution (0.666 mmol / L). 325 μL of MilliQ water and 125 μL of undiluted rat serum pre-incubated at 37 °C for approximately 10-15 min were reacted before adding 50 μL of the 0.666 mmol / L peptide solution. The mixture was incubated at 37 °C with shaking at 1000 rpm. Aliquots (25 μL) were taken at different time points (0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 5 h, 7 h, 24 h, 48 h) and the enzyme reaction was stopped with 225 μL of MeOH to precipitate all serum proteins. The Eppendorf tubes were directly centrifuged (14000 rpm) for 20 min at 4°C to pellet and remove precipitated proteins. The supernatants were transferred to injection vials and analyzed by LC-MS using a 0.1% formic acid in water and 0.1% formic acid in acetonitrile elution program (see Analytical Data section). The relative concentrations of the remaining peptides and cleavage products were calculated by integration of the corresponding peaks in the HPLC chromatogram / MS trace.

[0116] Control peptide solutions were prepared without rat serum. All peptide control solutions were found to be stable for 48 hours in water at 37°C. All serum stability experiments were performed in triplicate.

[0117] [Cell culture and transfection] HEK-293 cells were purchased from ATCC (American type culture collection, USA). They were cultured in DMEM (Gibco, Thermo Fisher Scientific, France) supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific, France) and 1% PS (Gibco, Thermo Fisher Scientific, France) in a humidified incubator at 37°C with 5% CO2.

[0118] For peptide screening assays and live imaging experiments, cells were transfected with mitoGCaMP2 and GCaMP2 plasmids using jet-PRIME reagent (Polyplus-transfection SA, France) according to the manufacturer's recommendations. These two plasmids express the mitochondria-targeted and cytosol-targeted GCaMP2 proteins, respectively.

[0119] [Live imaging] Live imaging experiments were performed on HEK-293 cells transfected with either mitoGCaMP2 or GCaMP2 plasmid. 500,000 cells per well were seeded in 1 ml of DMEM supplemented with 10% FBS and 1% PS in a 6-well microplate (NUNC, ref. 153066, Thermo Fisher Scientific, France). 48 h after seeding, cells were transfected with 2 μg of mitoGCaMP2 or GCaMP2 plasmid using jet-PRIME reagent according to the manufacturer's protocol. 48 h after transfection, the microplate was placed under a video microscope equipped with a humidified chamber at 37 °C and 5% CO2. Then, 6 mM pre-warmed MJ (37 °C) was added to the wells with or without 1 ml of DMEM containing 33 μM peptide 1a, supplemented with 10% FBS and 1% PS, without red phenol, and containing 0.15% DMSO and 5% ethanol. In parallel, wells containing only 1 ml of DMEM without red phenol and with 10% FBS, 1% PS, 0.1% DMSO and 5% EtOH were used as control conditions. Live imaging acquisition was started upon addition of MJ with or without peptide 1a. As a control condition, image acquisition was started after addition of 1 ml of DMEM without red phenol, with 0.1% DMSO and 5% EtOH, supplemented with 10% FBS and 1% PS. Movies were acquired every 2 min for 30 min using an inverted Zeiss Axio Observer Z1 (Zeiss, France) and a 20x / 0.4 objective (Zeiss, France). For each condition, three independent experiments were performed. Overall, five ROIs per condition were analyzed using Zen software (Zen 2.3 lite, Zeiss, France) and ImageJ software (version 1.52o, NIH, USA). Results are expressed as mean values±SEM using GraphPad Prism software (version 8.0.1).

