WO2006051224A1 - Peptides derives de la maurocalcine utilisables comme vecteurs pour l'adressage intracellulaire de molecules d'interet - Google Patents
Peptides derives de la maurocalcine utilisables comme vecteurs pour l'adressage intracellulaire de molecules d'interet Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to peptides derived from maurocalcine capable of entering the cells and to convey molecules of interest in these cells.
- translocation peptides also called CPP (CeI1-Penetrating Peptides) represent particularly interesting vectors (For a review, see in particular, Prochiantz, Curr Opin, Cell Biol. 2000, 12, 400-406, Lindgren et al., Trends Pharmacol Sci., 2000, 21, 99-102).
- these small molecules are able to cross cell membranes in a transporter and receiver-independent manner and to carry macromolecules impregnating proteins and nucleic acids, at low concentration, without energy, in an efficient manner (transduction 100% of cells), fast (of the order of 5 to 15 min), and this in all cell types, in vivo and in vitro.
- some of these peptides were able to cross the blood-brain barrier (Schwarze and Dowdy, Science, 1999, 285, 1569-1572).
- These peptide transporter-independent receptor independent vectors are different from other vectors comprising a PEG-coupled glycopeptide or peptide in which the positively charged peptide serves to condense DNA and PEG or the PEG.
- Sugar allows the targeting of the cells of interest, in particular by binding of the DNA / glycopeptide complex to the receptor for mannose or asialoglycoprotein (peptides CWCK 15 CK, CW (CK 3 ) 4 CK and CWK 5 CK 5 CK 5 C (SEQ ID NO: 26-28): Park et al., Bioconjugate Chem., 2002, 13, 232-239; Kwok et al., J. Pharm. Science, 2003, 92, 11741185).
- the currently known penetration peptides are grouped into two categories: * peptides derived from membrane translocation signal sequences of different proteins (Kaposi's sarcoma-derived fibroblastic growth factor (K-FGF) and immunoglobulin light chain (Ig) (v)), the mechanism of penetration of these peptides is not known, the central hydrophobic region is impli ⁇ in penetration but the structure of this region varies depending on the proteins (alpha helix (K-FGF) or beta sheet (Ig (v)).
- K-FGF membrane translocation signal sequences of different proteins
- Ig immunoglobulin light chain
- the smallest fragment of the homeoproteins capable of crossing the membranes and serving as a vector for other peptides or oligonucleotides is the peptide 43-58, called penetratin, corresponding to the helix 3 of the homeodomain (Derossi et al. J. Biol Chem., 1994, 269, 10444-10450 and International Application WO 97/12912).
- penetratin the peptide 43-58
- penetratin corresponding to the helix 3 of the homeodomain
- the most efficient fragment of the Tat protein is fragment 48-60 which corresponds to the entire basic region and includes the nuclear localization signal.
- a shorter fragment 47-57 is able to transport, in the form of a fusion protein, proteins of 15 to 120 kDa, in different cell types, in vitro and in vivo, and is able to cross the blood-glucose barrier. meningeal.
- the equine virus Tat peptide has a structure similar to that of a homeodomain.
- the maurocalcine (MCa) is a toxin of 33 amino acids isolated from the scorpion venom Scorpio maurus palmatus, corresponding to the sequence SEQ ID NO: 1 in the attached sequence listing.
- the corresponding cDNA encodes a precursor of 66 amino acids comprising 3 domains: an N-terminal signal peptide of 22 amino acids, followed by a propeptide of 11 amino acids, rich in negatively charged amino acids and terminated by a cleavage signal characteristic of prohormones (KR), and a C-terminal peptide of 33 amino acids corresponding to the mature peptide (maurocalcine).
- the maurocalcine has a strong homology with the toxin of two other scorpions: Pandinus imper imperox atox (IpTx A, SEQ ID NO: 9, 82% identity) and opicalcins 1 and 2 of Opistophtalmus carinatis (SEQ ID NO: 10 and 11, 91% and 88% identity, respectively, Figure IA). It also has a homology on a 6 amino acid motif comprising a succession of basic residues, followed by a serine or a threonine (K 1 QK 20 -K 22 R 23 R 24 -T 26 ), with the activator domain Dihydropyridine receptor loop II-III (DHPR), a voltage-dependent L-type calcium channel.
- Pandinus imper imperox atox IpTx A, SEQ ID NO: 9, 82% identity
- opicalcins 1 and 2 of Opistophtalmus carinatis SEQ ID NO: 10 and 11, 91% and 88% identity, respectively, Figure IA. It also has
- the maurocalcine is one of the most potent effectors of the ryanodine receptor type 1 (RyR1); it has been shown in particular to stimulate the binding of ryanodine to the RyR1 receptor, to induce significant changes in the opening of the calcium channel, characterized by the appearance of prolonged periods of subcondence, and that the extracellular addition of maurocalcine to myotube cultures, induces the release of calcium from the sarcoplasmic reticulum to the cytoplasm (Fajloun et al., FEBS Letters, 2000, 469, 179-185, Estève et al., J. BioL Chem., 2003 , 278, 37822-37831).
- maurocalcine or its analogs comprising the KKCKKR motif as an active ingredient for inducing immunosuppression or treating pathologies related to calcium channel dysfunction
- the three-dimensional structure of maurocalcine corresponds to an ICK (Inhibitor Knot Motif) pattern, present in many peptides of plants, animals or fungi; the ICK family encompasses peptides of different sequences and of various biological activities, such as animal toxins (snake venom or arachnid) and protease inhibitors of plant origin, such as the McoT I-II peptide (SGSDGGVCPKILKKCRRDSDCPGACICRGNGYCG (SEQ ID NO: 29)), (Zhu et al., The Faseb Journal, July 3, 2003, Heitz et al, Biochemistry, 2001, 40, 7973-7983).
- ICK Inhibitor Knot Motif
- maurocalcine consists more precisely of: (i) a compact nucleus linked by disulfide bonds (C 3 -C 17 , C 10 -C 2 ) and C 16 -C 32 and including 3 beta sheets (9-11 20-33 and 30-33, the sheets 20-33 and 30-33 being anti-parallel), and (ii) a loop emerging at the N-terminus (Mobash et al., Proteins, 2000, 40, 436-442). It is represented as a molecule comprising a positively charged face which could represent a surface of interaction with the RyR1 receptor (Mobash et al., Supra).
