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WO2011048443A1 - Peptides inhibant l'exocytose neuronale dérivés de la sous-unité c de v-atpase et compositions cosmétiques et pharmaceutiques contenant lesdits peptides - Google Patents

Peptides inhibant l'exocytose neuronale dérivés de la sous-unité c de v-atpase et compositions cosmétiques et pharmaceutiques contenant lesdits peptides Download PDF

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WO2011048443A1
WO2011048443A1 PCT/IB2009/055225 IB2009055225W WO2011048443A1 WO 2011048443 A1 WO2011048443 A1 WO 2011048443A1 IB 2009055225 W IB2009055225 W IB 2009055225W WO 2011048443 A1 WO2011048443 A1 WO 2011048443A1
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seq
subunit
polypeptide according
peptide
vamp2
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Oussama El Far
Michael Seagar
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Institut National de la Sante et de la Recherche Medicale INSERM
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Institut National de la Sante et de la Recherche Medicale INSERM
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/91—Injection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention refers to novel peptides derived from the carboxy end of the C subunit of theV-ATPase, useful as inhibitors of neuronal exocytosis, and to their use in therapeutic and/or cosmetic applications.
  • the basis or mechanism for the formation of facial wrinkles is the tensing of the muscles of the epidermis that drag the skin inwards. This muscular tension is the result of hyperactivity of the nerves innervating the facial muscles. Nerve hyperactivity is characterized by the uncontrolled and excessive release of neurotransmitters that excite muscle fibers. Because of this, the molecules that control neuronal exocytosis contribute to relaxing muscular tension, and consequently, to eliminating wrinkles.
  • Botulinum toxins are a family of bacterial neurotoxins produced by Clostridium Botulinum . 7 different serotypes are known (serotypes A, B, C, D, E, F and G) with an average molecular weight of 150 kDa. These toxins inhibit acetylcholine exocytosis in the neuromuscular junction (nerve-muscle synapse) of the skeletal muscle (Schiavo, G., et al).
  • botulinum toxins are proteases that degrade neuronal proteins involved in the exocytosis mechanism activated by the calcium ion .
  • botulinum toxin A the one most commonly used clinically and cosmetically [because of its applications in eliminating facial wrinkles and asymmetry, and to mitigate the symptomatology of spastic diseases] cleaves the neuronal protein SNAP-25.
  • This protein (SNAP-25) plays a key role in neurosecretion, as it is involved in the formation of a protein complex (known as SNARE complex or fusion complex), which directs and controls the release of acetylcholine accumulated in vesicles.
  • the nucleus of said fusion complex is made up of proteins SNAP-25 and syntaxin, located in the presynaptic plasma membrane, and protein synaptobrevin (or VAMP), located in the vesicular plasma membrane .
  • the main function of the fusion complex is to bring the vesicle loaded with neurotransmitter (i.e acetylcholine, glutamate, ...) nearer to the presynaptic plasma membrane and put it in contact with same. In this way, in response to an elevated concentration of calcium, the fusion of both plasma membranes is encouraged, thus producing the release of the neurotransmitter. Therefore, said vesicle docking and fusion protein complex (SNARE) is a key target in controlling neurosecretion. Cleaving any of the proteins that make up the fusion complex prevents its assembly, and therefore inhibits vesicle release and neuronal exocytosis.
  • botulinum toxins and, in particular, serotype A (BOTOX TM ) to inhibit neurosecretion, as well as their neuronal selectivity (they only act on neurons) is being widely used therapeutically to correct spastic ailments such as dystonias, strabismus, tics, blepharospasm, facial scoliosis, etc...
  • Botulinum toxin A (botulinum A) is, moreover, an effective agent for eliminating facial wrinkles and asymmetry.
  • the administration of BOTOX TM is the first effective non-surgical therapy to eliminate the signs of aging.
  • Therapeutic and cosmetic treatment with BOTOX TM consists of a localized injection of diluted pharmaceutical preparations (botulinum A-hemagglutinin complex, 500 kDa) in the areas where muscular tension is localized.
  • the paralytic effects of the toxin are reversible with an average duration of 6 months.
  • the treatment therefore, requires repeated injections of BOTOX TM .
  • the main problem with this treatment is the chance that it may trigger an immune reaction against the pharmaceutical preparation due to the fact that, because of its molecular size, it may be recognized by the patient's immune system.
  • the appearance of antibodies against botulinum A is a serious problem, as it contributes to a clear decrease in the treatment's effectiveness.
  • V0 V-ATPase
  • V0 c-subunit and the v-SNARE VAMP2 also called synaptobrevin, also target of several botulinum neurotoxins.
  • the different domain on the c-subunit likely to bind VAMP2 were mapped ,and, in order to explore the functional relevance of this interaction, various derived peptide isolated from c-subunit domain were tested.
  • the invention provides a solution to existing need , namely the provision of a soluble peptide with a sequence of twelve amino acids (POF16) as well as derived peptides corresponding to portion of the V0 c-subunit sequence (loop 3-4 domain) that VAMP2 binds to and which have the property to interfere with the neurotransmitter release.
  • POF16 twelve amino acids
  • derived peptides corresponding to portion of the V0 c-subunit sequence (loop 3-4 domain) that VAMP2 binds to and which have the property to interfere with the neurotransmitter release.
  • the invention provides a polypeptide comprising at least 6 consecutive amino acid selected in the amino acid sequence ranging from positions 117 to 128 of SEQ ID NO: l (Vo ATPase subunit C) or a function-conservative variant which is able to at least partially inhibit neuronal exocytosis.
  • the polypeptide has a length of 6 to 20 amino acids and contains an sequence made up of 6 to 12 adjacent amino acids contained in the carboxy end of the c-subunit of the membrane component of V-ATPase (called V0), which inhibits neuronal exocytosis.