[0120] Screening Assays The activity of the designed compounds was evaluated in HEK-293 cells transfected with mitoGCaMP2. 40,000 cells per well were seeded in 96-well microplates coated with poly-D-lysine (reference number 655946, Greiner Bio-One, France) in 200 μl of DMEM supplemented with 10% FBS and 1% PS. 24 hours after seeding, cells were transfected with 50 ng of mitoGCaMP2 plasmid per well using jet-PRIME reagent (Polyplus-transfection SA, France) according to the manufacturer's recommendations. 48 hours after transfection, a first measurement of fluorescence was performed using the microplate reader CLARIOstar® (BMG Labtech, France). This measurement represented the basal level of Ca2+ into mitochondria at the time of transfection. After washing with 100 μl of PBS, the cells were incubated with a mixture of pre-warmed (37° C.) MJ at a final concentration of 6 mM and compounds at the given final concentrations in PBS containing 0.1% DMSO and 5% EtOH. After 35 min in the cell incubator, a second measurement of fluorescence was performed using the microplate reader CLARIOstar®. This measurement represented the level of mitochondrial Ca2+ depending on the peptide activity. Compounds were tested in triplicate per microplate and in three or five independent experiments for each peptide. For dose-effect curves, compounds were tested in triplicate per microplate and in three independent experiments. Results are expressed as the ratio between the second and first measurements, which are normalized to the compound-free condition containing only PBS containing 0.1% DMSO and 5% EtOH. Results are expressed as the mean ± SD for histogram plots and dose-response curves using GraphPad Prism software (version 8.0.1).

[0121] [Mice used in this study] All mouse experiments were approved by the "Comité de l'Animal Experiencia" of the Languedoc-Roussillon region and the "Ministry of Research Supreme" (authorizations 2017032115087316 and 2016091313354892). All procedures were performed in accordance with French regulations on animal procedures (French Decrees 2013-118 and 2020-274) and specific guidelines of the European Union on the protection of animal welfare (Directive 2010 / 63 / EU). Mice were maintained at 40–60% humidity, an ambient temperature of 21–22 °C, and a 12 h dark / 12 h light cycle. Mouse experiments were performed on 12-week-old C57BL6 / J mice purchased from Janvier Labs (France).

[0122] [Sciatic nerve explant culture and CARS imaging] Twelve-week-old C57BL6 / J mice were euthanized using pentobarbital (54.7 mg / ml, 100 mg / kg, Centravet, France). First, the sciatic nerve was collected, washed with PBS, and the epineurium was removed. Then, 5 mm long nerves were placed in 500 μl of DMEM in a 24-well microplate (NUNC, Thermo Fisher Scientific, France), with 10% FBS containing 3 μM of compound with 0.1% DMSO or with 1% PS alone, and further incubated at 37 °C and 5% CO2 in a humidified chamber. Negative controls consisted of compound-free sciatic nerve explant cultures (only DMEM supplemented with 1% PS, with or without 10% FBS and 0.1% DMSO). Intact sciatic nerves were collected and immediately fixed in 4% PFA to serve as controls of healthy myelin sheaths for CARS imaging. After 24 h of culture, sciatic nerve explants were washed three times with PBS and fixed in 4% PFA aqueous solution (Electron Microscopy Sciences, Thermo Fisher Scientific, France) for 1 h at room temperature. All CARS images were acquired with a two-photon microscope LSM 7 MP connected to an OPO (Zeiss, France) complemented with a delay line. A 20x water immersion objective (W Plan Apochromat DIC VIS-IR, Zeiss, France) was used for image acquisition. Each acquisition was performed in three independent experiments. In each experiment, the percentage of damaged fibers was quantified per field using three ROIs per condition using Zen software (Zen 2.3 lite, Zeiss, France). Results are expressed as mean ± SD.

[0123] [Statistical analysis] Data were analyzed with Excel (Microsoft Office Standard 2016) and GraphPad Prism (version 8.0.1) software (Graphpad software) and expressed as mean ± SD or SEM as indicated in the figure legends. Statistical differences between means were tested using one-way ANOVA followed by Dunnett's multiple comparison test or two-way ANOVA followed by Tukey's multiple comparison test as indicated in the figure legends. Differences between values ​​were considered significant at *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns indicates no significant difference.

[0124] <Result> [Peptide synthesis 1-7] Peptide libraries 1, 2, which allow ala scanning, 3, 4, which were used for deletion studies, and 5, which was used for the first round of optimization, were purchased at Proteomic Solutions. Peptide libraries 6 and 7 were synthesized by the solid-phase Fmoc / tBu method using Rink amide resin. After completion of the elongation, the peptides were cleaved from the resin using TFA to obtain the desired compounds with yields of each synthetic peptide ranging from x% to x% and purity of at least 95% as judged by HPLC / MS analysis. All peptides 1-7, except for those in library 7', contain the Tat cell membrane-permeable peptide, which is used to ensure peptide internalization during in cellulo binding assays. To evaluate the effect of Tat on VDAC recognition, Tat was placed at the C-terminus or N-terminus of the HK fragment (Figure 4).

