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WO2013041238A1 - Utilisation de peptides kibra, d'un inhibiteur de hdac ou d'un inhibiteur du protéasome pour améliorer des troubles anxieux - Google Patents

Utilisation de peptides kibra, d'un inhibiteur de hdac ou d'un inhibiteur du protéasome pour améliorer des troubles anxieux Download PDF

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Publication number
WO2013041238A1
WO2013041238A1 PCT/EP2012/003974 EP2012003974W WO2013041238A1 WO 2013041238 A1 WO2013041238 A1 WO 2013041238A1 EP 2012003974 W EP2012003974 W EP 2012003974W WO 2013041238 A1 WO2013041238 A1 WO 2013041238A1
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sequence
peptide
seq
kibra
treating
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Inventor
Armin Schneider
Christian Plaas
Claudia Pitzer
Joachim KREMERSKOTHEN
Kerstin DUNING
Boris SKRYABIN
Hermann-Joseph PAVENSTÄDT
Lia SCARABOTTOLO
Loredana REDAELLI
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Westfaelische Wilhelms Universitaet Muenster
Sygnis Pharma AG
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Sygnis Bioscience GmbH and Co KG
Westfaelische Wilhelms Universitaet Muenster
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to KIBRA and peptides thereof, as well as peptide derivatives and peptide conjugates of said peptides, for use in methods of improving anxiety disorders. All of these compounds are considered capable of stabilizing protein kinase M zeta (in the following "PKM zeta", "PKMz” or the like) and thus allow said effect.
  • the invention relates to HDAC inhibitor and/or proteasome inhibitors for use in various medical methods.
  • PKM zeta is an atypical protein kinase and a molecule that is involved in memory maintenance (cf. e.g. Sacktor, 1993; Sacktor et ah, 2008). For example, inhibiting PKM zeta activity is linked to a decrease in the ability to maintain long-term memories and procedural knowledge, while a increase in PKM zeta levels enhances the capabilities for memory storage (Shema et ah, 2007; Shema et ah, 2011; Serrano et ah, 2008; Kraus et.ah, 2010).
  • PKM zeta is a brain specific variant of and is known to be identical to about the C- terminal half of the atypical protein kinase C ⁇ ("PKC zeta")D .
  • PKM zeta lacks the N- terminal auto-inhibitory domain of PKC zeta and is e.g. generated by an independent promoter within the gene (Hernandez, et ah, 2003).
  • PKM zeta mRNA is stored locally in dendrites and translated after sufficient synaptic stimulation (Osten et ah, 1996; Muslimov et ah, 2004).
  • PKM zeta is constitutively active after phosphorylation by PDKl (Kelly et ah, 2007).
  • KIBRA Kidney BRAin protein
  • WWC1 WW domain-containing protein 1
  • KIBRA has come into the focus of neuroscience since it was shown that a SNP of the ninth intron of the KIBRA gene (cf. the KIBRA rs 17070145 T allele, rather than the C allele) is associated with human episodic memory performance.
  • KIBRA has been implicated in human episodic memory performance by multiple genome-wide association studies (Papassotiropoulos et ah, 2006; Schneider et ah, 2010).
  • PDC zeta protein kinase C zeta
  • PKM zeta has been shown to interact with KIBRA ⁇ Yoshihama et al, 2009).
  • Anxiety disorders play an increasing role in modern societies.
  • Current treatments for anxiety disorders are comprised of the following: Cognitive behavioral therapy, Selective serotonin reuptake inhibitor (fluoxetine, paroxetinem escitalopram, sertraline), pregabalin, buspirone, duloxetine, imipramine, clomipramine, venlafaxine, moclobemide, propranolol and other beta-adrenoblockers, clonidine, guanfacine, prazosin, benzodiazepines (alprazolam, chlordiazepoxide, clonazepam, clorazepate, diazepam, lorazepam) ⁇ Davidson et al, 2010; Baldwin et al, 2011) .However, there is still a strong need for treatment regimes for said disorders.
  • the present invention relates inter alia to a peptide
  • FVRNSLERRSVRM (SEQ ID NO: 4);
  • PPFVRNSLERRSVRM (SEQ ID NO: 6);
  • FVRNSLERRSVRMKR (SEQ ID NO: 12);
  • FVRNSLERRSVRMKRPSS (SEQ ID NO: 20);
  • FVRNSLERRSVRMKRPSSVKS (SEQ ID NO: 28);
  • PPFVRNSLERRSVRMKRPSSVKS SEQ ID NO: 30
  • DSSTLSKKPPFVRNSLERRSVRMKRPSSVKS SEQ ID NO: 32
  • the present invention inter alia relates to HDAC inhibitors or proteasome inhibitors for use in
  • MCI mild cognitive impairment
  • xi a method for improving or treating a brain trauma
  • xiii) a method for treating an anxiety disorder.
  • Fig. 1 depicts that the presence of KIBRA elevates PKC/PKM zeta cellular protein levels: a, Co-immunoprecipitation of KIBRA and PKM zeta in COS-cells with aFLAG beads confirms strong interaction of both proteins b, Co-expression of myristoylated PKC zeta changes localization of a KIBRA-GFP fusion protein in SH-SY5Y cells, suggesting that the interaction is also of relevance in a cellular context c, PKM zeta and PKC zeta expressed alone or co-expressed with KIBRA in COS cells d, Endogenous PKC zeta protein levels are elevated by KIBRA co-expression.
  • Fig. 2 illustrates that PKM zeta is subject to proteasomal degradation: a, Quantification of KIBRA, PKM zeta and PKC zeta mRNA levels in rat primary cortical neurons after infection with KIBRA- or EGFP overexpressing AAV virus or in untreated cells by qPCR.
  • Fig. 3 shows that KIBRA binds only activated PKM zeta, and that PKM zeta remains active after binding to KIBRA.
  • a CREB peptide phosphorylation assay of either commercially available PKC zeta (positive control) or PKM zeta immunoprecipitated from COS cells when expressed alone or in the presence of KIBRA.
  • Fig. 4 depicts that binding and protection of PKM zeta is dependent on a short motif near the KIBRA C-terminus.
  • a Series of KIBRA deletion constructs for fine-mapping of the interaction site with PKM zeta. Starting from aa 882, increasing 20aa stretches of KIBRA were deleted and constructs were tested for interaction with FLAG-tagged P M zeta. Very weak interaction was observed for ⁇ 882-965, while ⁇ 882-985 failed to interact at all, suggesting that the interaction site lies between aa946 and 985.
  • PKM zeta-flag expression constructs were co-transfected with EGFP-fusion constructs containing the 956-975 PKM zeta binding motif. Amino acids from position 964 to 974 were mutated to Alanin. The arginine at position 965 is very important for binding, e, A synthetic biotin-labeled peptide containing the binding motif (position 948-978, DSSTLSKKPPFVRNSLERRSVRMKRPSSVKS (SEQ ID NO: 32)) is able to bind PKM zeta expressed in COS cells as shown by co-immunoprecipitation using streptavidin beads, f, The synthetic peptide 948-978 is able to disrupt a preformed KIBRA- PKM zeta complex.
  • IC50 in this assay is estimated to be around 60 nM.
  • the 20-amino acid binding motif is sufficient to mediate PKM zeta stability increase. Shown are PKM zeta levels after addition of CHX with co-expression of the EGFP-KIBRA-956-975 construct or an EGFP control vector.
  • Fig. 5 illustrates that hippocampal KIBRA knock-down by intrahippocampal delivery of KIBRA siRNA by AAV results in memory deficits.
  • a,b Open field test reveals no difference in general motor behavior (a, total pathlength), and exploratory drive (b, pathlength in center area),
  • c Acquisition phase in the Morris watermaze is similar between groups,
  • d In the probetrial after 24 h KIBRA knock-downs are significantly inferior to controls in recalling platform location (p ⁇ 0.05).
  • a, b Cell culture knock-downs of KIBRA mRNA in Hek cells (a) or primary cortical neurons (b). shRNA1267 was used for the further experiments.
  • a KIBRA-expressing construct was co-transfected with the siRNA.
  • c In vivo knock-down of KIBRA in the rat hippocampus shown by Western blot and quantification of bands.
  • Fig. 8 illustrates the localization of KIBRA in primary neurons in culture. Neurons were infected with a KIBRA-EGFP fusion constructs. KIBRA localizes differentially to different spines.