  • V0 V-ATPase
  • An additional object of the invention is a nucleic acid that essentially codes for the polypeptides provided by this invention.
  • Another additional object of this invention is a cosmetic composition that includes at least one peptide provided by the invention.
  • Another additional object of this invention is a pharmaceutical composition that includes at least one peptide provided by this invention, or alternatively, a vector containing a nucleic acid that codes for one of the peptides of the invention.
  • Another additional object of the invention is a combination of drugs that includes at least one of the peptides provided by the invention, along with, at least, one drug intended as a second therapeutic target which may be the same as or different from the therapeutic target at which the peptide provided by this invention is aimed.
  • V-ATPase c-subunit of V-ATPase
  • “Function-conservative variants” are peptides derived from the peptide of the invention in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70 % to 99 % as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
  • a “function-conservative variant” also includes a polypeptide which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75 %, most preferably at least 85%, and even more preferably at least 90 %, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.
  • Two amino acid sequences are "substantially homologous” or “substantially similar” when greater than 80 %, preferably greater than 85 %, preferably greater than 90 % of the amino acids are identical, or greater than about 90 %, preferably grater than 95 %, are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA, etc.
  • V-ATPase or Vacular proton ATPase, or “VATPase Vo” or “Vo VATPase” or “Vo” refers to large multi-molecular enzymatic complex expressed in all eukaryotic cells, with the primary function of proton pumping on cellular proximity. It is omnipresent in intracellular membrane compartments, including synaptic vesicles (Stevens and Forgac, 1997) where it generates vesicular proton gradients and membrane potential that underlie GABA/monoamine and glutamate uptake (Moriyama et al., 1992). V-ATPase is composed of two reversibly-associated sectors, a peripheral multi-subunit
  • V0 is composed of a rotor of six subunits (5 c-subunits and 1 c"-subunit) and single copies of a, d and e-subunits.
  • the V-ATPase c-subunit can be from any source, but typically is a mammalian (e.g., human and non- human primate, rodent,...) V-ATPase, and more particularly a rat V-ATPase.
  • ATPase c-subunit is provided by SEQ ID NO: l and the nucleic sequence of the ADNc is NCBI referenced as NM_130823.
  • the sequence of V-ATPase c-subunit is divided in different regions.
  • the term "preventing” or “prevention” refers to preventing the disease or condition from occurring in a patient which has not yet been diagnosed as having it.
  • treating refers to reversing, alleviating, inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • patient refers to any subject (preferably human) afflicted with or susceptible to be afflicted with i.e. a neuronal exocytosis-mediated pathological diseases and/or disorders or for the treatment of facial wrinkles and/or asymmetry.
  • cosmetically and/or pharmaceutically acceptable salts of the peptide of the invention include cosmetically and/or pharmaceutically acceptable salts of the peptide of the invention.
  • Cosmetically and/or pharmaceutically acceptable salts includes salts customarily used to form metal salts or salts formed by adding free acids or bases. The nature of the salt is not critical, as long as it is cosmetically and/or pharmaceutically acceptable.
  • Cosmetically and/or pharmaceutically acceptable salts of the peptide of the invention may be obtained from acids or bases, organic or inorganic, by conventional methods which are well known to technicians in these matters, by making the appropriate acid or base react with the peptide of the invention.
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other non desired reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically or cosmetically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • a first object of the present invention relates to a polypeptide comprising at least 6 consecutive amino acid selected in the amino acid sequence ranging from positions 117 to 128 of SEQ ID NO: l or a function-conservative variant which is able to at least partially inhibit neuronal exocytosis.
  • SEQ ID N° 1 is not a polypeptide of the invention.
  • This invention provides a peptide deriving from the carboxy end of the protein V- ATPase c-subunit. More specifically, the invention provides a polypeptide, herein designated as the polypeptide of the invention, which has a sequence of 6 to 12 adjacent amino acids contained in SEQ. ID. No. 1 [see the section regarding the SEQUENCE LIST].
  • the invention also includes peptides which are substantially homologous to the polypeptide of the invention.
  • the peptide of the invention has a length of 6 to 20 amino acids, and preferably from 6 to 12 amino acids.
  • said polypeptide comprises the amino acid sequence ranging from positions 117 to 128 of SEQ ID NO: l (SEQ ID N°2) .
  • said polypeptide comprises the amino acid sequence ranging from positions 119 to 128 of SEQ ID NO 1 : (SEQ ID N°3)
  • said polypeptide comprises the amino acid sequence ranging from positions 121 to 128 of SEQ ID NO:l (SEQ ID N°4)
  • said polypeptide comprises the amino acid sequence ranging from positions 123 to 128 of SEQ ID NO:l . (SEQ ID N°5)
  • said polypeptide comprises the amino acid sequence ranging from positions 117 to 126 of SEQ ID NO:l (SEQ ID N°6).
  • said polypeptide comprises the amino acid sequence ranging from positions 117 to 124 of SEQ ID NO:l (SEQ ID N°7).
  • said polypeptide comprises the amino acid sequence ranging from positions 117 to 122 of SEQ ID NO:l (SEQ ID N°8).
  • said polypeptide comprises the amino acid sequence ranging from positions 119 to 126 of SEQ ID NO:l (SEQ ID N°9).
  • said polypeptide comprises the amino acid sequence SEQ ID NO:2.
  • amino acids that make up the structural units of the peptide of the invention may have D- or L-configuration.
  • the amino acid from the amino end may have an acetylated terminal amino group, and the amino acid from the carboxyl end may have an amidated terminal carboxyl group.
  • this invention also includes derivatives of the peptide of the invention in which the amino -terminal end is acetylated and/or in those where the carboxy-terminal end is amidated.