- the inventors have now sought to define the minimum characteristics of the amino acid sequences derived from maurocalcine capable of serving as a vector for internalization and targeting of substances of interest, in particular macromolecules of interest such as proteins and acids. nucleic acids and particles comprising chemical molecules of interest.
- maurocalcin is a toxin with known pharmacological properties, it can not be used in vivo. Consequently, the inventors have also aimed to obtain peptide vectors derived from maurocalcine which, preferably, are not toxic in vivo, that is to say having no pharmacological activity on the surface.
- RyRl receiver especially because they do not bind to RyRl receiver.
- the present invention accordingly has for one object the use of a peptide vector for the intracellular targeting of a substance of interest, characterized in that said vector essentially consists of a peptide derived from maurocalcine corresponding to the sequence (I) following:
- - X 2 to X 25 each represent an amino acid or absent, X 7, X 10, X n, X i 2, X i 3, X 14, X 5, and X 16 is still present, and
- - Xio, Xn, X 3 and X 4 each represent lysine or arginine, and - Z and / or Z 'are absent or each represents a 1 sequence
- the invention includes the use of peptides derived from mauro ⁇ calcine such as natural or synthetic variants of maurocalcine, for example maurocalcine analogues.
- the invention also encompasses the use of chimeras between maurocalcine and a toxin comprising an ICK motif such as, for example, 1-oxalcin or 2-aminobutyric acid and
- amino acid means a natural or synthetic amino acid, namely: the 20 naturally occurring ⁇ -amino acids commonly found in the proteins (A, R, N, D, C, Q, E, G, H, I, L, K 3 M, F, P, S, T, W, Y and V), some amino acids rarely encountered in proteins (hydroxy-proline, hydroxylysine, methyllysine, dimethyllysine ..) amino acids that do not exist in proteins such as, for example, ⁇ -alanine, ⁇ -aminobutyric acid, homocystein
- the peptide as defined in the present invention is capable of penetrating into any type of cells, in vitro or in vivo, and of transporting substances of interest such as impermeable macromolecules (proteins, nucleic acids) and particles comprising chemical molecules of interest, in cell compartments, more particularly the cytoplasmic compartment and the nuclear compartment.
- substances of interest such as impermeable macromolecules (proteins, nucleic acids) and particles comprising chemical molecules of interest
- cell compartments more particularly the cytoplasmic compartment and the nuclear compartment.
- complexes between the peptide according to the invention and substances such as proteins whose molecular weight is up to at least 60 kDa and nanoparticles are transported in the cytoplasm and nucleus of the cells.
- This property can easily be verified by incubating said peptide bound to said substance in the presence of said cells and detecting the presence of said peptide and / or said substance in the cells, in particular by analysis of a specific labeling of said peptide and / or said substance, by any technique known to those skilled in the art, in particular by microscopy.
- said peptide is useful as a vector for intracellular targeting of substances capable of interacting with an intracellular target.
- substances are in particular pharmacologically active substances whose target is intracellular; these drugs include drugs or phytosanitary products.
- These substances may also be ligands of an intracellular component to be detected (endogenous or pathogenic molecule), in particular antibodies or antibody fragments (Fab, Fv, scFv), useful as intracellular molecular probe.
- Said substances include chemical molecules, especially macromolecules: proteins, peptides, peptides-acids nucleic acids (PNA), nucleic acids (plasmids, oligonucleotides, antisense, siRNA) and particles, in particular nanoparticles or liposomes comprising chemical molecules of interest, encapsulated or grafted (coupled) to said particles.
- PNA peptides-acids nucleic acids
- plasmids plasmids, oligonucleotides, antisense, siRNA
- particles in particular nanoparticles or liposomes comprising chemical molecules of interest, encapsulated or grafted (coupled) to said particles.
- said peptide has many applications in the field of biotechnologies, particularly nanobiotechnologies, in particular for the diagnosis and treatment of human or animal pathologies (biomedical applications) and as a tool for research in these fields.
- the substance to be carried is coupled to the peptide vector by any appropriate means, known in itself for associating a peptide with a substance (peptide, protein, nucleic acid or other molecule chemical).
- said substance to be transported is a peptide or a protein
- it is advantageously coupled to the peptide vector by a peptide bond.
- Said substance may also be coupled to the peptide vector, non-covalently, in particular via streptavidin-biotin complexes, for example the peptide vector is biotinylated and the substance of interest is coupled to streptavidin.
- Said substance and said peptide vector may also be incorporated into the same particle; they can in particular be coupled to nanoparticles or liposomes.
- intracellular molecular probe when used to detect an intracellular component (intracellular molecular probe), it can be advantageously coupled to an appropriate marker, to labeled particles, or to a labeled substance.
- the labeling is in particular a fluorescent marking or a magnetic marking, detectable by any technique known to those skilled in the art (fluorescence microscopy, flow cytometry, magnetic resonance imaging).
- Such intracellular molecular probes have applications in cell imaging in vitro and in vivo, especially in real time. They can in particular be used as diagnostic reagent for a genetic or acquired disease or infection by a microorganism and as a tool for research.
- X 15 is other than an arginine and lysine; this mutation makes it possible to abolish the link of said peptide to the RyR1 receptor and to abolish the pharmacological effects resulting from this binding.
- X 7 and / or X j 2 represent a cysteine. According to another advantageous embodiment of said use,
- X 2 , X 3 , X 4 , X 5 , X 6 , X 8 , X 23 , X 24 and X 25 are absent.
- X 17 to X 22 are present and X 21 and / or X 22 represent an arginine or a lysine, preferably X 21 represents a lysine. According to another advantageous embodiment of said use,
- Z ' represents arginine or lysine.
- Z corresponds to the following sequence (II):
- Z 2 J and / or Z 29 represent a cysteine; preferably when Z 2 i is cysteine then X 7 is also cysteine and when Z 29 is cysteine, then X 2 is also cysteine.
- X 7 , X 12 , Z 21 and Z 29 each represent a cysteine.