  • Fig. 9 depicts a full-length human KIBRA DNA and protein sequence, which can also be found in SEQ ID NOs: 1 and 2 of the sequence listing.
  • Fig. 10 illustrates certain KIBRA sequences related to the present invention, i.e. the sequences that can also be found as SEQ ID NOs: 3-42 of the sequence listing.
  • Fig. 10 additionally illustrates certain WWC2 and WWC3 sequences related to the present invention, i.e. the sequences that can also be found as SEQ ID NOs: 61 , 62, 65 and 66 of the sequence listing.
  • Fig. 11 and Fig. 12 illustrate Neuronal KIBRA knock-out results in anxiety symptoms.
  • Fig. 13 illustrates that stimulation with H-DAC inhibitors elevates the amount of KIBRA transcripts in cells
  • a) shows a qCR Experiment measuring the amount of KIBRA transcript in SHSY5Y cells treated with the HDAC-inhibitor Sodium Butyrate at a concentration of 5 mM in comparison to untreated cells.
  • Cells were seeded in 6-well plates at a density of 0,5 x 10 6 cells / well and stimulated for 48h. Cyclophiline served for normalization.
  • Fig. 14 depicts a full-length human WWC3 DNA and protein sequence, which can also be found in SEQ ID NOs: 59 and 60 of the sequence listing.
  • Fig. 15 depicts a full-length human WWC2 DNA and protein sequence, which can also be found in SEQ ID NOs: 63 and 64 of the sequence listing.
  • Fig. 14 depicts a full-length human WWC3 DNA and protein sequence, which can also be found in SEQ ID NOs: 59 and 60 of the sequence listing.
  • Fig. 15 depicts a full-length human WWC2 DNA and protein sequence, which can also be found in SEQ ID NOs: 63 and 64 of the sequence listing.
  • KIBRA 20-amino acid binding motif of KIBRA (KIBhu_956-975, according to SEQ ID NO 22), WWC3 (WWC3_936-955 5 according to SEQ ID NO 62) and WWC2 (WWC2hu_1031-1050 according to SEQ ID NO 66) is sufficient to mediate an increase of PKM zeta stability, while a KIBRA binding motif comprising a specific (R965A), single point mutation (KIBhu_965-975-R965A) is not able to increase stability of PKM zeta.
  • COS-1 cells were transiently transfected with equal amounts of plasmids expressing flag- PKM zeta and EGFP-fusions of afore mentioned binding motifs.
  • Cycolheximide (CHX) was added to stop Protein de-novo synthesis and PKM zeta levels were a) detected after 0, 24, 48 hours, using an anti-flag antibody, b) PKM zeta amounts at 0, 24 and 48 hour time points c) Analysis of PKM zeta levels, after normalization to actin, in a series of ELISA experiments confirmed the findings of b).
  • Fig 17. depicts the concentration dependent interaction capability of :WWC2 and WWC3 binding motifs to PKM zeta.
  • COS-1 cells were transiently transfected with flag-PKM zeta plasmid and harvested 48hrs after transfection. Cell lysates were pooled and then splitted again for incubation with different amounts of WWC1 , 2 and 3 20mere biotinylated peptides homologue to KIBRAhu956-975. The peptides were precipitated using streptavidine beads and the 2 nd Supernatants of the precipitations were probed in Western blot for PKM zeta coprecipitation using an anti-flag-antibody.
  • PKM zeta is coprecipitated when ⁇ ⁇ WWC1 -peptide or in minor amounts with ⁇ ⁇ WWC2 peptide is added to the cell lysate. All other probes are negative for PKM zeta coprecipitation.
  • COS-1 cells were transiently transfected with flag-PKM zeta and V5-KIBRA plasmid and harvested 48hrs after transfection. Cell lysates were pooled and then splitted again for incubation with different amounts of WWC1 , 2 and 3 20mere biotinylated peptides homologue to KIBRAhu956-975.
  • Flag-PKM zeta was precipitated using anti-flag-beads and the 2 nd Supernatants of the precipitations were probed in Western blot for V5-KIBRA coprecipitation using an anti-V5-antibody.
  • PKM zeta is precipitated in all probes while KIBRA is displaced from the complex when 10 ⁇ WWC1 -peptide or less effective with 10 ⁇ WWC2 peptide added to the cell lysates. All other probes are positive for KIBRA coimmunoprecipitation.
  • Fig. 18 illustrates intraneuronal delivery of synthetic KIB 956"975 peptide fragments via FAM-labelled cell penetrating peptide (CPP) moieties antp and TAT.
  • CPP cell penetrating peptide
  • Fig. 19 illustrates intraneuronal delivery in the murine hippocampus of synthetic KIB 956"975 peptide fragments via FAM-labelled cell penetrating peptide (CPP) moieties antp and TAT.
  • CPP cell penetrating peptide
  • A,B Both antp (A) and TAT (B) CPP moieties coupled to KIB 956"975 facilitate transduction of hippocampal neurons in vivo.
  • C Application of a FAM-labelled Hsp70-derived peptide sequence lacking a CPP moiety does not lead to intraneuronal peptide delivery.
  • Fig. 20 shows that chemically synthesized CPP-KJB 956"975 but neither CPP-KIB 956"975 R965A peptides nor non-CPP-tagged peptides containing the KIB 956"975 motif nor an Hsp70-derived, KIBRA-unrelated peptide transduces COS cells and subsequently binds plasmid-derived PKM zeta.
  • Fig. 21 shows that chemically synthesized peptides (KIB ) 2 but not (KIB 956"975 R965A) 2 which carry dimer repeats of the wildtype or mutant KIBRA motif, respectively, facilitate stabilization of endogenous neuronal PKM zeta protein levels after intracellularly delivery via antp or TAT cell penetrating moieties.
  • A Left panels: Western Blots showing reduced CHX-induced dissipation of PKM zeta protein levels after treatment with antp-KIB 956-975 (upper) or TAT-KIB 956"975 (lower) compared to KIB 956" 975 R965A peptides carrying similar CPP tags.
  • B Graphical representation of band intensities shown in A as determined by densitometric image analysis. Note stabilization of PKM zeta protein levels by both antp-KIB 956"975 and TAT-KIB 956"975 as compared to the respective R965A mutants.
  • B Upper panel: Western blots showing reduced CHX-induced dissipation of endogenous neuronal PKM zeta protein levels after treatment with AAV-EGFP-KIB 956"975 compared to AAV-EGFP-KIB 956"975 R965A.
  • Lower panel Immunodetection of housekeeping gene actin shows comparable total protein loading between lanes.
  • FIG. 23 Viral vector AAV-EGFP-KIB infects hippocampal neurons in vivo and enhances levels of neuronal PKM zeta protein.
  • A Illustration of AAV-mediated reporter gene expression in CA1 (left panel) and CA1 CA2/CA3 (right panel) region hippocampal neurons from expression cassette CBA-EGFP-KIB 956"975 .
  • B Western blots on protein preparations from dorsal hippocampus of AAV-treated mice at > 3 weeks after viral delivery. Upper panel: PKM zeta levels are increased in murine hippocampus treated with AAV-EGFP-KIB 956"975 compared to AAV-EGFP.
  • Intrahippocampal AAV-EGFP-KIB 956"975 enhances spatial memory in a task where animals are forced to memorize the two-dimensionally fixed location of a sector of a circular rotating disk entry to which is punished by a mild foot-shock.
  • A Learning curves of animals subjected to one day of 9 trials of avoidance learning are similar between groups treated with AAV-EGFP, AAV-EGFP-KIB 956"975 , or AAV-EGFP-KIB 956"975 R965A.
  • activated PKM zeta is stabilized by KIBRA by shielding the protein from proteasomal degradation.
  • said protection is the protection of activated, i.e. phosphorylated PKM zeta and that the protected form of PKM zeta retains its native activity, in particular, the shielded form of PKM zeta retains its kinase activity.
  • the present inventors have surprisingly found that a short peptide derived from the C-terminal section of KIBRA (i.e.
  • a short motif near the KIBRA C-terminus is sufficient for mediating said protein-protein interaction and for mediating such protection of PKM zeta.
  • the present inventors have obtained surprising evidence that the interaction between KIBRA and PKM zeta is not, at least not primarily, a kinase-target interaction.