  • polypeptides of the invention are those polypeptides that have sequences of amino acids shown in SEQ. ID No. 2 to SEQ. ID No. 9.
  • the polypeptide of the invention may undergo reversible chemical modifications in order to increase its bioavailability (including stability and fat solubility) and its ease in passing through the blood-brain barrier and epithelial tissue.
  • reversible chemical modifications include the esterification of the carboxylate groups of glutamic and aspartic amino acids with an acetyl-methyl group, by which the negative charge of the amino acid is eliminated and its hydrophobicity is increased.
  • reversible chemical modifications include the addition of a further peptidic sequence, which allows the increase of the membrane permability, such as a TAT peptide or Penetratin peptide (see - Charge-Dependent Translocation of the Trojan .A Molecular View on the Interaction of the Trojan Peptide Penetratin with the Polar Interface of Lipid Bilayers. Biophysical Journal, Volume 87, Issue 1, 1 July 2004,
  • polypeptide of the invention may be obtained through conventional methods of solid-phase chemical polypeptide synthesis, following Fmoc and/or Boc-based methodology (see Pennington, M.W. and Dunn, B.N. (1994). Peptide synthesis protocols.
  • the polypeptide of the invention may be obtained through conventional methods based on recombinant DNA technology, e.g., through a method that, in brief, includes inserting the nucleic acid sequence coding for the polypeptide of the invention into an appropriate plasmid or vector, transforming competent cells for said plasmid or vector, and growing said cells under conditions that allow the expression of the polypeptide of the invention and, if desired, isolating and (optionally) purifying the polypeptide of the invention through conventional means known to experts in these matters.
  • the nucleic acid sequence that codes for the polypeptide of the invention may be easily deduced from the correspondence that exists between the amino acids and the nucleotide codons that code for such amino acids.
  • an additional object of the invention is an isolated nucleic acid sequence that codes for the polypeptide of the invention.
  • said nucleic acid is selected from single-strand DNA, double-stranded DNA, and RNA.
  • Additional objects of this invention are plasmids and expression vectors that contain said nucleic acid sequence that codes for the polypeptide of the invention, as well as prokaryotic or eukaryotic cells that express the polypeptide of the invention.
  • the polypeptide of the invention is able to at least partially inhibit neuronal exocytosis, probably through a mechanism that involves interfering with the assembly of the fusion protein complex (SNARE) and/or its thermal destabilization and/or its zipping completion.
  • SNARE fusion protein complex
  • the invention also includes peptides which are functionally equivalent to the polypeptide of the invention or " function-conservative variant".
  • the expression “functionally equivalent” means that the peptide in question has at least one of the biological activities of the peptide of the invention, such as, for example, the ability to at least partially inhibit neuronal exocytosis
  • neuronal-exocytosis (neurosecretion) inhibiting capabilities of the polypeptides of the invention will become evident to the skilled person by implementing a simple test to evaluate the kinetic of said polypeptidepeptides in inhibiting the release of neurotransmitters (as in the example of this application in Nicotinic SCG neurons) This kinetic of the inhibition is measured as a reduction in the Excitatory Post Synaptic
  • Another test could be to assess the strength of said polypeptides in inhibiting the release of neurotransmitters induced by calcium in chromaffin cells permeabilized with a detergent (see Quetglas S, et al 2002). Briefly, calcium induced release of either tritiated norepinephrin or transfected growth hormon could be measured after cell membrane permeabilization..
  • the docapeptide of the invention [SEQ. ID. No. 2], at a concentration of 1.5 to 2 mM, blocked approximately 20% of the acetylcholine release from Nicotinic SCG neurons and up to 70% of the glutamate release in glutamatergic cortical pyramidal neurons.
  • said polypeptides may have a cyclic structure by adding a neutral polypeptide linker that should help reaching a sufficient length to allow a constrained presentation of the polypeptide of the invention.
  • a neutral polypeptide linker that should help reaching a sufficient length to allow a constrained presentation of the polypeptide of the invention.
  • the mechanism of action of the peptides of the is prima facie similar to that of botulinum toxins, thus affecting the formation and/or stability of the fusion protein complex; so that the polypeptides of the invention can be considered to have cosmetic/therapeutic applications identical or similar to those described for botulinum toxin. Therefore, the polypeptides of the invention may be regarded as efficacious, stable, safe and economical substitutes for botulinum toxins, both for the treatment of facial wrinkles and/or asymmetry and in the treatment of the symptomatology of spastic diseases, allowing to consider their use as neuroprotectors in the treatment of neurological disorders and neurodegenerative diseases.
  • polypeptides of the invention may thus be used for pathological neuronal exocyto sis-mediated cosmetic and/or therapeutic purposes.
  • a further object of the present invention relates to a nucleic acid molecule encoding polypeptides according to the invention.
  • a "coding sequence” or a sequence “encoding” an expression product, such as a R A, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme.
  • a coding sequence for a protein may include a start codon (usually ATG) and a stop codon. These nucleic acid molecules may be obtained by conventional methods well known to those skilled in the art, in particular by site-directed mutagenesis of the gene encoding the native protein.
  • said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.
  • a suitable vector such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.
  • a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted.
  • recombinant vectors may, for example, be cloning vectors, or expression vectors.
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) may be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • a DNA or RNA sequence e.g. a foreign gene
  • Any expression vector for animal cell may be used, as long as a gene encoding a polypeptide or chimeric derivative of the invention can be inserted and expressed.
  • suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4) and the like.
  • Plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.
  • viral vector examples include adenoviral, retroviral, herpes virus and AAV vectors.
  • recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • virus packaging cells include PA317 cells, PsiCRIP cells,
  • promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like.