- At least one amino acid selected from Z 30, Z 3 1, Z 32, Z 33, Z 34 and Z 35 represents a lysine or arginine; preferably Z 30 and / or Z 34 represent arginine or lysine.
- At least one amino acid selected from Z 22 , Z 23 , Z 24 , Z 25 , Z 26 , Z 27 and Z 28 represents a lysine or an arginine; preferably Z 27 and / or Z 28 represent arginine or lysine.
- Z 23, Z 24, Z 25, Z 26, Z 27, Z 28, Z 30, Z 3 i, Z 32, Z 33, Z 34 and Z 35 each represent a lysine or arginine. Even more preferably, Z 27 and Z 30 each represent a lysine or Z 28 and Z 34 each represent a lysine or arginine.
- At least Z 1 to Z 18 , Z 22 , and Z 31 or Z 34 are absent.
- the following residues are absent: Z 1 to Z 18 , Z 22 and Z 3 i or Z 34 ; Z 1 to Z 1 9 , Z 22 and Z 31 or Z 34 ; Z 1 to Z 2 o, Z 22 and Z 31 or Z 34 ; Z 1 to Z 26 and Z 3 ] or Z 34 , Z 1 to Z 28 and Z 31 or Z 34 .
- one of the following amino acid sequences is present: Z 19 to Z 2R, Z 23 to Z 30 and Z 32 to Z 35; Z1 9 to Z 21 , Z 23 to Z 33 and Z 35 ; Z 20 , Z 21 , Z 23 to Z 30 and Z 32 to Z 35; Z 20 , Z 21 , Z 23 to Z 33 and Z 35; Z2 1 , Z 23 to Z 30 and Z 32 to Z 35 ; Z 21 , Z 23 to Z 33 and Z 35 .
- the peptide of sequence (I) consists of amino acids D.
- Peptides according to the present invention are represented in particular by: a) chimeras between maurocalcine and impertoxine or maurocarcin and opicalcine, for example peptides of sequence SEQ ID NO: 24 and 25, b) peptides of 33 amino acids derived from maurocalcine in which:
- - X 1, X 7, X i 2, X 26, Z 2] and Z 29 are C, - Xi 0, Xn, X 13, X] 4, X 2I, X 22, Z 27, Z 30 and Z 28 or Z 34 represent K or
- R preferably at least Xi 0 , Xn, Xi 3 , X 2 i, Z 27 and Z 30 are K, even more preferably Xi 0 , Xn, Xi 3 , X 2 i, Z 27 and Z 30 represent K and Z 28 or Z 34 are K or R, preferably Z 28 is R or Z 34 is K.
- X 2 , X 3 , X 4 , X 5 , X 6 , X 8 , X 23 , X 24 , X 25 , Z 1 to Z 8, Z 22) and Z 31 or Z 34 are absent,
- X 9 represents S or G
- - Xi 9 represents a hydrophobic amino acid selected from: A, V, L, I, P, W, F and M.
- - Xi 6 day Xi8 "X2Q, Z 1 Z ⁇ 2Oj Z 23, Z 24; Z 25 , Z 26 , Z 32 , Z 33 , Z 35 , Z ', and optionally Z 28 , Z 31 , Z 34 represent A, C, D, E, F, G, K, L, M, P or N, preferably A or F; for example, Z 19 represents G, and Z 'represents R or K.
- said peptide vector is coupled to a suitable marker, especially a fluorochrome; the coupling may be covalent or non-covalent, especially via labeled streptavidin-biotin complexes or labeled particles.
- the peptide vector as defined above is coupled to particles, especially nanoparticles; advantageously said particles are labeled and / or they comprise a substance of interest, such as a pharmacologically active substance, useful in particular as a medicament or phytosanitary product, or a ligand substance of an intracellular component to be detected, useful as an intracellular molecular probe .
- a substance of interest such as a pharmacologically active substance, useful in particular as a medicament or phytosanitary product, or a ligand substance of an intracellular component to be detected, useful as an intracellular molecular probe .
- the sequence (I) of the peptide vector as defined above is fused to a heterologous peptide or polypeptide sequence of interest, so as to form a peptide or a chimeric protein.
- heterologous is meant a sequence other than that which is directly adjacent to the sequence (I), in the sequence of maurocalcine or a maurocalcine analogue.
- Said peptide or said chimeric protein are advantageously coupled to a suitable marker and / or to particles, said particles possibly being able to be labeled.
- the insertion of the sequence (I) in the peptide or the protein of interest is carried out at the NH 2 or COOH end or at an appropriate internal site, which site is chosen according to the structure of said protein. or said peptide.
- the present invention also relates to a composition
- a composition comprising a pharmacologically active substance whose target is intracellular and a peptide vector as defined above, as an intracellular targeting vector of said substance.
- said pharmacologically active substance and said peptide vector are in the form of a peptide or a chimeric protein as defined above.
- composition comprises particles comprising both said pharmacologically active substance and said peptide vector.
- said pharmacologically active substance is a medicament intended to be administered to a human or animal individual.
- the present invention also relates to the composition as defined above as a medicament.
- the present invention also relates to the use of a composition as defined above, for the preparation of a medicament, for the treatment of a pathology in humans or animals.
- the present invention also relates to a composition comprising a ligand of an intracellular component to be detected (intracellular probe) and a peptide vector as defined above, as intra-cellular addressing vector of said ligand.
- said peptide vector is coupled to an appropriate marker, to labeled nanoparticles, and / or to said ligand (protein or chimeric peptide), as specified above.
- said ligand is an antibody or a functional fragment of antibody directed against said component.
- the present invention also relates to the composition as defined above as a diagnostic reagent.
- the subject of the present invention is also a method for treating a pathology, characterized in that it comprises the administration of a composition as defined above, to an individual, by any appropriate means.
- the present invention also relates to an in vitro method for detecting an intracellular component, characterized in that it comprises:
- said cell sample comprises cells of a higher eukaryotic organism, possibly infected with a microorganism, or cells of a microorganism (bacterium, yeast, champi ⁇ gnon, parasite).
- the present invention also relates to an in vivo method for detecting an intracellular component, characterized in that it comprises:
- said detection reagent is a labeled reagent as defined above.
- the said reagent is for example coupled to a fluorochrome or to particles coupled to a fluorochrome.