[0125] Binding Assays The biological screening of peptides is based on the ability of methyl jasmonate (MJ) to bind and sequester HK-1 of the mitochondrial VDAC in a time- and dose-dependent manner (refs. 16, 20). To this end, we developed an in cellulo screening assay, in which HEK293 cells expressing cytoplasmic VDAC and HK25 are transfected with a cytoplasmic Ca2+-sensing probe, GCaMP2, or the same probe targeted to the mitochondrial matrix, mitoGCaMP2. The use of these probes allowed for real-time monitoring of cytoplasmic and mitochondrial Ca2+ levels, as shown in vivo above. The use of MJ, which removes HK from the VDAC, induced a Ca2+ release outside the mitochondria, measured through a decrease in mitoGCaMP2 fluorescence in the cells and an increase in GCaMP2 fluorescence. Compounds mimicking the NHKI sequence blocked this release in the presence of MJ, preserving mitochondrial and cytoplasmic fluorescence levels through binding to the VDAC. On the other hand, compounds with low VDAC activity result in a decrease in mitochondrial and an increase in cytoplasmic fluorescence, as observed with the addition of MJ.

[0126] To validate this assay, we performed time-lapse imaging of HEK-293 cells transfected with mitoGCaMP2 or its cytosolic form GCaMP2. Basal levels of Ca2+ in mitochondria or cytosol were stable for at least 30 min before treatment. Treatment with MJ (T=0) induced a significant decrease in mitoGCaMP2 fluorescence for at least 30 min (Figure 5A), indicating Ca2+ efflux from mitochondria.

[0127] Meanwhile, in the same time frame, GCaMP2 fluorescence was significantly increased (Figure 5B), indicating that cytosolic Ca2+ increased concomitantly with mitochondrial Ca2+ release. The fluorescence changes induced by MJ treatment were blocked by peptide 1a, which mimics the NHK-1 sequence (Figure 5A, MJ + peptide 1a condition) or cytosol (Figure 5B, MJ + peptide 1a condition), indicating that this peptide was able to block mitochondrial calcium release via VDAC over time.

[0128] This in cellulo system therefore constitutes a relevant assay for measuring the activity of compounds against VDAC-mediated mitochondrial Ca2+ release. Using this assay, IC50 values ​​of 15.6 ± 2 μM and 13.9 ± 3.1 μM for compounds 1a and 2a were measured, respectively (data not shown). In line with these data, these conditions were used to screen new peptides for activity at 10 μM. The position of the Tat peptide at the N- or C-terminus did not affect the activity of the NHKI peptide.

[0129] [Ala scan of peptides 1a and 2a] To identify the amino acids of peptides 1a and 2a involved in VDAC recognition, we performed an Ala scan on both peptides. We synthesized 12 derivatives of each peptide in which all amino acids were replaced with alanine-delivered series 1b-m and 2b-m (Figure 6A, 6B). The two series, which differ in the position of Tat at the N-terminus or C-terminus in series 1 and series 2, respectively, were tested at 10 μM concentration using the binding assay described above. The two series behave comparably. Indeed, the replacement of leucine 6, 7 and phenylalanine 11 in NHK1 with alanine in compounds 1e-f, 1i, 2e-f, and 2i causes a decrease in affinity for VDAC. The replacement of leucine 14 with alanine in compound 1l causes a similar decrease, which is not seen in the corresponding compound 2l.

[0130] [Deletion study of peptides 1a and 2a] This ala scan was pursued by a deletion study of the NHK1 sequence to identify a minimal sequence useful for proper binding to VDAC (Figure 6C, 6D). N-terminal deletions were examined through the synthesis of seven peptides 3a-g based on 1a, and C-terminal deletions were explored by an equivalent set of peptides 4a-g derived from 2a. The two series were tested at a concentration of 10 μM. As evidenced by compounds 3a-c, the N-terminal portion of NHK1 spanning residues 1-3 appears to be non-essential. Confirming the results of the ala scan, deletion of leucines 6 and 7 in compounds 3f-g is detrimental to the interaction with VDAC. Deletion of the last three C-terminal amino acids (ELK) of compound 2a induced a decrease in fluorescence, as evidenced by compounds 4a-c. Surprisingly, compound 4d retained a remarkable affinity for VDAC, again highlighting the key role of phenylalanine 11 in the interaction with VDAC. Thus, the hydrophobic sequence of the NHK1 peptide, AQLLAYYF (SEQ ID NO:89), constitutes the core of the interaction with VDAC, as evidenced by a combination of ala scan and deletion studies.