  • the invention also includes gene delivery systems comprising a nucleic acid molecule of the invention, which can be used in gene therapy in vivo or ex vivo.
  • This includes for instance viral transfer vectors such as those derived from retrovirus, adenovirus, adeno associated virus, lentivirus, which are conventionally used in gene therapy.
  • This also includes gene delivery systems comprising a nucleic acid molecule of the invention and a non-viral gene delivery vehicle.
  • non viral gene delivery vehicles include liposomes and polymers such as polyethylenimines, cyclodextrins, histidine/lysine (HK) polymers, etc.
  • a subject of the present invention is also a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule according to the invention.
  • transformation means the introduction of a "foreign” (i.e. extrinsic or extracellular) gene, DNA or R A sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
  • a host cell that receives and expresses introduced DNA or RNA bas been "transformed”.
  • eukaryotic cells in particular mammalian cells, and more particularly human cells, will be chosen.
  • cell lines such as CHO, BHK-21, COS-7, CI 27, PER.C6 or HEK293 could be used, for their ability to process to the right post-translational modifications of the derivatives.
  • V-ATPase c-subunit derivatives of the invention can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the derivative expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractionated precipitation, in particular ammonium sulphate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention.
  • the invention provides a cosmetic composition that includes a cosmetically effective amount of at least one peptide of the invention, along with at least one cosmetically acceptable adjuvant.
  • the peptides of the invention may be applied by means any medium that produces contact between the peptide and the location where it is to act in a mammal's body, preferably in humans.
  • the cosmetically effective amount of peptide that are applied, as well as the dosage for the treatment of facial wrinkles and/or asymmetry with the peptides and/or cosmetic compositions of the invention will depend on numerous factors, including the age and condition of the person desiring treatment, the severity of the wrinkles and/or facial asymmetry, the method and frequency of application and the particular peptide to be used.
  • the presentation of the cosmetic compositions containing the polypeptides of the invention may be in any form that is suitable for application, e.g., solid, liquid or semisolid, such as creams, ointments, gels or solutions, and the application of these compositions may be by any suitable means, preferably topically, so they will include the cosmetically acceptable adjuvants necessary to make up the desired form of administration.
  • the peptides of the invention are encapsulated in liposomes, along with (optionally) another or other (COOH) peptide(s), which are added to the other components of the cosmetic preparation.
  • an additional object of this invention is the use of the peptides of the invention in the preparation of cosmetic compositions for the treatment of facial wrinkles and/or asymmetry.
  • the invention also provides a method for the cosmetic treatment of facial wrinkles and/or asymmetry in mammals, preferably humans, which consists of applying a cosmetically effective amount of at least one peptide of the invention to the mammal that has facial wrinkles and/or asymmetry, along with (optionally) one or more (COOH) peptides, preferably in the form of a cosmetic composition containing it.
  • a further object of the present invention relates to the use of polypeptides of the invention for the treatment of spastic diseases, for example, dystonias, strabismus, blepharospasm, facial scoliosis, tics, etc.; and/or as neuroprotectors in the treatment of neurological disorders and/or neurodegenerative diseases.
  • spastic diseases for example, dystonias, strabismus, blepharospasm, facial scoliosis, tics, etc.
  • neuroprotectors in the treatment of neurological disorders and/or neurodegenerative diseases.
  • neurological disorders are acute neurological diseases, for example, those that take place in the first stages of cerebral ischemia. It is a known fact that during an ischemic process an uncontrolled release of the neurotransmitter glutamate takes place in the affected area. This neurotransmitter interacts with specific neuronal membrane receptors causing a massive influx of calcium ions inside the neuron. The intracellular calcium causes the release of more glutamate, thus triggering a chain reaction. Moreover, the massive, prolonged influx of calcium inside the neurons causes their death, which translates into the formation of necrotic tissue in the ischemic zone. Clearly, the progress of the ischemic damage can be stopped, at least partially, if the uncontrolled glutamate exocytosis is controlled.
  • the polypeptides of the invention because of their ability to inhibit exocytosis, may be suitable for preventing and/or slowing down the neuronal death that is characteristic of an ischemic process, and so would be useful in the treatment of neuropathologies that occur because of excessive glutamate exocytosis, such as, for example, senile dementia, Alzheimer's-related dementia, AIDS-related dementia, epilepsy, amiotrophic sclerosis, multiple/lateral sclerosis, etc.
  • application in the treatment of neurological diseases would be similar to the one described for botulinum toxin A (see Clarke, C.E. (1992). Therapeutic potential of botulinum toxin in neurological disorders. Quart. J. Med. 299, 197-205. 18).
  • polypeptides of the invention could therefore form part of a combined therapy (aimed at several therapeutic targets) with the objective of more effectively stopping neurodegeneration.
  • An additional object of this invention is a pharmaceutical composition which includes a therapeutically effective amount of at least one polypeptide of the invention, along with at least one pharmaceutically acceptable excipient.
  • said pharmaceutical composition also contains one or more (COOH) peptides.
  • the pharmaceutical composition of the invention may contain a therapeutically effective amount of a vector that contains at least one nucleic acid sequence that codes for a polypeptide of the invention, along with at least one adjuvant and/or a pharmaceutically acceptable excipient. Said vector may be used in gene therapy.
  • a “therapeutically effective amount” is meant a sufficient amount of the chimeric derivative of the invention to treat pathological neuronal exocytosis-mediated pathological diseases and/or disorders, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily dosage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the active products of the invention may be administered for the treatment of pathological neuronal exocytosis, manifested, for example, by spastic diseases, neurological disorders or neurodegenerative diseases, through any medium that produces contact between the polypeptide and the place where it is to act in a mammal's body, preferably in humans.