  • PKM zeta binds to the C-terminus of KIBRA and binding and protection are mediated by the same motif.
  • the present inventors have also shown that knock-down of KIBRA is accompanied by decreased PKM zeta protein levels. Without intending to be bound by theory, the present inventors consider that a key mechanism in memory maintenance is the stabilization of synaptic PKM zeta levels by binding to KIBRA or the KIBRA-derived peptides and the like of the present invention.
  • the present inventors propose that KIBRA is a second component in the synaptic tag element constituted by PKM zeta. PKM zeta would be normally rapidly degraded after its initial local dendritic synthesis unless KIBRA is available for binding and preservation of this tag.
  • KIBRA could not only be a regulator of the degree to which a particular spine, dendrite segment, or neuron is allowed to stabilize new input signal information or not, but also has a quantifiable (data not shown) effect on the amount and density of spines encountered on dendrite segments. While the mode of regulation of KIBRA levels or its translocation is not fully understood at present, the present inventors already have evidence (not shown) that different learning paradigms can increase neuronal KIBRA levels. This model furthermore complements existing theories how PKM zeta level are maintained at spines for long time periods by positive feedback-loops, through which active PKM zeta would enhance its own translation by relief of a translational block ⁇ Sacktor 2011).
  • Such a self-sustaining regulation is postulated to lead to a switch-like behavior stabilizing the activated state of the system after the critical threshold level has been reached.
  • local KIBRA levels could either set the threshold of PKM zeta activity required to enter the positive feedback-loop leading to self-sustained activity, or act as a second component of the molecular decision process which spines to enhance.
  • the present invention relates to a peptide
  • FVRNSLERRSVRM (SEQ ID NO: 4);
  • PPFVRNSLERRSVRM (SEQ ID NO: 6);
  • FVRNSLERRSVRMKR (SEQ ID NO: 12);
  • FVRNSLERRSVRMKRPSS (SEQ ID NO: 20);
  • PPFVRNSLERRSVRMKRPSS (SEQ ID NO: 22);
  • FVRNSLERRSVRMKRPSSVKS (SEQ ID NO: 28);
  • SPFVRNTLERRTLRY QSCR (SEQ ID NO 62), or a peptide derivative thereof or a peptide conjugate of the peptide or peptide derivative, for use in a method of treating an anxiety disorder.
  • Anxiety disorders are well-known to the skilled person.
  • the current DSM-IV in its revision DSM-IV-TR (American Psychiatric Association, 2000) recognizes different types of anxiety disorders: Generalized anxiety disorder, Panic disorder, Agoraphobia without history of panic disorder, Specific phobia, Social phobia, Obsessive-compulsive disorder, Posttraumatic stress disorder, Acute stress disorder, Anxiety disorder due to a general medical condition (for example Parkinson's disease (Dissanayaka et ah, 2010)).
  • Parkinson's disease Parkinson's disease (Dissanayaka et ah, 2010)
  • Anxiety and fear may also occur situatively, and be not diagnosed as an illness falling under the categories above, but nonetheless require treatment.
  • the single largest category of anxiety disorders is that of phobic disorders, where the anxiety or fear is triggered by a very specific situation or stimulus. Prevalence is estimated between 3 and 5% over one year - the lifetime risk for a phobia is approximately 3.5%. People suffering from a phobia do typically understand that their fear is out of proportion to the real danger a stimulus poses, but can not act rationally against their phobia. Phobias are divided into the following subclasses: Social phobia is anxiety caused by presence of other people generally, or in specific situations. Social phobia is further subclassified into generalized social phobia or social anxiety disorder and specific social phobia. The symptoms range from discomfort to psychosomatic manifestation.
  • Specific phobias are fears of a single specific trigger (often spiders, water, milk, snakes, dogs, flying, heights etc) (LeBea et ai, 2010). Agoraphobia is a generalized fear of entering wide open spaces, or simple fear of leaving home at all.
  • Posttraumatic stress disorder is a severe anxiety disorder that usually develops after exposure to traumatic events. Examples for such events are being in danger of death, being subjected to violence, being sexually assaulted, or being psychologically traumatized. PTSD has been found to be especially common in military personnel ⁇ Creamer et ai, 2011). People suffering from PTSD often relive traumatic events through nightmares or flashbacks, and avoide stimuli associated with the event. In addition, symptoms are increased arousal, anger, and hypervigilance. DSM-IV-TR diagnostic criteria state that symptoms should last more than one month, and cause significant impairment in social, occupational, or other areas of daily functioning. Any of the above types, categories and subclasses of anxiety disorders are envisaged to be treated in accordance with the present invention.
  • the anxiety disorder is generalized anxiety disorder (also known as GAD).
  • GAD Generalized anxiety disorder
  • GAD is an anxiety disorder that characterized by excessive, uncontrollable and often irrational worry about common events or things that is not in proportionate to the event or thing in question (Andrews et al, 2010). GAD often interferes with daily functioning. There is some regional difference in the prevalence of generalized anxiety, estimated between 3 - 5% in countries like the US, Canada, Italy, or Australia.
  • the diagnostic criteria for GAD according to DSM-IV are: Excessive worry occurring in more than half of the days in a 6-month period, difficulty controlling the worries, worries are associated with 3 or more of the following: restlessness, being easily fatigued, difficulty concentrating, irritability, muscle tension, sleep disturbance.
  • the focus of the worries should not be contained in other entities such as social phobia, or PTSD, the worries should interfere with social or professional functioning, and the anxiety is not caused by medical conditions, drugs, psychotic disorders, or a developmental cause.
  • the peptide for use of the first aspect may have a sequence selected from the group consisting of SEQ ID NOs 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42.
  • the peptide for use of the first aspect may have a sequence selected from the group consisting of SEQ ID NOs 66 and 62.
  • the invention relates to a peptide for use having a sequence as disclosed in FIG. 10.
  • the peptide of the invention has a sequence of 13 to 40, more preferably 15 to 40, such as 15 to 31 or 20 to 30, consecutive amino acids of SEQ ID NO: 42.
  • such peptide comprises the sequence FVRNSLERRSVRM (SEQ ID NO: 4).
  • such peptide comprises the sequence PPFVRNSLERRSVRMKRPSS (SEQ ID NO: 22).
  • the peptide of the invention has a sequence of n to 40 consecutive amino acids of SEQ ID NO: 42 and comprises a sequence selected from the group consisting of SEQ ID NOs 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, wherein in each case, n corresponds to the total number of amino acids of the particular SEQ ID NO.
  • the peptide of the first aspect may comprise or have a sequence of the amino acids from between residue 946, 948, 956 or 958 respectively, and residue 970, 972, 975 or 985, respectively of the human KIBRA.
  • Individual preferred peptides are e.g.
  • a "peptide” usually has from 5 to 40 amino acids, such as 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, and preferably from about 13 to about 35 amino acids, such as from about 15 to about 31 amino acids, such as from 20 to 30 amino acids, such as 25 amino acids.
  • amino acids such as 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, and preferably from about 13 to about 35 amino acids, such as from about 15 to about 31 amino acids, such as from 20 to 30 amino acids, such as 25 amino acids.
  • peptides can easily be obtained by the skilled person, such as by chemical synthesis, production in host cells, or combinations thereof.
  • a peptide of the invention is capable of binding to PKM zeta.
  • a peptide of the invention is capable of preventing PKM zetan from degradation, particularly from proteasomal degradation.
  • a "peptide derivative” of the invention can be derived from a peptide of the invention.
  • such "peptide derivative” of the invention may be derived from a peptide of the invention in various manners. For example, it may be derived from said peptide in that it has a sequence that has a certain percentage of sequence identity with that of the corresponding peptide, comprises a sequence of said peptide, has certain residues in common with said peptide, and/or shares a certain activity with said peptide.
  • a peptide derivative of the invention may itself be a peptide or may also be a polypeptide.
  • the term "polypeptide” as used herein includes reference to a protein.
  • a "polypeptide” as a peptide derivative of the invention has at least 41, such as at least 42, 45, 50, 60, 70, 80, 90, particularly at least 100 amino acids.
  • a polypeptide does not contain more than 2000, such as not more than 500, 450, 400, 350, 300, 250, 200, or 150 amino acids.