[0131] [Binding Optimization] Therefore, sequence-truncated peptides 3c and 4d were retained for a second optimization aimed at replacing amino acids suspected to be involved in the interaction with VDAC with their equivalent counterparts. Thus, compound 3c provides a new series of compounds 5a-h in which the unique threonine at position 12 of the NHK1 sequence was replaced with tyrosine, aspartic acid, asparagine and valine to study the importance of the hydroxyl moiety that threonine carries. In the same series, leucine 14 was replaced with valine, isoleucine, phenylalanine and tryptophan to evaluate the influence of beta-branched or aromatic amino acids. The same substitution was applied to leucines 6 and 7 of compound 4d, with leucine, tryptophan and tyrosine used as surrogates for phenylalanine 11 (data not shown).

[0132] Substitution of threonine 12 in 5a-d was not efficient and even detrimental when a negatively charged aspartic acid was introduced (5b). Conversely, substitution of leucine by tryptophan slightly enhanced the interaction with VDAC, as shown by compounds 5h and 6d, but to a more significant level in 6h, where leucine 7 was replaced. Finally, substitution of leucine 14 in compound 5h with tryptophan significantly improved activity. Thus, the interaction of NHK1 with VDAC is mainly mediated by hydrophobic residues, and tryptophan, considered the most hydrophobic residue according to the hydrophobicity scale of amino acids, enhances such interactions (Refs. 35, 36).

[0133] To take advantage of these results, modifications resulting in a positive effect were combined to provide compounds 5i-z and 6l-r, tested at 3 μM and compared with 3c and 4d, respectively (Figure 6). In the series of compounds 5i-z and 6l-r, compounds 3c and 4d serve as benchmarks. Lowering the reference concentration from 10 μM to 3 μM allowed the VDAC to be kept in a partially closed state, allowing a better discrimination between compounds that block calcium efflux. Individual substitutions of the hydrophobic leucines 6 or 7 by tryptophan were accompanied by a moderate increase in affinity, but combining the two substitutions in a single peptide gave a larger effect, as shown for compounds 5x, 5z, and 6q. Nevertheless, the analysis of the substitution of phenylalanine 11 is less clear, although the leucines in 5l, 6q or the tyrosines in 5t, 5m, 6m, 6o are similarly accommodated in this position. Finally, the substitution of leucine 14 by tryptophan also brings a beneficial effect for compounds 5l, 5t, 5x, and 5z.

[0134] [Optimizing bonding by strengthening spiral folding] The last modification we introduced was aimed at examining the helical fold adopted by the NHK1 sequence (Ref. 37). To this end, we introduced a sequence of alternating alanine and α-aminoisobutyric acid (Aib, U), both of which are α-helix inducers, at the N-terminal side of the HK-1 sequence delivering peptide series 7. Furthermore, this modification is expected to reduce the susceptibility of the compound to proteolytic cleavage. ioFurthermore, we introduced the 3-CF3Ph[Tz]U dipeptide as an N-terminal capping, which was shown in previous studies to enhance peptide insertion into the membrane (Ref. 38), because hydrophobic interactions conditioned proper VDAC interactions (Figure 8A).