  • the therapeutically effective amount of the active product of the invention [peptides or vectors (constructions)] that should be administered, as well as the dosage for the treatment of a pathological condition with the peptides and/or pharmaceutical compositions of the invention, will depend on numerous factors, including the age and condition of the patient, the severity of the disturbance or disorder, the method and frequency of administration and the particular peptide to be used.
  • compositions that contain the peptides or vectors (constructions) of the invention may be in any form that is suitable for administration, e.g., solid, liquid or semi-solid, such as creams, ointments, gels or solutions, and these compositions may be administered by any suitable means, for example, orally, parenterally or topically, so they will include the pharmaceutically acceptable excipients necessary to make up the desired form of administration.
  • suitable means for example, orally, parenterally or topically
  • a review of the different pharmaceutical forms for administering medicines and of the excipients necessary for obtaining same may be found, for example, in the "Tratado de Farmacia Gal nica" (Treatise on Galenic Pharmacy), C. Faul i Trillo, 1993, Luz n 5, S.A. Ediations,
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral- route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the 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.
  • 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 comprising compounds of the invention as free base 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 and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypetides of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the 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.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. 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.
  • the polypeptide of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • parenteral administration such as intravenous or intramuscular injection
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.
  • the peptides of the invention could form part of a combined therapy for the purpose of more effectively stopping neurodegeneration.
  • the invention provides a pharmaceutical composition that includes at least one peptide of the invention; along with (optionally) another or other neuronal-exocytosis inhibiting compound(s), and along with at least one drug intended for another therapeutic target, selected from the group formed by a neuronal glutamate receptor blocker, a calcium chelator, an anti-oxidant, a free-radical destroyer and their combinations.
  • said composition that is useful in combined therapy may contain at least one peptide of the invention, along with (optionally) another or other neuronal exocytosis inhibiting compound(s) and a neuronal glutamate receptor blocker.
  • said composition could contain at least one peptide of the invention, along with (optionally) another or other neuronal exocytosis inhibiting compound(s), a neuronal glutamate receptor blocker, a calcium chelator, an anti-oxidant and/or a free-radical destroyer.
  • An additional object of this invention is the use of the peptides of the invention or of vectors that contain at least one sequence that codes for a polypeptide of the invention, in the preparation of a medicine for the treatment of pathological neuronal exocytosis- mediated pathological diseases and/or disorders, such as, for example, spastic diseases, neurological disorders and/or neurodegenerative diseases.
  • the invention provides a method for the treatment in mammals of pathological neuronal exocytosis-mediated pathological diseases and disorders such as, for example, spastic diseases, neurological disorders and/or neurodegenerative diseases, which consists of administering to said mammal suffering from said pathological disease or disorder a therapeutically effective amount of at least one peptide of the invention, or of a vector containing at least one DNA sequence that codes for a peptide of the invention, preferably in the form of a pharmaceutical composition that contains it.
  • said pharmaceutical composition contains, in addition to the peptide or peptides of the invention, one or more (COOH) peptides.
  • V0 c-subunit loop 3.4 peptide inhibits neurotransmission at L5-L5 excitatory synapses
  • oligonucleotides were purchased from MWG Biotech (Germany). Glutathione Sepharose was from GE Healthcare and Ni-NTA agarose from QIAGEN. Peptides (L3.4: GVRGTAQQPRLF; L3.4s: GQATVQPLGRRF) were synthesized by Activotec (Southampton, UK). All proteins purifications were performed in the presence of protease inhibitors (Complete, Roche).
  • VAMP2 constructs were either full length or contained syntaxin transmembrane domain VAMP2-STXTM. Except for two hybrid studies where full length VAMP2B was used, all VAMP2B constructs lacked a transmembrane domain (TM) (amino acids 2-92). All constructs were generated by standard polymerase chain reaction (PCR) using a commercial Y2H adult rat brain cDNA library (Origine).
  • PCR polymerase chain reaction
  • VAMP2 construct encompassing residues 2-92 (forward 5'- GCGAATTCTCGGCTACCGCTGCCAC-3' + Reverse 5'- GCGTCGACTTAGTTTTTCCACCAGTATTTGCG-3') was amplified and inserted between EcoRl and Sail sites of pGEX4T-l (GVA).
  • VAMP2 2-31 Formward 5'- GCGAATTCTCGGCTACCGCTGCCAC-3' + Reverse 5'-
  • VAMP2 28-76 (Forward 5'- CGAATTC AGT AAC AGGAGACTGC AGC-3 ' + Reverse 5'-GCGTCGACTTACTGG GAGGCCCCTGCCTG-3') and VAMP2 28-92 (Forward 5'-
  • GCGTCGACTTAGTTTTTCCACCAGTATTTGCG-3' were amplified and inserted between EcoR I and Sal I restriction sites into pGEX-5X-l (respectively GV-2-31, GV- 28-76 and GV-28-92). His tagged VAMP2-2-92 (His-VAMP2) was also obtained by inserting VAMP2 in pET28a using EcoRl and Sail sites.
  • VAMP2-STXTM Forward 5'-GCGAATTCTCGGCTACCGCTGCCAC-3' + Reverse 5'- GCGTCGACCTATCCAAAGATGCCCCCGATGGTGGAGGCGATGATGATGCCCA GAATCACACAGCAAATGATGATCATGATGTTTTTCCACCAGTATTTGCG-3'.
  • the reverse primer covers the TM of syntaxin and residues 86 to 92 of VAMP2.
  • HSV tag (QPELAPEDPED) was introduced as follow at the 5' of pET28a (Novagen) "multiple cloning site" to generate pET28-HSV- Nter: 5' phosphorylated primers encoding HSV tag sequence ( Forward 5'- GATCCCAGCCTGAACTCGCTCCAGAGGATCCGGAAGATG-3' Reverse 5'- AATTCATCTTCCGGATCCTCTGGAGCGAGTTCAGGCTGG-3') were annealed and ligated to BamHl, EcoRl open pET28a.