  • said peptide derivative or peptide conjugate is selected from any of the below A, B, C, D and E:
  • vii) having a sequence that is at least 60 % identical to a sequence as defined above, comprising the amino acid residues R, S, R at the positions corresponding to positions 965, 967, 969 of the full-length sequence of human KIBRA (SEQ ID NO: 2), and being capable of binding to PKM zeta; and/or viii) having a sequence that is at least 70 % identical to a sequence as defined above and comprising the amino acid residues F, R, at the positions corresponding to positions 958, 965 of the full-length sequence of human KIBRA (SEQ ID NO: 2), optionally additionally comprising the amino acid residues S, R, at the positions corresponding to positions 967, 969 of the full-length sequence of human KIBRA (SEQ ID NO: 2); and/or
  • ix) being capable of binding to PKM zeta and having a sequence that is at least 55 % identical to a sequence as defined above and comprising the amino acid residues F, R, at the positions corresponding to positions 958, 965 of the full- length sequence of human KIBRA (SEQ ID NO: 2), optionally additionally comprising the amino acid residues S, R, at the positions corresponding to positions 967, 969 of the full-length sequence of human KIBRA (SEQ ID NO: 2) ⁇
  • v a sequence interacting with the postsynaptic density, particularly with a PDZ motif.
  • the peptide derivative of A) viii) and ix) as defined above is not a peptide comprising the sequence FVRNSLERRSVRM, wherein said peptide has a sequence selected from the group consisting of:
  • FVRNSLERRSVRM (SEQ ID NO: 4);
  • PPFVRNSLERRSVRM (SEQ ID NO: 6);
  • FVRNSLERRSVRMKR (SEQ ID NO: 12);
  • FVRNSLERRSVRM KRPSS (SEQ ID NO: 20);
  • PPFVRNSLERRSVRM KRPSS (SEQ ID NO: 22);
  • FVRNSLERRSVRMKRPSSVKS SEQ ID NO: 28
  • PPFVRNSLERRSVR KRPSSVKS SEQ ID NO: 30
  • DSSTLSKKPPFVRNSLERRSVRMKRPSSVKS SEQ ID NO: 32
  • DSDSSTLSKKPPFVRNSLERRSVRMKRPSSV S SEQ ID NO: 34
  • the peptide derivative of A) viii) and ix) as defined above is not a peptide derivative of a peptide as defined in claim 1 as filed herewith.
  • the peptide derivative of A) viii) and ix) as defined above is not a peptide derivative as defined in claim 2 A) i) to vii), as filed herewith.
  • the peptide derivative of A) viii) and ix) as defined above is not a peptide derivative as defined in claim 2 B), as filed herewith, as far as it refers back to a peptide as defined in claim 1 as filed herewith and/or to a peptide derivative as defined in claim 2 A) i) to vii), as filed herewith.
  • the peptide derivative of A) viii) and ix) as defined above is not a peptide derivative as defined in claim 2 C), as filed herewith, as far as it refers back to a peptide as defined in claim 1 as filed herewith and/or to a peptide derivative as defined in claim 2 A) i) to vii), as filed herewith.
  • the peptide conjugate as defined above is not a peptide conjugate of a peptide as defined in claim 1 as filed herewith.
  • the peptide conjugate of D) as defined above is not a peptide conjugate as defined in claim 2 D), as filed herewith, as far as it refers back to a peptide as defined in claim 1 as filed herewith and/or to a peptide derivative as defined in claim 2, A) i) to vii), as filed herewith.
  • the peptide conjugate of E) as defined above is not a peptide conjugate as defined in claim 2 E), as filed herewith, as far as it refers back to a peptide as defined in claim 1 as filed herewith and/or to a peptide derivative as defined in claim 2 A) i) to vii), as filed herewith.
  • a peptide derivative having a sequence that is "at least 55% identical" to a given other sequence has a sequence that is at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%; 65%, 66%, 67%; 68%; 69%; 70%, 71%, 72%, 73%, 74%; 75%, 76%, 77%; 78%; 79%; 80%; 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said other sequence.
  • said peptide derivative is capable of binding to P M zeta.
  • a peptide derivative having a sequence that is "at least 60% identical" to a given other sequence has a sequence that is at least 61%, 62%, 63%, 64%; 65%, 66%, 67%; 68%; 69%; 70%, 71%, 72%, 73%, 74%; 75%, 76%, 77%; 78%; 79%; 80%; 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said other sequence.
  • said peptide derivative is capable of binding to PKM zeta.
  • a peptide derivative having a sequence that is "at least 70% identical" to a given other sequence has a sequence that is at least 71%, 72%, 73%, 74%; 75%, 76%, 77%; 78%; 79%; 80%; 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said other sequence.
  • said peptide derivative is capable of binding to PKM zeta.
  • a peptide derivative having a sequence that is "at least 80% identical" to a given other sequence has a sequence that is at least 81%), 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said other sequence.
  • said peptide derivative is capable of binding to PKM zeta.
  • reference to a sequence in SEQ ID NO: 2 also includes reference to a corresponding sequence in Figure 9.
  • Percentages identity can easily be determined by the skilled person. As a non-limiting example, for a peptide of 20 amino acids, 70 % identity to said sequence according to the first aspect means that 14 out of 20 amino acids are identical.
  • Peptide derivatives of the present invention may e.g. easily be obtained by the skilled person, e.g. by means of one or more amino acid substitution(s), addition(s) or deletion(s) or any combination thereof in amino acids of the peptides of the invention.
  • the peptide derivative preferably comprises conservative substitutions or semi-conservative substitutions, i.e. substitutions by a member of a family of amino acids that are related in their side chains and chemical properties. Examples of such families, all of which are well- known to the skilled person, are families of amino acids with acidic side chains, basic side chains, non-polar aliphatic side chains, non-polar aromatic side chains, uncharged polar side chains, small side chains, bulky side chains and the like.
  • the peptide derivative of the invention preferably contains from 13 to 500 amino acids, such as from 13 to 400, from 13 to 300, particularly from 13 to 200, from 20 to 200, from 13 to 100, from 20 to 100, more preferably from 13 to 50, from 15 to 50, from 20 to 50, from 13 to 45, from 13 to 40, even more preferably from 13 to 30, from 15 to 35, from 20 to 40, such as from 15 to 30, from 15 to 25 or from 17 to 23 amino acids - or any range in between or combination between those numbers.
  • 13 to 500 amino acids such as from 13 to 400, from 13 to 300, particularly from 13 to 200, from 20 to 200, from 13 to 100, from 20 to 100, more preferably from 13 to 50, from 15 to 50, from 20 to 50, from 13 to 45, from 13 to 40, even more preferably from 13 to 30, from 15 to 35, from 20 to 40, such as from 15 to 30, from 15 to 25 or from 17 to 23 amino acids - or any range in between or combination between those numbers.
  • a "peptide derivative” may also be derived from a peptide as defined above e.g. in that it comprises a sequence of one or more peptides or peptide derivatives of the invention.
  • the latter includes cyclic derivatives and multimers of a peptide of the invention and/or of a peptide derivative of the invention.
  • the invention also relates to a peptide derivative being a multimer of a peptide of to the invention or of a peptide derivative described above.
  • the invention also relates to a peptide derivative being a cyclic derivative of a peptide of to the invention or a peptide derivative described above.
  • a functionality of a peptide of the invention is maintained.
  • a peptide derivative disclosed herein is capable of binding to PKM zeta.
  • a peptide derivative disclosed herein is capable of preventing PKM zeta from degradation, particularly from proteasomal degradation.
  • "a peptide being capable of binding to PKM zeta” is understood to also qualify as, and hence to also be, "a peptide being capable of binding to PKC zeta”.
  • a peptide being capable of binding to PKM zeta is also understood to include “a peptide being capable of binding to PKM zeta and/or PKC zeta”.
  • PKM zeta may be replaced by "PKC zeta” and vice versa, or may be replaced by "PKM zeta and/or PKC zeta”.
  • PKC zeta Numerous ways of determining capability of binding of a peptide or peptide derivative to a protein such as PKM zeta are known to the skilled reader. Suitable qualitative and quantitative assays e.g. include, but are not limited those involving co-precipitation or surface plasmon resonance (SPR).