- said organism is higher eukaryotic, in particular a human being, an animal or a plant.
- the present invention also relates to a peptide or a chimeric protein as defined above, said peptide or said chimeric protein being optionally labeled.
- the subject of the present invention is also a peptide of sequence (I) as defined above, with the exception of peptides SEQ ID NO: 1 to 11 and 26 to 29; said peptide may advantageously be labeled.
- the subject of the present invention is also particles coupled to a peptide vector as defined above; said particles and / or said vector may advantageously be labeled.
- said particles are nanoparticles.
- the present invention also relates to a polynucleotide encoding the peptide vector, or the peptide or the fusion protein as defined above. According to the invention, the sequence of said polynucleotide
- DNA or RNA corresponds to that of the cDNA coding for said peptide vector or for said peptide or said fusion protein.
- the sequence of said polynucleotide may comprise a translocation signal peptide in the endoplasmic reticulum so as to produce a peptide / peptide vector or fusion protein secreted into the extracellular medium.
- Said signal peptide may especially be that of maurocalcine or any peptide or protein capable of being secreted in the extracellular medium.
- sequence of said polynucleotide may also comprise the 11 amino acid sequence of the propeptide of maurocalcine or another peptide of the ICK family, inserted 5 'of the cDNA sequence encoding said peptide vector.
- the subject of the present invention includes, in particular: a) expression cassettes comprising at least one polynucleotide as defined above, under the control of appropriate regulatory sequences for transcription and optionally for translation ( promoter, activator, intron, initiation codon (ATG), stop codon, polyadenylation signal), and b) recombinant vectors comprising a polynucleotide according to the invention.
- these vectors are expression vectors comprising at least one expression cassette as defined above.
- the present invention further relates to prokaryotic or eukaryotic host cells modified with at least one polynucleotide or a recombinant vector as defined above.
- the present invention further relates to a transgenic non-human mammal, characterized in that all or part of its cells are modi ⁇ fiées by a polynucleotide or a recombinant vector as defined above.
- the subject of the present invention is also a transgenic plant, characterized in that all or part of its cells are modified by a polynucleotide or a recombinant vector as defined above.
- Many nucleic acid vectors into which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or integration into the chromosomal material of the host), as well as the nature of the host cell.
- viral vectors such as adenoviruses, retroviruses, lentiviruses, AAVs and baculoviruses, in which the sequence of interest has been inserted beforehand; said sequence (isolated or inserted in a plasmid vector) may also be associated with a substance enabling it to cross the membrane of the host cells, such as a transporter such as a nano transporter or a preparation of liposomes, or of cationic polymers, or introduce it into said host cell using physical methods such as electroporation or microinjection.
- these methods can advantageously be combined, for example by using electroporation associated with liposomes.
- polynucleotides, the recombinant vectors and the trans ⁇ formed cells as defined above are particularly useful for the production of the peptides and fusion proteins according to the invention.
- the polynucleotides according to the invention are obtained by conventional methods, known in themselves, following the standard protocols such as those described in Ou ⁇ rent Pr oto cols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA). For example, they can be obtained by amplification of a nucleic sequence by PCR or RT-PCR, by screening of genomic DNA libraries by hybridization with a homologous probe, or by total or partial chemical synthesis. Recombinant vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se.
- peptides and their derivatives are prepared by conventional techniques known to those skilled in the art:
- the peptides derived from maurocalcine and the fusion peptides can be synthesized in solid phase, according to the Fmoc technique, originally described by Merrifield et al. (J. Am Chem Soc., 1964, 85: 2149-) and purified by reverse phase high performance liquid chromatography,
- peptides derived from maurocalcine may also be produced from corresponding cDNAs, obtained by any means known to those skilled in the art; the cDNA is cloned into a eukaryotic or prokaryotic expression vector and the protein or fragment produced in the host cells modified by the recombinant vector are purified by any appropriate means, in particular by affinity chromatography.
- FIG. 1 shows the sequence and structure of mauro ⁇ calcine.
- A Alignment of the maurocalcine and two other analogous toxin sequences, also active on the ryanodine receptor: Popicalcine 1 and Imperoxin A. All the toxins have the same number of positively charged amino acids or basic amino acids ( 12 amino acids charged positively over a total of 33 amino acids).
- B Three-dimensional structure of mauro ⁇ calcine, established using WebLab ViewerPro TM software. Left panel: basic face including positively charged residues (G 1 , K 8 , Kn, K 14 , Ki 9 , K 20 , K 22 and K 30 ). Right panel: hydrophobic face that illustrates the absence of positively charged residues on the opposite side of the molecule.
- the peptide backbone is shown by a ribbon, while the side chains of the positively charged residues are indicated by balls and rods represented in scale.
- FIG. 2 shows that the MCa complex t / Strept-Cy3 transduced efficiently many different cell cultures.
- Cells of hippocampal CA1 neurons, HEK293 cells and undifferentiated L6 cells were incubated for 30 min with 100 nM biotinylated maurocalcine, complexed with cyanine labeled 3 streptavidin (MCa b / Strept-Cy3). , then the cells were fixed and analyzed by confocal microscopy. Differentiated L6 cells were similarly treated. Four times smaller panels represent the control experiments, in which the cells were treated with a mixture of non-biotinylated MCa and Strept-Cy3.
- Figure 3 shows that complexes between R24A and L7A variants of biotinylated maurocalcine and cyanine labeled streptavidin efficiently transduce cell cultures.
- Cultures of HEK293 cells were incubated for 30 min with 333 nM R24A, L7A or maurocalcine, biotinylated and complexed with cyanine labeled 3 streptavidin (MCa t / Strept-Cy3), and then the cells were fixed. and analyzed by confocal microscopy.
- FIG. 4 shows that the penetration of the MCa b / Strept-Cy3 complexes is dose-dependent.
- A. Flow Cytometry Analysis of the Penetration of MCai / Strept-Cy3 Complexes at the Concentrations Indicated (10 nM, 33 nM, 100 nM, 333 nM and 1 ⁇ M). The cells were treated with trypsin (1 mg / ml), before the flow cytometry analysis.
- B Mean cellular fluorescence, as a function of the concentration of MCa b / Strept-Cy3 complexes.