  • V0 c-subunit Full length V0 c-subunit (residues 2-155) was amplified and inserted between Sacl and Sail of pEGFPC3 and pET28a-HSV-Nter to generate pEGFPc3-c-2-155 and pET28-HSV-c-2-155 respectively. All truncated c-subunit constructs were amplified by PCR and cloned between EcoRl and Sail sites in pEGFPc2, pET28a-HSV-Nter, or EcoRl and Xhol for the yeast two hybrid vectors (Origine): pGilda
  • pEGFPc-2-c2-155 E-c-2-155
  • pGilda-c2-155 BD-c2-155
  • pGilda- c-2-76 BD-c-2-76
  • pGilda-c-2-36 BD-c-2-36
  • pGilda-c-37-155 BD-c37-155
  • pGilda- c-77-155 BD-c-77-155
  • pGilda-c-117-155 BD-c-117-155
  • BD-c-2-155 full length
  • BD-c-2-76 from the N-terminus until the end of TM2
  • BD-c-2-36 from the N-terminus until the end of TM1
  • c-37-155 from the beginning of loop 1.2 until the C-terminus
  • c77-155 from the beginning of loop 2.3 until the C-terminus
  • c- 117-155 from the beginning of loop 3.4 until the C-terminus).
  • c- 117-155 with scrambled loop 3.4 sequence GQATVQPLGR F/ ggtcaggccactgtccagcctctgggccgattc
  • L3.4s (c-117-155-L3.4s) was generated by PCR suing the following primers (Forward 5'- gcgaattcggtcaggccactgtccagcctctgggccgg-3' + Reverse 5'- gcgtcgacCTACTTTGTGGAGAGGATTAG-3') on a matrix of a full length c-subunit construct with scrambled loop 3.4 sequence pET28a-HSV-c-2-155-L3.4s (c-2-155-L3.4s). This later construct was made as follows using three consecutive PCRs: Using Platinum
  • Taq DNA polymerase (Pfx) (Invitrogen) and as amplification matrix pET28a-HSV-c-l- 155, overlapping PCR fragments were generated using a) 1- EcoRl flanked c-subunit forward primer (5'-GCGAATTCGCTGACATCAAGAACAACCC) and 2- reverse encoding the scrambled L3.4 sequence (5'- gaatcgccggcccagaggctggacagtggcctgaccAGCATCTCCGACAATGCC-3') b) 3- Forward primer encoding the scrambled L3.4 sequence (5'- ggtcaggccactgtccagcctctgggccgattcGTGGGCATGATCCTGATCC-3') and 4- a Sail flanked reverse T7 terminator primer (5 '-GCTAGTTATTGCTCAGCGG) that primes 3' to the vector multiple cloning site.
  • Pfx 1- EcoRl flanked c-sub
  • Syntaxin la / SNAP25 heterodimer (co-expressed from pGEX-KG and pET28a respectively) were provided by G. Schiavo (London Research Institute, London).
  • PH-L3.4 and PH-L3.4s were constructed as follow: Using Bbsl and Sail restriction sites, L3.4 and scrambled L3.4s encoding phosphorylated complementary oligonucleotides were introduced between -strands 6 and 6' of the PH domain of human cytohesinl in pET21d [Bedet, 2006 #46].
  • PH-L3.4 Forward primer (5'- aggccGGTGTCCGGGGCACTGCCCAGCAGCCTCGACTGTTCg-3'); Reverse primer (5'- tcgacGAACAGTCGAGGCTGCTGGGCAGTGCCCCGGACACCg-3').
  • PH-L3.4s Forward primer (5'- aggccGGTCAGGCCACTGTCCAGCCTCTGGGCCGACGGTTCg- 3'); Reverse primer
  • VAMP2 and c-subunit were expressed in Rosetta2 bacteria (Novagen). VAMP2 constructs were expressed using classical protocols. Crude supernatants of ultracentrifuged "French press" bacterial lysates were stored at -20°C. Bacterial strains expressing c-subunit constructs were cultured in pH 5.7 phosphate-buffered LB medium and protein expression was induced with 1 mM IPTG for 4-5 hours. Bacterial pellets were stored at -20°C until protein purification.
  • Bacterial pellets of His tagged proteins were French pressed in 20 mM Tris pH 8.0, 500 mM NaCl, 10%> glycerol buffer and purified on Ni-NTA beads in the presence of 0.1 %> Triton X-100. A last wash was made by adding to the French press buffer 50 mM imidazole and 0.5 mM DTT. Proteins were eluted in French press buffer containing 500 mM imidazole and 0.5 mM DTT and dialyzed overnight against 25 mM Hepes pH 7.4, 140 mM KC1 and 0.5 mM DTT. Protein aliquots were stored at -80°C.
  • t-SNAREs were co-expressed and purified using standard methods and were either eluted or cleaved from their tags by thrombin.
  • Bacterial pellets of GST tagged VAMP2-2-92 (GV ) and VAMP2-2-92-W89, 90A (GV- W89, 90A) were French pressed in 25 mM Tris pH 8.0, 150 mM NaCl buffer and purified on glutathione Sepharose beads in the presence of 0.1% Triton X-100. Proteins were eluted in Tris 50 mM pH 8.0, 10 mM reduced glutathione and frozen at -20°C until used.
  • VAMP2 constructs were freshly purified on glutathione beads from frozen cleared bacterial extracts. Per tube, a 1 ml mixture of ⁇ ⁇ GST and GST- VAMP2 constructs (GVA, GV-2-31, GV-28-76, GV-28-92, GV-W89,90A, GV-W89,90P or GV-W90A) was immobilized.