  • a peptide that is capable of binding to PKM zeta is intended to mean a peptide that binds to PKM zeta with a dissociation constant K d , where l/Kj is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%; 90%, 95%, 96%, 97%, 98%, 99% of 1 K d of the K d of a peptide according to the invention, particularly the K d of a peptide corresponding to the respective peptide derivative or of the d of a peptide having SEQ ID NO: 22 (or KIBRA residues 956-975).
  • a peptide that is capable of binding to PKM zeta binds to PKM zeta even better than a peptide according to the invention, such as one having a sequence of SEQ ID NO: 4.
  • a peptide that is capable of binding to PKM zeta may be a peptide that binds to PKM zeta with a dissociation constant 3 ⁇ 4 where 1 K d is at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% of 1/K d of the 3 ⁇ 4 of of a peptide according to the invention, particularly of the Kj of a peptide corresponding to the respective peptide derivative or of the Kj of a peptide having SEQ ID NO: 22.
  • the peptide derivatives disclosed herein may alternatively or additionally be peptide derivatives i) comprising D-amino acids having an inverse sequence of a sequence of a peptide disclosed herein or of a sequence of a peptide derivative disclosed herein, and being capable of binding to PKM zeta; and/or ii) being a cyclic derivative of a peptide disclosed herein or of a peptide derivative disclosed herein,; and/or iii) being a multimer of a peptide disclosed herein or of a peptide derivative disclosed herein.
  • the peptide derivative may also be derived from a peptide disclosed herein in that it has a similar three-dimensional structure or surface structure, respectively.
  • the invention also employs a peptide derivative comprising D-amino acids having an inverse sequence of a sequence of a peptide disclosed herein or of a peptide derivative disclosed herein, and being capable of binding to PKM zeta.
  • a peptide derivative comprising, preferably consisting of, D-amino acids that have an inverse sequence of a sequence defined above.
  • the peptide derivative disclosed herein may be characterized in that it consists of D-amino acids having an inverse sequence of a sequence defined herein, and is capable of binding to PKM zeta.
  • the latter peptide derivatives may also be referred to as a retro- inverso peptides / peptide derivatives of a peptide or peptide derivative disclosed herein or as a peptides / peptide derivatives having a retro-inverso form of a peptide or peptide derivative disclosed herein.
  • a "peptide derivative" herein may also be derived from a peptide disclosed herein e.g.
  • peptide derivative in that it comprises a sequence of one or more peptides or peptide derivatives disclosed herein.
  • the latter includes cyclic derivatives and multimers of a peptide disclosed herein and/or of a peptide derivative disclosed herein.
  • a peptide derivative being a multimer of a peptide herein or of a peptide derivative described herein.
  • the invention also employs a peptide derivative being a cyclic derivative of a peptide disclosed herein or a peptide derivative disclosed herein.
  • the peptide, peptide derivative or peptide conjugate herein may be a chimeric peptide, chimeric peptide derivative or chimeric peptide conjugate.
  • Chimeric peptides etc. are not particularly limited and are generally well-known to the skilled person.
  • the peptides, peptide derivatives and peptide conjugates disclosed herein may easily be obtained by the skilled person - e.g. by methods involving chemical synthesis, production in host cells, or combinations thereof.
  • the peptides and peptide derivatives disclosed herein may comprise one or more covalent modification(s).
  • Covalent modifications are well known to the skilled person and are not particularly limited.
  • the present invention may furthermore employ a peptide conjugate.
  • such peptide conjugate comprises a peptide or peptide derivative disclosed herein.
  • a "peptide conjugate” preferably refers to a conjugate that comprises a peptide or peptide derivative disclosed herein as well as an additional moiety. Said additional moiety is not particularly limited. Preferably, the additional moiety is covalently bound to a peptide or peptide derivative disclosed herein. Said additional moiety, for purposes herein, may also refer to more than one such moiety.
  • peptide conjugate may also comprise more than one such moiety, such as two or more moieties of a given type of moieties and/or two or more moieties of different types of moieties.
  • Said moieties are preferably selected from cell penetration moieties, such as moieties for crossing the blood brain barrier; detectable labels; polymers; membrane anchoring moieties; and sequences (such as peptide sequences) interacting with the postsynaptic density, such as sequences interacting with a PDZ motif.
  • the peptide conjugate herein is preferably characterized in that it comprises: i) a cell penetration moiety; and/or ii) a detectable label; and/or iii) a polymer; and/or iv) a membrane anchoring moiety, and/or v) a sequence interacting with the postsynaptic density.
  • the peptide conjugate disclosed herein may comprise any of the above i) to v) or any combination thereof.
  • a peptide conjugate herein is characterized in that it comprises a cell penetration moiety.
  • a cell penetration moiety refers to a moiety that it is capable of entering into a cell. Preferably, it is capable of doing so even if comprised in a peptide conjugate disclosed herein.
  • Said cell penetration moiety preferably is a cell penetration peptide (CPP) or a derivative thereof.
  • CPP cell penetration peptide
  • Cell penetration peptides are well-known in the art.
  • Non-limiting examples of preferred cell penetration peptides to be used in accordance with the present invention include cell penetration peptides having a sequence selected from the group consisting of the HIV-gp41 fusion sequence, the Kaposi FG signal sequence, the human integrin b3-signal sequence, RRRRRRR (SEQ ID NO: 43), RRRRRRRR (SEQ ID NO: 44), RRRRRRRRR (SEQ ID NO: 45), RRRRRRRRRR (SEQ ID NO: 46), a nuclear localization sequence, a HIV Tat- 1 -derived sequence, a SynBl sequence, a buforin sequence, a magainin sequence, a sequence of the N-terminal repetitive domain of maize gamma-zein, a sequence of a lactoferrin-derived peptide, an antennapedia-derived sequence such as a penetratin sequence, the Isl-1 homeodomainm, KLALKLALKALKAALKLA (SEQ ID NO: 47), a transportan sequence, a
  • a cell penetration moiety herein is also capable of mediating trans-cellular transport such as the transport over the blood brain barrier. Preferably, it is capable of doing so even if comprised in a peptide conjugate disclosed herein.
  • the cell penetration moiety herein preferably is a moiety capable of mediating trans-cellular transport.
  • the cell penetration moiety herein preferably is enabling the peptide conjugate disclosed herein to cross the blood brain barrier.
  • Such moiety may also be referred herein as "a moiety for crossing the blood brain barrier”.
  • the cell penetration moiety is a moiety for crossing the blood brain barrier.
  • said cell penetration moiety may also be a derivative of a cell penetration moiety, such as a derivative of a CPP.
  • Said derivatives include but are not limited to i) a D-form of a cell penetration peptide, ii) a retro-inverso form of a cell penetration peptide (i.e. a CPP comprising, preferably consisting of, D-amino acids that have an inverse sequence of a sequence of a CPP), iii) a cell penetration peptide comprising one or more covalent modification(s) as described herein with relation to the peptides and peptide derivatives disclosed herein, iv) a small molecule mimicking a cell penetration peptide, e.g. a small molecule comprising a polyguanidine backbone.
  • a peptide conjugate may comprise a detectable label.
  • a detectable label is not particularly limited. Numerous detectable labels are well-known to the skilled person.
  • said detectable label is selected from the group consisting of the following tags: FLAG-tag, GST-tag, HA-tag, His-tag, V5-tag, myc-tag, Isopep-tag, BCCP, calmodulin-tag, maltose binding protein tag, Nus-tag, glutathione-S-transferase-tag, green fluorescent protein tag, S-tag, Softag 1, Softag 3, streptavidin tag, biotin tag, SBP- tag, Ty tag, ACP-tag, CBP-tag, glutathione-S-transferase-tag (GST-tag), snap-tag, tandem affinity purification tag (TAP -tag), thioredoxin tag, GFP, YFP, and BFP.
  • a peptide conjugate may comprise a polymer.
  • the polymer is preferably selected from the group consisting of PEG (polyethylene glycol), mPEG (methoxypolyethylene glycol), cyclodextrin, polyglutamate, polyaspartate and HPMA (N-(2- hydroxypropyl)methacrylamide).
  • a peptide conjugate may comprise a membrane anchoring moiety.
  • the membrane anchoring moiety is preferably selected from the group consisting of a GPI-anchor, an isoprenyl-anchor (such as a farnesyl anchor or geranylgeranyl anchor), a myristoyl-anchor and a palmitoyl-anchor.