[0135] Series 7a-g were tested at 10 μM and 3 μM in the screening assay (Figure 8A-B). Among this series, 7b, 7d, 7f, and 7g showed significantly higher activity than compounds 3c and 5x when tested at 10 μM (Figure 8A), whereas only 7f and 7g still showed significantly higher activity at 3 μM (Figure 8B). Therefore, to precisely define their activity, 7f and 7g were tested in a dose-response manner in the screening assay (data not shown). Their IC 50 were evaluated to be 2.6 ± 0.6 μM and 1.7 ± 0.2 μM, respectively. To summarize this SAR study, the AQLLAYYF sequence of HK (SEQ ID NO: 36) contains key residues involved in the interaction with HK. More specifically, a hydrophobic patch composed of leucines 6, 7 and phenylalanine 11 governed the interaction, and their replacement by tryptophan enhanced the interaction. Furthermore, the different steps of optimization led to the identification of a novel inhibitor of VDAC-mediated mitochondrial Ca uptake, as shown by dose-response experiments performed for compounds 1a, 5x, and 7g in the screening assay. 2+ There is a 10-fold increase in activity against efflux (Fig. 9). Furthermore, helical wheel projections of a different series of compounds placed the residues involved in VDAC interaction on the same face of the helix (data not shown).

[0136] [Circular dichroism] Compounds 3c, 5x, and 7a–f in PBS buffer were analyzed by circular dichroism. Compounds 3c, 5x, and 7a are unstructured, whereas compounds 7b–f show negative maxima at around 210 and 220 nm, consistent with peptides partially structured in an α-helix (data not shown). To evaluate the contribution of Aib introduced at the N-terminus in the structure of the NHK1 peptide, compounds 3c', 7a', 7b', and 7c' were synthesized without the Tat sequence (data not shown). In PBS solution, the AU repeat sequence introduced at the N-terminus of the NHK sequence allows the compounds to gradually fold as helices, as observed for compounds 7b' and 7c' (data not shown). The analysis was performed in methanol, since the CD spectrum cannot be extended to the entire wavelength range of interest in PBS solution. Compound 3c adopts a random coil structure in PBS, while it starts to fold as a helix due to the kosmotropic effect of methanol. Nevertheless, compound 7d′ containing three Aibs was the most structured in the series, confirming the trend observed in PBS with regard to folding depending on the amount of Aibs introduced into the sequence (data not shown).

[0137] Proteolytic stability assay Although Tat has been shown to be a suitable CPP for the delivery of bioactive cargoes such as NHK1-derived peptides, its use is limited due to its low serum stability (Refs. 39, 40). Indeed, the half-life of the Tat sequence in serum is less than 6 min (Refs. 41, 42). Moreover, serum is composed of a mixture of enzymes that cannot distinguish between different cleavage sites. Preliminary experiments confirmed this instability, since a solution of peptide 3c in 25% rat serum left only 10% of 3c after 5 min (data not shown). Moreover, multiple cleavage sites would generate so many fragments that their concentration would be below the detection limit of the LC-MS instrument, making their identification impossible. Therefore, it is generally more convenient to use defined proteolytic enzymes to identify preferred cleavage sites.

[0138] Among the enzymes available in our laboratory, the serine endopeptidase elastase (EC 3.4.21.36), present in the pancreas as well as in serum, was chosen considering its remarkable primary specificity for alanine and leucine at position P1, two amino acids present in the AQLLAYYF sequence (SEQ ID NO: 89), which must remain intact for VDAC recognition but are absent in TAT (Refs. 43, 44). Therefore, since the NHK1 sequence is important for proper binding to VDAC, we focused on studying the NHK1 sequence without Tat and exposed compounds 3c', 5x', 7a' and 7d'-f' to elastase for 2 h in Tris.HCl buffer at pH 8 (data not shown).

[0139] Of this series of compounds, peptides 3c' and 5x', containing the NHKI sequence, which induced the highest activity after the first optimization step, were completely degraded in less than 30 min. Addition of an AUAU patch to the N-terminus of these peptides 7c' and 7e' had no effect on improving metabolic stability (data not shown). However, alanine substitution at position 8 by Aib in 7a' and 7d' improves stability against elastase. Moreover, compound 7f', N-terminally capped with a triazole derivative, was the most stable compound, despite the conservation of alanine at position 8 (data not shown). It is noteworthy that the main cleavage site for elastase was at the C-terminus of alanine 8, since the compounds with reduced half-lives (3c', 5x', 7c', and 7e') contained this alanine. Thus, the enzyme fragments conform to elastase specificity, and replacement of alanine at position 8 by Aib improved stability.

[0140] To verify whether this stabilization is maintained in more complex media, compounds 7a', 7d', and 7f', which show the highest stability against elastase, as well as compound 7g', an analog of 7f' with Aib at position 8 instead of alanine, were tested in rat serum containing hundreds of peptidases (Reference 43). Compounds 3c' and 5x' were used as references (Figure 10).