  • GVA ⁇ ⁇ GST and GST- VAMP2 constructs
  • GV-2-31, GV-28-76, GV-28-92, GV-W89,90A, GV-W89,90P or GV-W90A was immobilized.
  • pre-transfected HEK 293 cells from a 10 cm culture dish were solubilized in 1 ml of 25 mM Tris pH 7.5, 150 mM NaCl, 1% CHAPS supplemented with protease inhibitors (Complete, Roche).
  • the chromogenic HRP substrate TMB (3,3 ' ,5,5 ' -tetramethylbenzidine) was then used to detect VAMP2 binding using anti GST (GE healthcare) and an HRP coupled secondary antidody (Jackson ImmunoResearch) .
  • the synaptic vesicle enriched supernatant was then adjusted to 10 mM Tris-HCl pH 8.5, 60 mM sucrose, 140 mM KC1, 2 mM MgC12, 50 ⁇ EGTA (assay buffer). ATP-dependent proton transport was monitored by the quenching of acridine orange (AO) fluorescence.
  • AO acridine orange
  • Lipids were from Avanti Polar Lipids. 850 nmoles of dried lipids (85% (mol/mol) 1- palmitoyl, 2-oleoyl phosphatidylcholine (PC) + 15% 1,2-dioleoylphosphatidylserine (PS)) were resuspended in 25 mM HEPES-KOH pH 7.4, 140 mM KC1, 0.25 mM DTT,1.5% (w/v) sodium cholate (Sigma). Liposomes were obtained by rapid dilution and extensive dialysis in the resuspension buffer.
  • v-SNAREs and t-SNAREs liposomes were carried out essentially as previously described (Weber et al, 1998).
  • Lipids were from Avanti Polar Lipids. Dried lipids were resuspended in VAMP2 or syntaxin 1 / SNAP25 heterodimer solutions in presence of 1.5% (w/v) sodium cholate (Sigma), at a 1/100 (mol/mol) protein-to-lipid ratio.
  • Lipid compositions were 85% (mol/mol) 1 -palmitoyl, 2-oleoyl phosphatidylcholine (PC), 15% 1,2- dioleoylphosphatidylserine (PS) for the acceptor v-SNARE vesicles and 83% PC, 15% PS, 1.5% (mol/mol) N-(7-nitro-2- 1 ,3-benzoxadiazol-4-yl)- 1 ,2-dipalmitoyl phosphatidylethanolamine (NBD-PE), and 1.5% (mol/mol) N-(lissamine rhodamine B sulfonyl)-l,2-dipalmitoyl phosphatidylethanolamine (Rhodamine-PE) for the donor t-SNARE liposomes.
  • PC 2-oleoyl phosphatidylcholine
  • PS 1,2- dioleoylphosphatidylserine
  • Liposomes were obtained by rapid dilution and extensive dialysis in 25 mM HEPES-KOH pH 7.4, 140 mM KC1, 1 mM DTT in the presence of Bio Beads (Biorad), unincorporated proteins and aggregates were removed by a 4 hour centrifugation (250000xg) on a discontinuous Optiprep (Abcys) gradient.
  • Loop 3.4 L3.4
  • L3.4s scrambled synthetic c-subunit peptides
  • Bafilomycin Al was purchased from Euromedex (France) and was bath applied (final concentration, 5 ⁇ ). All paired-pulse protocols were performed at a frequency of 20 Hz. Synaptic responses were averaged following alignment of the presynaptic action potentials using automatic peak detection (Detectivent 4.0, N. Ankri INSERM). Data are means ⁇ SEM.
  • EPSP recording and injection of peptides were performed as described previously (Mochida et al, 1994).
  • EPSPs were recorded at 0.1, 0.25 Hz or 0.5 Hz.
  • the peak amplitudes were normalized to the values before injection.
  • VAMP2 interacts with VP c-subunit
  • Yeast two hybrid (Y2H) methods have not been widely used to probe interactions between proteins with transmembrane (TM) domains.
  • detection of V0 c-subunit as a partner for ⁇ integrin (Skinner and Wildeman, 1999) and identification of a full length (FL) syntaxin 1 clone in a screen using ⁇ SNAP as bait (O El Far - unpublished results) encouraged us to pursue Y2H analysis of in vivo interactions between SNAREs and V0 subunits. Therefore, we used the LexA Y2H system to probe for interactions between c-subunit and VAMP2.
  • VAMP2-stxTM interacted with c-subunit, while FL syntaxinl did not ( Figure IB).
  • V0 c-subunit is an extremely hydrophobic protein with four TM domains connected by very short loops and discrete N and C-termini. Analysis of native proteins was unsuccessful since both commercially available and specifically designed anti-peptide antibodies against exposed conserved regions of V0 c-subunit, failed to recognise rat proteins (data not shown). We therefore expressed GFP-tagged full length c-subunit (E-c-2-155) in HEK 293 cells. Pulldown assays detected the specific binding of multimeric forms of E-c-2-155 to bacterially- expressed GST-VAMP2-2-92 (GVA), but not to GST, immobilised on glutathione beads (). These data indicate that c-subunit can associate with VAMP2 and that the TM region of VAMP is not necessary for interaction, consistent with Y2H data.
  • VAMP2 Mapping of c-subunit interaction sites on VAMP2 indicated that only VAMP constructs containing the calmodulin (CaM) binding motif at amino acids 76-92 (Quetglas et al, 2000) bound to c-2-155 . Furthermore mutation of juxtamembrane tryptophans (W89W90) known to be involved in CaM binding (Quetglas et al, 2002) completely abolished c-subunit binding. To consolidate binding data, we used soluble His- tagged VAMP2-ATM (His-VAMP2) to displace c-subunit binding to immobilized GVA.