  • a peptide conjugate may comprise a sequence interacting with the postsynaptic density, such as a sequence interacting with a PDZ motif.
  • the sequence interacting with the postsynaptic density is preferably selected from or comprises a PSD97 or PSD95 motif.
  • the peptide, peptide derivative or peptide conjugate herein comprises the amino acid residue R at the position corresponding to position 965 of the full-length sequence of human KIBRA (SEQ ID NO: 2).
  • the peptide, peptide derivative or peptide conjugate herein may alternatively or additionally comprise the amino acid residues S and R, respectively at the positions corresponding to positions 967 and 969, respectively, of the full-length sequence of human KIBRA (SEQ ID NO: 2 or Figure 9, respectively).
  • the peptide, peptide derivative or peptide conjugate herein comprises the amino acid residue F at the position corresponding to position 958 of the full-length sequence of human KIBRA (SEQ ID NO: 2).
  • the peptide, peptide derivative or peptide conjugate herein comprises the amino acid residues F, R at the positions corresponding to positions 958, 965 of the full-length sequence of human KIBRA (SEQ ID NO: 2).
  • the peptide, peptide derivative or peptide conjugate herein additionally comprises the amino acid residues S, R at the positions corresponding to positions 967, 969 of the full-length sequence of human KIBRA (SEQ ID NO: 2).
  • a phrase such as “comprises the amino acid residues F, R at the positions corresponding to positions 958, 965" is to be understood as equivalent to a phrase “comprises the amino acid residue F at the position corresponding to position 958 and comprises the amino acid residue R at the position corresponding to position 965".
  • the native human KIBRA protein sequence referred to for purposes of the present invention is a full-length human KIBRA protein sequence, and is depicted in SEQ ID NO: 2 or Figure 9, respectively.
  • reference to a sequence in SEQ ID NO: 2 also includes reference to a corresponding sequence in Figure 9.
  • the native, full length human WWC3 amino acid sequence is shown in SEQ ID NO 60 and Fig. 14, and the native, full length human WWC2 amino acid sequence is depicted in SEQ ID NO 64 or Fig. 15, respectively.
  • the peptide derivative or peptide conjugate is selected from full-length KIBRA and a derivative or conjugate thereof, particularly a conjugate comprising a moiety selected from the group consisting of i) a cell penetration moiety, particularly a moiety for crossing the blood brain barrier; and/or
  • KIBRA a sequence interacting with the postsynaptic density, particularly with a PDZ motif. especially wherein said full-length KIBRA has the sequence of SEQ ID NO: 2.
  • the present invention also relates to a complex between PKM zeta or PKC zeta and a peptide, peptide derivative or peptide conjugate as defined above for use in a method of treating an anxiety disorder.
  • Said complex may have been formed in vitro or in vivo. It may e.g. be an isolated complex that has been purified from its environment. The presence of such complex may easily be determined by the skilled person by a number of ways, including methods involving co- immunoprecipitation.
  • the present invention also relates to an isolated nucleic acid for use in a method of treating an anxiety disorder
  • a) encodes the peptide as defined above; the peptide derivative as defined above; or a peptide conjugate as defined above,
  • nucleic acid having a sequence selected from the group consisting of:
  • iii an isolated nucleic acid having a sequence of 39 to 600 nucleotides that comprises a sequence as defined in i);
  • nucleic acid having a sequence that is at least 60 % identical a sequence as defined in i) and encodes a peptide or polypeptide that is capable of binding to PKM zeta;
  • nucleic acid having a sequence of 39 to 1500 nucleotides that comprises a sequence as defined in i) and encodes a peptide or polypeptide that is capable of binding to PKM zeta;
  • c) is capable of hybridizing under stringent conditions to a nucleic acid according to any one of a) and/or b).
  • the isolated nucleic acid of said aspect of the invention encodes the peptide; the peptide derivative or the peptide conjugate as defined above.
  • the conjugate comprises a cell penetration moiety, especially a moiety for crossing the blood brain barrier.
  • the nucleic acid of said aspect comprises or has a sequence encoding a peptide having a sequence selected from the group consisting of SEQ ID NOs 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42.
  • the nucleic acid of said aspect comprises or has a sequence encoding a peptide having a sequence selected from the group consisting of SEQ ID NOs 62 and 66.
  • the nucleic acid of said aspect has a sequence selected from SEQ ID NOs: 61 and 65.
  • the nucleic acid of said aspect comprises or has a sequence encoding a peptide having a sequence of 13 to 40, more preferably 15 to 40, such as 15 to 31 or 20 to 30, consecutive amino acids of SEQ ID NO: 42.
  • the nucleic acid of said aspect may comprise or have a sequence encoding a peptide having the sequence of the amino acids from between residue 946, 948, 956 or 958 respectively, and residue 970, 972, 975 or 985, respectively of the human KIBRA.
  • Individual preferred isolated nucleic acids are e.g.
  • the isolated nucleic of said aspect has a sequence of 39 to 1500 nucleotides. In other preferred embodiments, the isolated nucleic acid of the invention has a sequence of 39 to 600 nucleotides.
  • a nucleic acid of said aspect may have at least 45, such as at least 51 , 54, 60, 63, 69, 75, 81, 84, 90, 120, 135, 150, 180, 210, 240, 270, or at least 300 nucleotides.
  • a nucleic does not contain more than 6000, such as not more than 1500, 1350, 1200, 1050, 1000, 900, 800, 750, 700, 600, 500, 450, 400, 350, or 300 nucleotides, or even not more than 270, 240, 210, 180, 150, 135 or 120 nucleotides.
  • the isolated nucleic acid of said aspect of the invention is an isolated nucleic acid having a sequence selected from the group consisting of: i) an isolated nucleic acid having a sequence selected from the group consisting of: SEQ ID NOs 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and a sequence of 39 to 120 consecutive nucleotides of SEQ ID NO: 41 ; ii) an isolated nucleic acid having a sequence that is at least 80 % identical to a sequence as defined in i); iii) an isolated nucleic acid having a sequence of 39 to 600 nucleotides that comprises a sequence as defined in i); iv) an isolated nucleic acid having a sequence that is at least 60 % identical a sequence as defined in i) and encodes a peptide that is capable of binding to PKM zeta; v) an isolated nucleic acid having a sequence selected from the group consisting of: i) an isolated nu
  • the nucleic acid according to i) has a sequence of 39 to 120, more preferably 45 to 120, such as 45 to 69 or 60 to 90, consecutive amino acids of SEQ ID NO: 42.
  • an isolated nucleic acid having a sequence "of 39 to 600 nucleotides” has from 39 to 300, from 60 to 600, from 60 to 300, from 60 to 150 nucleotides, from 39 to 150 nucleotides.
  • said nucleic acid has from 39 to 150, from 45 to 150, from 60 to 150, from 39 to 135, from 39 to 120, even more preferably from 39 to 90, from 45 to 105, from 60 to 120, such as from 45 to 90, from 45 to 75 or from 54 to 69 nucleotides.
  • an isolated nucleic acid having a sequence that is "at least 60% identical" to a given other sequence is at least 61%, 62%, 63%, 64%; 65%, 66%, 67%; 68%; 69%; 70%, 71%, 72%, 73%, 74%; 75%, 76%, 77%; 78%; 79%; 80%; 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), or 99% identical to said other sequence.
  • said isolated nucleotide encodes a peptide, peptide derivative or peptide conjugate that is capable of binding to PKM zeta.
  • an isolated nucleic acid having a sequence "of 39 to 1500 nucleotides” preferably has a sequence of from 39 to 1200, from 39 to 900, from 39 to 600, from 60 to 600, from 60 to 300, from 60 to 150 nucleotides or any range or combination in between.
  • said isolated nucleotide encodes a peptide, peptide derivative or peptide conjugate that is capable of binding to PKM zeta.
  • nucleic acid of 60 nucleotides that is 70 % identical to a sequence of 60 nucleotides means, that 42 out of said 60 nucleotides are identical.
  • isolated nucleic acids of the present invention include those having a sequence that encodes the same peptide as any other isolated nucleic acid of the present invention. That is to say, due to the well-known degeneracy of the genetic code, an isolated nucleic acid of the invention may significantly differ in sequence to other isolated nucleic acids of the invention, as long it encodes the same peptide as any other isolated nucleic acid of the present invention - or as long as it encodes for a peptide or peptide derivative of the invention, respectively.