[0141] Data obtained with elastase showed that compound 3c' was readily processed by proteolytic enzymes present in rat serum, with only 8% remaining after 1 h (Figure 10). Compound 7f' disappeared at a comparable rate, suggesting that capping the peptide with a triazole group is not sufficient in serum. However, replacement of the alanine at position 8 with Aib in 7g resulted in 54% of the compound being maintained after 24 h. Compound 7a' showed slower degradation over time, with approximately 65% ​​of the compound remaining after 24 h of incubation, whereas 7d', an analog of 7a' containing an AUAU patch at the N-terminus, showed the highest serum peptidase resistance with 75% of the compound remaining (Figure 10). Enzyme fragments were identified by high-resolution tandem mass spectrometry.

[0142] In conclusion, these stability studies showed that alanine at position 8 appears to be the preferred site for enzymatic cleavage of NHKI-derived peptides. Indeed, the addition of Aib at position 8 improved the stability of NHKI against serum proteases. This stability was further enhanced by capping the N-terminus with a 3-CF3-Ph[Tz]U derivative or an AUAU patch.

[0143] [Ex vivo activity on sciatic nerve explant cultures] Next, we tested the activity of NHKI-derived compounds on sciatic nerve explant cultures, where Schwann cells demyelinate through a mechanism involving mitochondrial Ca2+ release via VDAC1 (Refs. 44, 45, 9). Coherent anti-Stokes Raman scattering (CARS) nonlinear microscopy was used to image and quantitate intact myelin in sciatic nerve explants. This imaging method does not require specific labeling and is suitable for the analysis of myelin sheaths (Refs. 47, 48). In intact sciatic nerves imaged using CARS, the myelin sheaths generated by SCs form a continuous line surrounding the axons, except at the nodes of Ranvier (Figure 11). 24 hours after incubation of the nerve in cell culture medium, spontaneous demyelination occurs, characterized by the formation of spheres (Figure 11). Demyelination was quantified by measuring the percentage of damaged fibers, i.e., fibers showing sphere formation, relative to the total number of fibers imaged.

[0144] In a first set of experiments, the most active compounds of the screening assay, namely 7d and 7g, and the related compounds 3c and 5x used as controls, were tested at 3 μM in serum-free medium (data not shown). After 24 h in serum-free medium, compound 3c showed the same percentage of damaged fibers as the negative control, while all other compounds significantly reduced the levels of damaged fibers (data not shown). Consistent with previous experiments, the optimized compounds 7d and 7g were significantly more active than the reference compounds 3c and 5x. Moreover, these optimized compounds notably showed the same myelin sheath pattern as in intact nerves (data not shown). The results obtained in serum-free medium conditions correlate with those obtained from the screening assay, since the optimized compounds have enhanced activity on blocking mitochondrial Ca2+ release.

[0145] In the next set of experiments, the same compounds were tested at 3 μM in medium supplemented with serum (Figure 11A-C). Among all compounds tested, only treatment with compounds 7d and 7g significantly reduced nerve fiber damage, indicating that these peptides are effective in blocking demyelination and are stable in serum for a sufficient period of time to be effective. Remarkably, these two compounds were able to significantly preserve the myelin sheath at levels similar to those in intact nerves (Figure 11A-C).

[0146] In conclusion, the above results confirmed the higher proteolytic stability of compounds 7d and 7g, i.e., the positive effect of the A8U substitution at position 8 used in combination with the AUAU patch or the N-terminal triazole moiety.