- FIG. 5 shows that the penetration of the complexes between the biotinylated maurocalcine variants and the cyanine labeled streptavidin is dose-dependent.
- A. Flow cytometric analysis of the penetration of the complexes between R24A, L7A or maurocalcine, biotinylated variants and Strept-Cy3 cells were treated with trypsin (1 mg / ml), before flow cytometric analysis.
- B Mean cellular fluorescence, as a function of complex concentration.
- FIG. 6 represents the transduction kinetics of the MCa b / Strept-Cy3 complex in undifferentiated L6 cells over a period of one hour.
- A Confocal image of the Cy3 fluorescence of an undifferentiated L6 cell, 3 sec, 3 min and 12 min after the addition of 100 nM MCa b / Strept-Cy3 and 23 min after washing the complex (35 min in total).
- B Position of the different regions of interest (ROI) on the image of the light transmitted by the cell (differential interference contrast, upper panel) and corresponding position of the confocal image of the Cy3 fluorescence.
- ROI regions of interest
- FIG. 7 illustrates the subcellular localization of the MCa b- Strep-Cy5 complexes, analyzed by immunofluorescence by confocal microscopy and compared with the location of concanavalin A (A), tubulin alpha (B), and as a function of time (C ).
- A Subcellular localization of MCa b -Strep-Cy5 complexes in HEK293 cells, compared with a plasma membrane marker. The cells were incubated for one hour in the presence of MCab-Strep-Cy5 complexes (333 nM). The plasma membrane is labeled with concanvaline and nuclei with propidium iodide. The images represent a single confocal plane.
- C Changes in Cell Distribution of MCab-Strep-Cy5 Complexes After Cellular Translocation. Confocal fluorescence images of Cy3 (MCa b -Strep-Cy3 complexes) and To-PRO (nuclei) of HEK293 cells incubated with 333 nM complexes, for 2 hours, 4 hours and 24 hours. Progressive labeling of the nuclei by the MCa b -Strep-Cy5 complexes is observed. - Figure 8 shows that the entry into the cell MCa complex t / Strept ⁇ Cy3 does not require energy.
- FIG. 9 illustrates the flow cytometry analysis of the effect of heparin and / or trypsin treatment on the cellular penetration of the MCa b -Strep-Cy5 (A) complexes and the cellular toxicity of the complexes (FIG. B).
- A Incubation of MCa b- Strep-Cy5 cells and complexes with 10 ⁇ g / ml of heparin reduces penetration of the complex (left panel). The average fluorescence value of Cy5 is 307 without treatment, while in the presence of heparin, it goes down to 78.
- the penetration of the MCa b -Strep-Cy5 complexes is tested at a concentration of 1 ⁇ M.
- FIG. 10 illustrates the effect of the increase of the extracellular concentration in K + on the cellular penetration of the MCa b -Strep-Cy3 (A 5 B) complexes.
- A Flow Cytometry Analysis of the Effects of Increasing Concentrations in KCl on Cellular Penetration of MCa b -Strep-Cy3 Complexes. The panels on the right correspond to the controls (cellular fluorescence without complex (top) and with Strep-Cy3 (bottom)), in the presence of 145 mM KCl. The KCl gradient has no effect on control values.
- the left panels illustrate the effect of 5 mM (top, average fluorescence value of 145), 125 mM (medium, average fluorescence value of 40) and 145 mM KCl (bottom, mean fluorescence value of 17) , respectively.
- FIG. 11 shows the interaction of MCa with membrane lipids (A 5 B).
- A Measurement of surface pressure of monomolecular films of GD3 and DPPC. Kinetics of changes in surface pressure induced by the application of 1 ⁇ M MCa. The results illustrate the interaction of MCa with GD3 but not DPPC. The initial surface pressure was of the order of 10 mN.m -1 .
- FIG. 12 illustrates the effect of complex MCa t, / Strept-Cy3 on the RyRl receiver.
- A Stimulation of [ 3 H] -ryanodine Binding with 100 nM MCa b / Strept-Cy3 Complex. Specific binding of tritiated ryanodine to sarcoplasmic reticulum vesicles was measured as described in the material and methods.
- - represents the binding in the absence of MCa or MCa b / Strept-Cy3 complex.
- FIG. 13 illustrates the effect of the biotinylated and Strep-Cy3 complexed L7A and R24A variants of the maurocarcin on the RyR1 receptor. Stimulation of binding of [3 H] -ryanodine was measured at increasing concentrations of complexes. Specific binding of tritiated ryanodine to sarcoplasmic reticulum vesicles was measured as described in the material and methods.
- FIG. 14 illustrates the use of maurocalcine for cellular penetration of nanoparticles.
- A. HEK293 cell cultures were incubated for 1 hour with 100 nM maurocalcine coupled to nanoparticles (Qdot®, QUANTUMDOT CORPORATION), and then the cells were fixed and analyzed by confocal microscopy. Cultures of cells treated under the same conditions with Qdots coupled with streptavidin serve as control.
- the maurocalcine, the maurocalcine peptides according to the present invention and their biotinylated derivatives were prepared by solid phase peptide synthesis (Merrifield, Science, 1986, 232, 341-347), using an automatic synthesizer ( Model 433A, APPLIED BIOSYSTEMS).
- N- ⁇ -Fmoc-L-Lys (Biotin) -OH was provided by NEOSYSTEM (SNPE group).
- the N- ⁇ -Fmoc-L amino acid derivatives, the 4-hydroxymethylphenyloxy resin and the reagents used for peptide synthesis were provided by PERKIN ELMER LIFE SCIENCES.
- the peptide chains were sequentially assembled on 0.25 meq of hydroxymethylphenyloxy resin (1% crosslinking, 0.89 meq amine group / g), using 1 mmol of N- ⁇ -Fmoc-L amino acid derivatives.
- the side chain protecting groups are as follows: trityl for cysteine and asparagine residues; tert-butyl for the residues of serine, threonine, glutamic acid and aspartic acid; pentamethylchromane for arginine residues, and tert-butyloxycarbonyl for lysine residues.
- N- ⁇ -amino groups were deprotected by treatment with piperidine / N-methylpyrrolidone (18 and 20% v / v), respectively for 3 and 8 minutes.