  • the isolated nucleic acid of said aspect of the present invention is an isolated nucleic acid capable of hybridizing under stringent conditions to a nucleic acid according to any of the nucleic acids described in the embodiments above.
  • stringent conditions are preferably intended to refer to hybridization performed at 68 °C (preferably over night) in a solution comprising 1 mM EDTA; 0.5 M NaH 2 P0 4 (pH 7.2); 7% SDS (also referred to herein as "High SDS Hybridization Solution”) - or may be conditions of similar stringency as easily determined by the skilled person.
  • two washing steps with 2*SSC at 65 °C for at least 30 minutes may be included and may be followed by a final 30 minutes washing step in 0.1 *SSC at 65 °C.
  • the isolated nucleic acids of said aspect are preferably not found as such in nature, but instead have to be produced or synthesized or e.g. have to be obtained from naturally occurring nucleic acids by means of techniques used in molecular biology or genetic engineering.
  • the present invention relates to a recombinant vector for use in a method of treating an anxiety disorder, wherein said vector comprises a nucleic acid as defined above, particularly wherein said vector is derived from a viral vector, especially wherein said viral vector originates from a virus selected from the group consisting of retroviruses, lentiviruses, foamyviruses, adenoviruses and adeno-associated viruses, in particular from adeno-associated viruses.
  • said vector is derived from a viral vector.
  • said viral vector originates from a virus selected from the group consisting of retroviruses, lentiviruses, foamyviruses, adenoviruses and adeno-associated viruses. Even more preferably, the vector originates from adeno- associated viruses.
  • the present invention relates to a host cell for use in a method of treating an anxiety disorder, the host cell comprising a nucleic acid and/or a vector as defined above.
  • said host cell is selected from the group consisting of immortalized eukaryotic cells (including but not limited to SHSY5Y, HEK and COS cells), primary cells (including but not limited to rat hippocampal cells and cortical cells), yeast cells (including but not limited to Pichia pastoris cells or Saccharomyces cerevisiae cells) or bacterial cells (including but not limited to E. coli cells, such as XL-1 blue), and insect cells.
  • Host cells are preferably suitable or beneficial for the production and or purification of protein. A multitude of such cells is known to the skilled person, numerous examples of which are commercially available.
  • said host cell is a non-human host cell.
  • the present invention relates to an HDAC inhibitor for use in a method selected from the group consisting of:
  • MCI mild cognitive impairment
  • xi a method for improving or treating a brain trauma
  • xiii) a method for treating an anxiety disorder.
  • HDACs histone deacetylases
  • HDAC inhibitors including those mentioned below are known in the art and in clinical trials.
  • the HDAC inhibitor is selected from the group consisting of Suberoylanilide Hydroxamic Acid (SAHA), Entinostat (MS-275), Tacedinaline (CI-994), BML-210, M344, Mocetinostat (MGCD0103), Belinostat (PXD101), Panobinostat (LBH-589), and sodium butyrate, more preferably wherein the HDAC inhibitor is selected from the group consisting of Suberoylanilide Hydroxamic Acid (SAHA) and sodium butyrate.
  • SAHA Suberoylanilide Hydroxamic Acid
  • MS-275 Entinostat
  • Tacedinaline CI-994
  • BML-210 BML-210
  • M34414 Mocetinostat
  • MGCD0103 Mocetinostat
  • PXD101 Belinostat
  • Panobinostat LH-589
  • sodium butyrate more preferably wherein the HDAC inhibitor is selected from the group consisting of Suberoylanilide Hydroxamic Acid (SAHA
  • said method further comprises the use of a proteasome inhibitor, as defined below.
  • the invention further relates to a proteasome inhibitor for use in a method selected from the group consisting of: i) a method for improving cognition
  • MCI mild cognitive impairment
  • xi a method for improving or treating a brain trauma
  • xiii) a method for treating an anxiety disorder.
  • Proteasome inhibitors including those as defined below are known in the art.
  • proteasome inhibitor is selected from the group of: Bortezomib (Velcade), Carfilzomib, Marizomib (NPI-0052; salinosporamide A), PS-519, CEP- 18770, MLN-9708, ONX 0912, Clioquinol (5-chloro-7-iodo-quinolin-8-ol), EGCG ((-)-epigallocatechin-3- gallate), PR-924, ISPS- 101 , 5AHQ (5-amino-8-hydroxyquinoline), chloroquine, PR957, MG132, Epoxomycin, Lactacystin, more preferably wherein the proteasome inhibitor is selected from the group consisting of MG132, and Epoxomycin.
  • the proteasome inhibitor is selected from the group consisting of MG132, and Epoxomycin.
  • said method further comprises the use of an HDAC inhibitor as defined above.
  • Said method for improving memory preferably includes, but is not limited to a method for improving declarative memory; a method for improving episodic memory; a method for improving semantic memory; a method for improving non-declarative memory; and a method for improving long term memory.
  • Said method for improving motor learning and/or sensory motor recovery or for treating diseases which benefit from improved motor learning and/or improved sensory motor recovery relates to, but is not limited to a method for improving the treatment of or for treating diseases and conditions like Stroke and/or rehabilitation after stroke, spinal cord trauma and/or rehabilitation after spinal cord trauma, rehabilitation after major surgery, rehabilitation after orthopedic surgery, rehabilitation after brain surgery.
  • Said method for treating or improving neuropsychological deficits after stroke includes, but is not limited to method for treating or improving neuropsychological deficits such as agnosia, apraxia, anosognosia, prosopagnosia, hemineglect.
  • Said method for improving or treating a memory deficit includes, but is not limited to a method for improving or treating memory deficits following stroke (ischemic, hemorrhagic, subarachnoidal hemorrhage); a method for improving or treating memory deficits accompanying Parkinson's disease; a method for improving or treating memory deficits accompanying Huntington's disease; a method for improving or treating memory deficits accompanying Schizophrenia: a method for improving or treating memory deficits accompanying depression; a method for improving or treating memory deficits following radiation or chemotherapy to the brain; a method for improving or treating memory deficits following perinatal hypoxic conditions; a method for improving or treating memory deficits ensuing from infection of the brain (such as meningitis, viral encephalitis, limbic encephalitis, Hashimoto's encephalopathy, cerebral vasculitis); a method for improving or treating memory deficits accompanying tumours in the brain (such as lymphomas, metastatic tumours, gliomas); a method for improving or treating memory deficit
  • Said method for improving or treating a dementia deficit includes, but is not limited to a method for improving or treating alcohol dementia, Wernicke-Korsakoff Syndrome; a method for improving or treating dementia following hypothyroidism; a method for improving or treating dementia accompanying HIV-infection; a method for improving or treating vascular dementia; a method for improving or treating Lewy-body dementia; a method for improving or treating Morbus Binswanger; a method for improving or treating fronto-temporal dementia (FTD); a method for improving or treating Alzheimer's disease.
  • Said method for improving or treating a brain trauma also includes a method for improving or treating a mild (brain) trauma, e.g. boxer's syndrome (dementia pugilistica).
  • Said cancer preferably includes, but is not limited to carcinomas derived from epithelial cells (such as breast cancer, prostate cancer, lung cancer, pancreatic carcinoma, colon carcinoma), sarcomas derived from connective tissue (such as osteosarcoma, chondrosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma), lymphomas and leukemias derived from hematopoietic (blood-forming) cells (such as Hogdkin lymphoma, multiple myeloma), germ cell tumors derived from pluripotent cells (such as seminoma), blastoma derived from immature cells or embryonic tissue (such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma).
  • epithelial cells such as breast cancer, prostate cancer,
  • cognition is a term subsuming a broad variety of mental activities that are associated with the general processing of information. This information processing is central to consciousness, perception, learning and memory, associative thinking, reasoning based on rational judgment and deduction logic, problem-solving, conceptual thinking, knowing, planning, imagination, language, music, emotionality, humor, or motor learning. In other words, cognition refers to all higher functions of the central nervous system. The most intensely studied of these phenomena are learning and memory, partially owing to their amenability to animal studies. Memory itself can be sub-classified into different systems. One of those uses the time span of storage, and therefore propagates the existence of short-term and long-term memory.