[0147] <Consideration> Two molecular models based on the structure of VDAC1 bound to HK1 and HK2 have been proposed (Refs. 46, 47). Both models have a similar shape with the HK located at the top of the pore, closing the channel. The 25 residues that make up the HK N-terminal helix are sandwiched between the N-terminal VDAC helix and the wall of the barrel. Mutation of the serine in HK2 to a nonpolar leucine increased the stability of the mutant and its binding to VDAC. Ala-scanning combined with deletion studies allows the identification of the AQLLAYYF sequence (SEQ ID NO: 89) and its leucines and phenylalanines as crucial for VDAC interaction. Substitution of these three amino acids with more hydrophobic ones such as tryptophan enhanced the interaction with VDAC, supporting the hypothesis of a relationship between hydrophobicity and binding ability (Ref. 48). The N-terminal sequence of HK adopts a helical structure (Ref. 37), and we considered the possibility that leucines 6, 7 and phenylalanine 11 constitute a hydrophobic patch located on the same face of the helix. Therefore, we attempted to strengthen the helical structure by adding helix inducers such as Aib to the HK sequence. As expected, such substitutions induced a helical structure, which was more pronounced in organic solvents such as methanol than in buffer, and was accompanied by an increase in binding affinity to the pore. Nevertheless, this result was counterintuitive with respect to the molecular model of blocking the N-terminal helix in the water-filled pore. Indeed, the porin channel is mostly positively charged, whereas the negatively charged residues E66, E73, K74, D78, E189, and E203, located on the cytoplasmically exposed loop of VDAC, have been identified as essential for HK binding (Ref. 49), although little is known about the residues in the N-terminal region of HK involved in binding (Ref. 50). In fact, the N-terminal helix of HK, which is essential for proper interaction with VDAC, is composed mostly of hydrophobic residues and cannot directly bind to the charged VDAC residues. Nevertheless, various studies have highlighted E73 as a critical residue for HK binding (Ref. 51), which is supported by the E73Q mutation that abolishes HK1 binding. E73 is in an unusual position on the outer surface of the β-barrel, pointing towards the membrane (Refs. 52, 3, 4).E73 was also identified by photoaffinity approaches as a special binding site for cholesterol and neurosteroids (Ref. 53). In this case, steroid binding to VDAC does not affect its conductance capacity, but suggests that the steroid binding site is likely involved in channel dimerization or hexokinase-mediated signaling. Evidence that cholesterol loading affects HK binding to VDAC led to the development of a cholesterol hydroxamic acid derivative, olesoxime. Recently, it was shown that the highly hydrophobic olesoxime does not enter the water-filled VDAC pore but interacts at the protein-lipid interface (Ref. 54). Thus, as has been suggested for the HK helix, which is thought to be inserted within the lipid bilayer (Ref. 50), compound 7f, which bears a hydrophobic 3-CF3-Ar[Tz] tag, may exhibit similar behavior interacting with the hydrophobic exterior of the β-barrel of VDAC. Furthermore, various small molecules featuring a molecular pattern similar to that present in compound 7f can interact with VDAC-1, and their binding was measured by microscale thermophoresis (Refs. 55, 56). The hydrophobic nature of the stabilizing helix developed in this study facilitates direct interaction of the helix at the membrane interface between the membrane and VDAC. Thus, the HK helix can be defined as a membrane anchor that initiates the HK / VDAC interaction, as recently proposed in a model supported by electrophysiological measurements (Ref. 57). In this context, such a helix serves as a tool for the development of cross-linking probes that can correctly position the NHK1 sequence on the VDAC interface.

[0148] [Example 2] AAV9 virus expressing the HK peptide 5z (AAV9-HK peptide) was generated. HEK293 cells were infected with control AAV9, AAV9-HK peptide, or not infected. After 2 days, cells were incubated with the fluorescent dye Rhod-2, which fluoresces in calcium in mitochondria. After 15 min, infected cells were incubated with methyl jasmonate (6 mM) and non-infected cells were incubated with methyl jasmonate (6 mM) + 5z peptide (5 μM) for 40 min. Pictures were taken every 5 min to image the Rhod-2 dye.

[0149] Methyl jasmonate induced a decrease in Rhod-2 fluorescence in mitochondria of cells infected with the control virus, similar to the decrease seen in uninfected cells in previous experiments, but not in cells infected with viruses expressing the 5z peptide or treated with the 5z peptide (Figure S12).

[0150] This indicates that AAV9 viruses expressing the 5z peptide, like peptide 5z, prevent mitochondrial calcium release in the presence of methyl jasmonate, making AAV9 expression an efficient way to sustain expression of anti-demyelinating peptides in target cells.