- Fmoc amino acid derivatives were coupled (20 min), like their active esters of hydroxybenzotriazole, in N-methylpyrrolidone (in excess of a factor of 4).
- the resin containing the peptide (approximately 1.8 mg) was treated for 2 to 3 hours at room temperature, with continuous stirring, with a mixture of trifluoroacetic acid / H 2 O / thioanisol / ethanedithiol (88: 5/5/2, v / v) in the presence of phenol crystals (2.25 g).
- the peptide mixture was then filtered, and the filtrate was precipitated by addition of cold butyl methyl ether.
- the crude peptide was pelleted by centrifugation (3000 g for 10 min), and the supernatant was removed.
- the reduced peptide was then dissolved in 200 mM Tris-HCl buffer, pH 8.3, to a final concentration of 2.5 mM and mixed in the open air for 50 to 72 hours at room temperature in order to allow its oxidation and its folding.
- a second purification step of the maurocalcine, the peptides derived from the maurocalcine according to the invention, and their biotinylated derivatives was carried out by ion exchange chromatography, on a carboxymethylcellulose matrix, using phosphate buffers. 10 mM (buffer A) and 590 mM (buffer B), pH 9.0 (linear gradient from 0 to 60% buffer B at a rate of 1 ml / min, for 1 hour).
- Soluble streptavidin-cyanine 3 and streptavidin-cyanine were mixed with 4 molar equivalents of biotinylated maurocalcine (1 mM) or peptide derived from biotinylated maurocalcine, in buffer phosphate (PBS in mM: NaCl 136, Na 2 HPO 4 1.47, KCl 2.6, CaCl 2 1, MgCl 2 0.5, pH 7.2), for 2 hours at 37 ° C and in the dark ,
- the L6 line of rat myogenic cells (clone C5, EACC) is cultured in DMEM medium supplemented with 15% fetal bovine serum (LIFE TECHNOLOGIES) and 1% penicillin-streptomycin (Invitrogen).
- the differentiation of the L6 line was induced by replacement of the culture medium with differentiation medium (DMEM + 5% horse serum), when the cells become confluent.
- the neurons of the hippocampal region CA1 are prepared from hippocampi of newborn mice (1 to 2 days post partum), dissected, freed from the meninges and placed in HBSS buffer (INVITROGEN). Then, they are incubated in dissociation medium (HBSS, 1% penicillin / streptomycin (GIBCO), 2000 IU / ml DNase and 1% (w / v) trypsin / EDTA) for 7 min at 37 ° C.
- dissociation medium HBSS, 1% penicillin / streptomycin (GIBCO), 2000 IU / ml DNase and 1% (w / v) trypsin / EDTA
- the supernatant is removed and the tissue is washed with HBSS medium containing 1% penicillin / streptomycin.
- the tissue is gently triturated in HBSS medium, 1% penicillin / streptomycin, 10% fetal bovine serum, 2000 IU / ml DNase I, using a plastic pipette, until a homogeneous suspension.
- the cell pellet is resuspended in Neurobasal / B27 medium (GIBCO) containing 0.5 mM L-glutamine and 1% penicillin / streptomycin.
- GEBCO Neurobasal / B27 medium
- the cell cultures are seeded at a density of 10 cells / cm, in culture dishes previously treated with 20 ⁇ g / ml of poly-L-lysine, for 2 hours at 37 ° C. After 2 days of culture, cytosine arabinoside (3 ⁇ M) is added to the cultures to limit the proliferation of non-neuronal cells, and 24 hours later, half of the medium is changed. Then the culture medium is changed every 2 days.
- Embryonic cells of human kidney line HEK 293,
- the cells were incubated with maurocalcine and the peptides derived, biotinylated and complexed with streptavidin-cyanine 3 or streptavidin-cyanine 5, at the final concentration of 100 nM in PBS, in the dark and at room temperature. ambient, for 30 minutes to 1 hour.
- the cells After 3 washings in PBS, the cells are fixed at room temperature, in a solution of paraformaldehyde (4%), for 10 min, in the dark, washed in PBS and incubated for one hour with concanavalin conjugated to FITC (MOLECULAR PROBES, 5 ⁇ g / ml) to label the plasma membrane and TO-PRO-3 iodide (MOLECULAR PROBES, 1 ⁇ M), to mark the nucleus.
- FITC concanavalin conjugated to FITC
- TO-PRO-3 iodide MOLECULAR PROBES, 1 ⁇ M
- the living cells were incubated with maurocalcine and the peptides derived, biotinylated and complexed with streptavidin-cyanine 3, at the final concentration of 100 nM in PBS, at room temperature in freshly changed culture medium, on the right microscope stage (Eclipse 600 FN, Nikon, equipped with a water immersion objective (x 40), aperture 0.8) and a confocal head (PCM 2000, Nikon), or with a LEICA TCS-SP2 microscope, equipped with an objective (x 100), according to the "XYZt" mode.
- the cell suspension was centrifuged at 500 g and the cells resuspended in PBS containing 1 ⁇ g / ml propidium iodide (SIGMA).
- SIGMA propidium iodide
- the MCa b -Strept-Cy 3 complex is used in place of the MCa b -Strept-Cy 5 complex.
- the assay was performed on living cells using a flow cytometer (FACScalibur, BECTON DICKINSON). The data were recorded and analyzed using appropriate software (CellQuest, BD BIOSCIENCES). Living cells were sorted by size and granulosity (forward / side scattering) out of a total of 10,000 events. 2) Results a) Analysis of the penetration
- the ability of the peptides derived from maurocalcine according to the present invention to transduce different types of cells and to transport macromolecules is studied using complexes between biotinylated peptides and streptavidin coupled to cyanine-3.
- the biotinylated-streptavidin-Cy3 maurocalcine complexes (MCa b / Strept-Cy3) serve as control.
- the primary rat hippocampal neurons, and the cells of the HEK293 and L6 lines are incubated for 30 min, at room temperature, with 100 nM or 333 nM of biotinylated peptide maurocalcin derivative complexed at streptavidin-Cy3 or 1010 nM or 333 nM MCa t / Strept-Cy3, as a control.
- the cells are fixed and the fluorescence is observed by confocal microscopy.