  • CaM calmodulin binding motif at amino acids 76-92
  • W89W90 juxtamembrane tryptophans
  • L3.4 peptides might inhibit V-ATPase proton pump activity and induce a default in vesicle loading with transmitter, leading to "firing blanks" (ie. fusion of synaptic vesicles that are empty or have reduced contents).
  • L3.4 peptide inhibits neurotransmitter release in cholinergic rat superior cervical ganglion (SCG) neurons
  • SCG neurons provide a well-established culture system in which many agents that perturb neurotransmitter release mechanisms have already been studied.
  • SCG neurons have extremely short axons, thus somatically- injected peptides reach nerve terminals very rapidly.
  • this experimental system allows direct evaluation of the kinetics of effects, providing an indication as to which step in exocytosis is perturbed when a protein-protein interaction is disrupted (Ma and Mochida, 2007).
  • L3.4 peptide does not affect vesicle recycling and thus are consistent with the view that it perturbs the interaction of VAMP2 and c-subunit of V-ATPase V0 at a late step of exocytosis, close to fusion. L3.4 peptide inhibits SNARE dependent in vitro membrane fusion
  • L3.4 peptide mimics a c-subunit cytoplasmic linker sequence
  • empty vesicles might still fuse without generating an EPSP, thus a reduced EPSP could result from blockade of the proton pump.
  • bafilomycin Al In the strong synapses of peripheral SCG neurons, bafilomycin Al only modified EPSPs after prolonged stimulation, while L3.4 peptide reduced EPSPs much more rapidly, even with lower stimulation frequencies. This observation is consistent with the idea that bafilomycin affects a step in vesicle recycling (eg. reduced loading with transmitter due to a defective proton gradient) while L3.4 peptide inhibits a downstream step closer to vesicle fusion. As expected in the weaker synapses of central glutamatergic neurons the onset of inhibition by bafilomycin Al was faster and induced gradual inhibition of EPSCs similar to that observed with L3.4. However, the effects of bafilomycin Al and L3.4 on short term plasticity were completely different.
  • L3.4 increased the paired pulse ratio (PPR) by over 62% while bafilomycin Al produced the opposite effect with a PPR decrease of about 23%.
  • high concentrations of L3.4 peptide had no effect on bafilomycin-sensitive proton transport by V-ATPase in vitro, in a membrane fraction enriched in synaptic vesicles. All these results indicate that the inhibition of neurotransmitter release by L3.4 peptide is not a consequence of perturbing V-ATPase proton pump activity.
  • L3.4 peptide induced very rapid inhibition (onset at around 1 min) which is typical of agents that perturb neurotransmission at late steps in exocytosis (eg. SNARE interacting Synprint peptide from voltage-gated N-type calcium channel (Mochida et al., 1996), P/Q type calcium channel blockers (Mochida et al., 2003). Consistent with this interpretation the L3.4 peptide induced a slightly slower speed of rise and decay of the EPSP, suggesting that the kinetics of synaptic vesicle fusion might be modified in the presence of the peptide.
  • VAMP2 W89W90 in Ca2+-dependent exocytosis from chromaffin and PC12 cells.
  • the tryptophan motif has been implicated in SNARE complex dimers (Fdez et al, 2008).
  • VAMP W89A,W90A mutant in VAMP KO neurons induced a 2-fold decrease in evoked release compared to wild-type rescue but does not affect SNARE assembly nor binding of complexin or synaptotagmin to the SNARE complex (Maximov et al, 2009).
  • V0 sector of the V- ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton
  • beta(l) integrin binds the 16-kDa subunit of vacuolar H(+)-ATPase at a site important for human papillomavirus E5 and platelet-derived growth factor signaling. J Biol Chem 274, 23119-23127.
  • SNAP-29 A general SNARE protein that inhibits SNARE disassembly and is implicated in synaptic transmission. Proceedings of the National Academy of Sciences of the United States of America 98, 14038-14043.

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Abstract

La présente invention concerne des peptides dérivés de l'extrémité carboxy de la protéine de sous-unité C de V-ATPase, utiles en tant qu'inhibiteurs de l'exocytose neuronale, et leur utilisation dans des applications cosmétiques et/ou thérapeutiques.
PCT/IB2009/055225 2009-10-20 2009-10-20 Peptides inhibant l'exocytose neuronale dérivés de la sous-unité c de v-atpase et compositions cosmétiques et pharmaceutiques contenant lesdits peptides Ceased WO2011048443A1 (fr)

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EP2649985A1 (fr) 2012-04-13 2013-10-16 Lipotec, S.A. Composés inhibant l'exocytosis neuronale (III)
EP2649984A1 (fr) 2012-04-13 2013-10-16 Lipotec, S.A. Composés qui inhibent l'exocytosis neuronale
EP2649983A1 (fr) 2012-04-13 2013-10-16 Lipotec, S.A. Composés inhibant l'exocytosis neuronale (II)
WO2013153191A1 (fr) 2012-04-13 2013-10-17 Lipotec, S.A. Composés inhibant l'exocytose neuronale (ii)
CN106047862A (zh) * 2016-06-07 2016-10-26 上海欧易生物医学科技有限公司 用于降低酵母双杂交文库假阴性率的反转录方法
US9566227B2 (en) 2012-04-13 2017-02-14 Activen Cosmetic composition comprising a muconopeptide
US10149812B2 (en) 2012-04-13 2018-12-11 Activen Cosmetic composition comprising a muconopeptide
WO2019238683A1 (fr) 2018-06-13 2019-12-19 Aziende Chimiche Riunite Angelini Francesco - A.C.R.A.F. S.P.A. Peptides ayant une activité inhibitrice sur l'exocytose neuronale

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