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Claims

1. Amino acid sequence: Alanine (A) - Glutamine (Q) - X １ - X ２ - X ３ - Tyrosine (Y) - Tyrosine (Y) - X ４ (SEQ ID NO: 1), and X １ is leucine (L) or tryptophan (W), X ２ is leucine (L) or tryptophan (W), X ３ is alanine (A), D-isomer alanine (A Ｄ ) or α-aminoisobutyric acid (U), and X ４ is phenylalanine (F), leucine (L) or tyrosine (Y), The HK-derived peptide does not consist of the amino acid sequence shown in SEQ ID NO: 95, and the HK-derived peptide does not contain the amino acid sequence shown in SEQ ID NO: 96, the HK-derived peptide.

2. Amino acid sequence: alanine (A) - glutamine (Q) - X １ - X ２ - X ３ - tyrosine (Y) - tyrosine (Y) - X ４ - threonine (T) - glutamic acid (E) - X ５ - lysine (K) (SEQ ID NO: 2), and X １ is leucine (L) or tryptophan (W), X ２ is leucine (L) or tryptophan (W), X ３ is alanine (A), D - alanine (A Ｄ ) or α - amino - isobutyric acid (U), and X ４ is phenylalanine (F), leucine (L) or tyrosine (Y), X ５ is leucine (L) or tryptophan (W), The HK-derived peptide according to claim 1.

3. X ３ The HK-derived peptide according to claim 1 or 2, wherein X is α-aminoisobutyric acid (U).

4. 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids, the HK-derived peptide according to claim 1.

5. The HK-derived peptide according to claim 1, comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO:

53.

6. The HK-derived peptide according to claim 5, comprising, or consisting of, the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO:

29.

7. The HK-derived peptide according to claim 1, to which the sequence AU AU (SEQ ID NO: 54) or the sequence AU (SEQ ID NO: 55) is attached.

8. Dipeptide 3-CF ３ Ph[Tz]U is bound to the N-terminus of the HK-derived peptide, and the dipeptide 3-CF ３ Ph[Tz]U has the following formula 【Chemical 1】 The HK-derived peptide according to claim 1.

9. The HK-derived peptide according to claim 1, to which a cell membrane permeable sequence is attached.

10. The HK-derived peptide according to claim 9, wherein the cell membrane permeable sequence is tat (SEQ ID NO: 58).

11. A vector comprising the HK-derived peptide according to claim 1.

12. The HK-derived peptide according to claim 1 or the vector according to claim 11 for use in therapy.

13. The HK-derived peptide according to claim 1 or the vector according to claim 11 for use in the treatment of peripheral demyelinating diseases, myocardial diseases, cancer, diabetes, lupus-like diseases, non-alcoholic fatty liver diseases, chemically induced neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease, neurodegenerative diseases such as ALS.

14. The HK-derived peptide for use in the use according to claim 13, wherein the peripheral demyelinating disease is selected from the group consisting of Refsum disease, abetalipoproteinemia, Tangier disease, Krabbe disease, metachromatic leukodystrophy, Fabry disease, Dejerine-Sottas syndrome, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsy (HNPP), familial amyloid neuropathy, hereditary sensory neuropathy type II (HSN II), hereditary porphyria, muscular dystrophy, Dejerine-Sottas syndrome, diabetic neuropathy, immune-mediated neuropathy, acute motor neuropathy, acute sensory neuropathy, acute autonomic neuropathy, Miller Fisher syndrome, chronic polyneuropathy, vasculitis or peripheral demyelinating diseases associated with inflammation of blood vessels in the peripheral nerves, peripheral demyelinating diseases associated with monoclonal gammopathy, peripheral demyelinating diseases associated with tumors or neoplasms, peripheral demyelinating diseases caused by drugs, peripheral demyelinating diseases caused by infections, peripheral demyelinating diseases caused by disruption of nutritional balance, peripheral demyelinating diseases caused by kidney diseases, hypothyroidism neuropathy, peripheral demyelinating diseases caused by alcohol and toxins, peripheral demyelinating diseases caused by trauma or compression, and idiopathic peripheral demyelinating diseases.

15. A pharmaceutical composition comprising the peptide according to claim 1 or the vector according to claim 11.

16. A pharmaceutical composition for the treatment of peripheral demyelinating diseases, comprising the HK-derived peptide according to claim 1 or the vector according to claim 11.

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