- the rate and relative intensity at which the fluorescence increases in each compartment are consistent with the direction of progression of the MCa t / Strept-Cy3 complexes in the cell, namely: from the extracellular space to the plasma membrane, from the membrane plasma to the cytoplasm, and then cytoplasm to the nucleus.
- the signal recorded in the ROI-I compartment is likely overestimated due to fluorescence contamination of the probe in the cytoplasm.
- the relative intensity of cytoplasmic fluorescence should be more accurate.
- the passage of cytoplasm to the nucleus is very weak, indicating that this transition is much less favored than the other two.
- the assay is performed as described in Example 1.
- the complexes were prepared in a heparin solution (bovine intestinal heparin, SIGMA) and the cells were washed twice with the heparin solution, before and after incubation with the complexes prepared in the same heparin solution, b?) results
- the cell penetration was analyzed by flow cytometry, on cells incubated with the complexes MCa b / Strept-Cy3, then treated with trypsin, so as to eliminate the complexes bound to the plasma membrane, via of lipids, specific receptors or glycosaminoglycan heparan sulfate (HPSG).
- FIG. 9A shows that heparin reduces the penetration of maurocalcine into cells, hindering its interaction with glycosaminoglycan heparan sulfate or by altering these interaction properties with negatively charged lipids, e, binding to its basic side .
- FIG. 9A also shows that the fraction of MCa1 / Strept-Cy3 that is associated with the outer surface of the plasma membrane, in particular by attachment to heparan sulfate, is minimal since the effect of trypsin treatment on cell penetration is weak (compare left panel and right panel).
- MCa b / Strept-Cy3 is not saturable, which is compatible with a mechanism of diffusion of maurocalcine and derived peptides.
- FIG. 9B shows an absence of incorporation of propidium iodide by the living cells incubated in the presence of MCa b / Strept-Cy5 complex (1 ⁇ M complex concentration); this absence signifies a notable absence of cellular toxicity of maurocalcine in the presence of streptavidin-Cy5.
- the penetration of maurocalcine and derived peptides is sensitive to membrane potential C 1 ) protocol .
- the MCa b -Strept-Cy complexes were prepared in solutions containing different NaCl / KCl ratios (145: 5 NaCl / KCl composition, KCl 5 to 145, 2.5 CaCl 2 , 1.2 MgCl 2 , glucose 10). , HEPES 10, pH 7.4).
- the cells were washed twice with the NaCl / KCl solution used for the preparation of the complexes, and then incubated with maurocalcine and the peptides derived, biotinylated and complexed with streptavidin-cyanine 3 (MCab-Strept-Cy 3 complexes), with the final concentration of 100 nM in the same NaCl / KCl solution, in the dark and at room temperature, for 1 hour. After 2 washes in the same NaCl / KCl buffer, fluorescence of cyanine 3 was analyzed on living cells, using a flow cytometer (FACScalibur, BECTON DICKINSON).
- Disialoganglioside NeuAc ⁇ 2-8NeuAca2-3Galbl-aGlcbl-Cer (GD3) and Dipalmitoylphosphatidylcholine (DPPC) are provided respectively by MATREYA INC and SIGMA.
- Figure HB shows that the interaction of maurocalcin with GD3 is dose-dependent. The interaction is detectable at concentrations of 100 nM maurocalcine and reaches a maximum at 750 nM. The concentration producing 50% of the maximum effect is 500 nM.
- MCa mauro ⁇ calcine
- the representation of the surface electrostatic potential shows that maurocalcine has a basic face involving G 1 and all lysines and a hydrophobic face and indicates that maurocalcine is a highly charged molecule with a large dipole moment (Fig. 1B).
- GD3 / MCa interactions neutralize the basic face of MCa and promote the interaction of its hydrophobic face with the inner part of the membrane.
- GD3 could transiently translocate from the outer surface of the membrane to the inner side to release MCa which could then establish new electrostatic interactions with other negatively charged lipids or proteins, due to an environment richer in negative charges.
- [3 H] -ryanodine linked to heavy sarcoplasmic vesicles was measured by filtration through Whatmann GF / B, followed by three washes with 5 ml ice-cold wash buffer (15O mM NaCl, 2O mM Hepes, pH 7.4
- the [ 3 H] -ryanodine retained on the filters is measured by liquid scintillation
- the non-specific binding is measured in the presence of labeled ryanodine (20 ⁇ M)
- the results are presented in the form of means ⁇ standard deviation. experiment is performed in triplicate and repeated at least twice c) Measurement of Ca 2+ release
- the release of Ca 2+ by the heavy vesicles of the sarcoplasmic reticulum was measured using a Ca 2+ sensitive dye, Antipyrylazo III. Absorbance was measured at 710 nm, using a diode beam spectrophotometer (MOS-200, Optical System, BIOLOGIC).
- the heavy vesicles of the sarcoplasmic reticulum (50 ⁇ g) were actively loaded with Ca 2+ at 37 ° C in 2 ml of buffer containing 100 mM KCl, 7.5 mM sodium pyrophosphate, 20 mM MOPS, pH 7.0 supplemented with 250 ⁇ M antipyrylazo III, 1 mM ATPMgCl 2 , 5 mM phosphocreatine and 12 ⁇ g / ml creatine phosphokinase (Palade, J. Biol Chem, 1987, 262, 6142-6148). Calcium loading begins with sequential additions of 50 ⁇ M and 20 ⁇ M CaCl 2 .
- Figure 12 shows that MCa t , / Strept-Cy3 complexes retain the ability to stimulate binding of [ 3 H] -ryanodine to the heavy vesicles of the sarcoplasmic reticulum (panel A) and to induce Ca release. 2+ from these vesicles (panel B).
- FIG. 13 shows that among the maurocalcine variants capable of entering the cells and transporting substances of interest, the L7A mutant is less active on the RyR1 receptor, MCa t , / Strept-Cy3 whereas the mutant R24A is inactive.
- the biotinylated maurocalcine was coupled to streptavidin-conjugated nanoparticles (Qdot®, QUANTUMDOT CORPORATION) according to the protocol recommended by the supplier. Streptavidin-conjugated nanoparticles alone (Qdot Streptavidin Conjugate, QUANTUMDOT CORPORATION) have been used as a control.
- the nanoparticles have a diameter of 10 to 15 nM and are each coupled to 5 to 7 molecules of streptavidin.
- HEK293 cell cultures were incubated for 1 hour with 100 nM maurocalcine coupled to nanoparticles (Qdot®, QUANTUMDOT CORPORATION) or nanoparticles coupled to streptavidin alone, then the cells were fixed and analyzed by confocal microscopy, as described. in example 1.
- Figure 14 shows that maurocalcine allows cell penetration of nanoparticles.
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Abstract
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Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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CA002586811A CA2586811A1 (fr) | 2004-11-12 | 2005-11-14 | Peptides derives de la maurocalcine utilisables comme vecteurs pour l'adressage intracellulaire de molecules d'interet |
EP05817457.4A EP1814905B1 (fr) | 2004-11-12 | 2005-11-14 | Peptides derives de la maurocalcine utilisables comme vecteurs pour l'adressage intracellulaire de molecules d'interet |
JP2007540681A JP5416901B2 (ja) | 2004-11-12 | 2005-11-14 | 興味のある分子の細胞内位置決めのためのベクターとして用いられるマウロカルシン由来ペプチド |
US11/719,171 US8207293B2 (en) | 2004-11-12 | 2005-11-14 | Peptides derived from maurocalcine used as vectors for intracellular addressing of molecules of interest |
AU2005303623A AU2005303623A1 (en) | 2004-11-12 | 2005-11-14 | Peptides derived from maurocalcine used as vectors for intracellular addressing of molecules of interest |
IL183022A IL183022A0 (en) | 2004-11-12 | 2007-05-06 | Peptides derived from maurocalcine used as vectors for intracellular addressing of molecules of interest |
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FR0412045 | 2004-11-12 | ||
FR0412045A FR2877946B1 (fr) | 2004-11-12 | 2004-11-12 | Peptides derives de la maurocalcine utilisables comme vecteurs pour l'adressage intracellulaire de molecules d'interet |
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EP (1) | EP1814905B1 (fr) |
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CN (1) | CN101094863A (fr) |
AU (1) | AU2005303623A1 (fr) |
CA (1) | CA2586811A1 (fr) |
FR (1) | FR2877946B1 (fr) |
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Cited By (2)
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EP2537858A1 (fr) | 2011-06-20 | 2012-12-26 | Commissariat à l'Énergie Atomique et aux Énergies Alternatives | Petites peptides à pénétration cellulaire efficace dérivées de la maurocalcine de toxine de scorpion |
WO2014141124A1 (fr) | 2013-03-13 | 2014-09-18 | Institut National De La Sante Et De La Recherche Medicale | Composé d'activation de voie erk pour prévenir ou traiter une résistance à la leptine |
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US9777274B2 (en) | 2013-03-15 | 2017-10-03 | Techulon Inc. | Antisense molecules for treatment of staphylococcus aureus infection |
JP2016515381A (ja) | 2013-03-15 | 2016-05-30 | テチュロン インコーポレイテッド | 黄色ブドウ球菌感染の治療のためのアンチセンス分子 |
CN111875670A (zh) | 2013-06-12 | 2020-11-03 | 法瑞斯生物技术有限公司 | 对天然cxcr4具有拮抗活性的肽 |
US11559580B1 (en) | 2013-09-17 | 2023-01-24 | Blaze Bioscience, Inc. | Tissue-homing peptide conjugates and methods of use thereof |
WO2018170480A1 (fr) | 2017-03-16 | 2018-09-20 | Blaze Bioscience, Inc. | Conjugués peptidiques d'écotropisme du cartilage et leurs méthodes d'utilisation |
CN105087492B (zh) * | 2015-07-16 | 2021-04-13 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 培养原代海马神经元的方法 |
CA2994865A1 (fr) | 2015-09-09 | 2017-03-16 | Fred Hutchinson Cancer Research Center | Peptides localisant le cartilage |
CA3005928A1 (fr) * | 2015-12-11 | 2017-06-15 | Fred Hutchinson Cancer Research Center | Peptides pour therapie renale |
US11548923B2 (en) | 2017-01-18 | 2023-01-10 | Fred Hutchinson Cancer Center | Peptide compositions and methods of use thereof for disrupting TEAD interactions |
AU2018283161A1 (en) | 2017-06-15 | 2020-01-02 | Blaze Bioscience, Inc. | Renal-homing peptide conjugates and methods of use thereof |
CA3086040A1 (fr) | 2017-12-19 | 2019-06-27 | Blaze Bioscience, Inc. | Complexes d'agent d'adressage tumoral et d'immuno-oncologie de peptide de penetration cellulaire et leurs procedes d'utilisation |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2537858A1 (fr) | 2011-06-20 | 2012-12-26 | Commissariat à l'Énergie Atomique et aux Énergies Alternatives | Petites peptides à pénétration cellulaire efficace dérivées de la maurocalcine de toxine de scorpion |
WO2012176138A2 (fr) | 2011-06-20 | 2012-12-27 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Petits peptides efficaces pénétrant les cellules, dérivés de la toxine maurocalcine issue du scorpion |
US9439975B2 (en) | 2011-06-20 | 2016-09-13 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Small efficient cell penetrating peptides derived from the scorpion toxin maurocalcine |
WO2014141124A1 (fr) | 2013-03-13 | 2014-09-18 | Institut National De La Sante Et De La Recherche Medicale | Composé d'activation de voie erk pour prévenir ou traiter une résistance à la leptine |
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IL183022A0 (en) | 2007-09-20 |
CN101094863A (zh) | 2007-12-26 |
US20090142266A1 (en) | 2009-06-04 |
JP2008519592A (ja) | 2008-06-12 |
AU2005303623A1 (en) | 2006-05-18 |
CA2586811A1 (fr) | 2006-05-18 |
JP5416901B2 (ja) | 2014-02-12 |
EP1814905B1 (fr) | 2013-04-17 |
US8207293B2 (en) | 2012-06-26 |
FR2877946A1 (fr) | 2006-05-19 |
EP1814905A1 (fr) | 2007-08-08 |
FR2877946B1 (fr) | 2011-02-11 |
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