  • Non-declarative memory is procedural memory, which stores information about motor execution (e.g. swimming).
  • declarative or explicit memory refers to memories that can be consciously recalled, and is subdivided into episodic and semantic memory.
  • Episodic memory is the memory for biographical events ordered into time and space.
  • Semantic memory is the memory of facts about the outside.
  • Agnosia is a neurological condition which results in an inability to know, to name, to identify, and to extract meaning from visual, auditory, or tactile impressions (Rhawn 2000).
  • Apraxia is a movement disorder involving the control and sequencing of skilled fine and gross motor skills in the absence of impaired motor functioning or paralysis and is commonly associated with injuries to the parietal lobe (Rhawn, 2000).
  • Anosognosia is a physiological rooted condition, like damage to the frontal or parietal lobe due to illness and disease, in which a person who suffers disability seems unaware of the existence of his or her disability.
  • Around 10% - 18% of acute hemi paretic stroke patients suffer from anosognosia (Baier et al., 2005).
  • Prosopagnosia also called face blindness
  • face blindness is an impairment in the recognition of faces, which is often accompanied by other types of recognition impairments like place recognition, car recognition or facial expression of emotion, though sometimes it appears to be restricted to facial identity.
  • prosopagnosia can create serious social problems. Patients suffering from prosopagnosia often have difficulty recognizing family members, close friends, and even themselves. Most of the documented cases are due to brain damage suffered after maturity from head trauma, stroke, and degenerative diseases (cf. www.faceblind.org/research/index.html). Hemineglect - also called spatial neglect or hemi-inattention - is a disorder of space-related behaviour. It is characterized by failure to explore the side of space contralateral to a brain lesion, or to react or respond to stimuli or subjects located on this side (Karnath, 2002)).
  • Morbus Binswanger or Binswanger's disease is a form of vascular dementia characterized by diffuse white matter lesions, accompanied with varying clinical findings which typically include a progressive dementia, depression and "subcortical" dysfunction such as gait abnormalities, rigidity and neurogenic bladder (Olsen, 1998).
  • Morbus Alzheimer or Alzheimer's disease (AD) is a well-known, but incurable, neuro-degenerative disease characterised by the loss of neurons and synapses in the cerebral cortex and subcortical regions. Biochemically it is defined by accumulation of abnormally folded A-beta and tau proteins.
  • Fronto-Temporal-Dementia is an early-onset dementia caused by degeneration of the frontal lobe and may extend back to the temporal lobe.
  • Vascular dementia refers to a subtle, progressive decline in memory and cognitive functioning. It occurs when the blood supply carrying oxygen and nutrients to the brain is interrupted by a blocked or diseased vascular system.
  • Vascular Dementia is the most common form of dementia in elderly in Asia and the second most common form of dementia in the US and Europe (cf. www.helpguide.org/elder/vascular_dementia.htm).
  • Lewy-Body Dementia or Dementia with Lewy bodies (DLB) is the second commonest cause of neurodegenerative dementia in older people.
  • Alcohol dementia Wernicke-Korsakoff syndrome or alcoholic encephalopathy is a manifestation of thiamine (vitamin Bi) deficiency, usually secondary to alcohol abuse and characterized by confusion, nystagmus, ophthalmoplegia, anisocoria, ataxia, sluggish papillary reflexes and severe memory loss (cf. www.nlm.nih.gov/medlineplus/ency/article/000771.htm).
  • AIDS end-stage acquired immunodeficiency syndrome
  • HIV infection is associated with neurocognitive deficits and anatomic and functional brain abnormalities.
  • Evidence supports a key role for the virus.
  • MCI Mild Cognitive Impariment
  • Age-associated cognitive decline - or normal (non-pathological, normative, usual) cognitive ageing - is an important human experience which differs in extent between individuals (Deary, et al., 2009). Mild Brain Trauma is also referred to as concussion, is considered the most underreported, underdiagnosed and underestimated head trauma injury. Cumulative and repetitive head trauma caused by repetitive blows to the head over a long period of time often lead to dementia pugilistica (boxer's syndrome) a form of chronic traumatic brain injury mostly accompanied by some or all of the following symptoms: intolerance to loud noise, depression, confusion, unsteady walk, improper behavior, disorientation, double vision, poor concentration, speech problems, loss of memory, headache, dizziness (cf. www.depression-guide.com/dementia-pugilistica.htm).
  • Morbus Parkinson or Parkinson's disease is a progressive neurological disorder characterised by a large number of motor and non-motor features that can impact on function to a variable degree.
  • the pathology of the disease is characterized by the accumulation of alpha-synuclein into inclusions (Lewy bodies) in neurons, and from insufficient formation and activity of dopamine in certain neurons in parts of the midbrain.
  • Patients with PD are at almost six fold increased risk for dementia (Aarsland, et al., 2001).
  • Morbus Huntington or Huntington's disease is a neurodegenerative disorder caused by an autosmal dominant mutation on either of an individual's two copies of the Huntingtin gene As the disease progresses, memory deficits tend to appear.
  • Schizophrenia is a chronic, severe, and disabling brain disease. Approximately 1 percent of the population develops schizophrenia during their lifetime. Developmental neurobiologists funded by the National Institute of Mental Health (NIMH) have found that schizophrenia may be a developmental disorder resulting when neurons form inappropriate connections during fetal development (NIH Publication No. 02-35 17). Depression is a state of low mood and aversion to activity. Many people suffering from this chronic illness lose their appetites, their ability to sleep normally, their sex drive, and the very ability to enjoy the simplest pleasure.
  • Hypoxic conditions may cause HIE, hypoxic-ischemic encephalopathy, a serious condition that causes significant mortality and long-term morbidity.
  • Hypoxic-ischemic encephalopathy is characterized by clinical and laboratory evidence of acute or subacute brain injury due to asphyxia, i.e., hypoxia or acidosis (cf. emedicine.medscape.com/article/973501-overview).
  • hypothyroidism is a condition in which the thyroid gland does not produce enough thyroid hormone
  • the adequate functioning of both the maternal and fetal thyroid glands play an important role to ensure that the fetal neuropsycho- intellectual development progresses normally (Glinoer, 2000).
  • the hallmark of encephalopathy is an altered mental state. Depending on the type and severity of encephalopathy, common neurological symptoms are progressive loss of memory and cognitive ability, subtle personality changes, inability to concentrate, lethargy, and progressive loss of consciousness.
  • Wilson's disease is a rare inherited disorder in which excessive amounts of copper accumulate in the body. The build-up of copper leads to damage in the kidneys, brain, and eyes.
  • PKC zeta deficient mice display an increased Ras-induced lung carcinogenesis suggesting a role for this kinase as a tumor suppressor by repression of the interleukin-6 promoter in vivo (Galvez et al., 2009).
  • these findings that an increased PKC zeta/PKM zeta activity maintains normal cell differentiation and suppresses tumor and cancer formation suggest that an increased PKC zeta/PKM zeta activity maintains normal cell differentiation and therefore is an attractive approach to suppress formation of tumors and cancer.
  • PKC zeta/PKM zeta preventing PKC zeta/PKM zeta from degradation is considered to extend the availability of these peptides kinases and therefore to increase the activity of PKC zeta/PKM zeta and, hence, is considered to be an attractive approach to maintain cell differentiation and suppress cancer formation.
  • the present invention preferably also relates to such method per se or to the use of a peptide, peptide derivative, peptide conjugate, complex, nucleic acid, vector and/or host cell of the invention in such method - and not only to a peptide, peptide derivative, peptide conjugate, complex, nucleic acid, vector and/or host cell for use in such method.
  • the present invention explicitly involves methods for the treatment of a subject, such as a method for treating a subject in need thereof comprising administering to said subject a peptide, peptide derivative, peptide conjugate, complex, nucleic acid, vector and/or host cell of the invention in an effective amount.
  • a method for treating a subject in need thereof comprising administering to said subject a peptide, peptide derivative, peptide conjugate, complex, nucleic acid, vector and/or host cell of the invention in an effective amount.
  • said method is selected from the preferred methods described in the context with said aspects.
  • said anxiety disorder is generalized anxiety disorder (GAD) as defined above.
  • GAD generalized anxiety disorder
  • said therapy is preferably carried out in combination with a treatment selected from the group consisting of cognitive behavioral therapy, and treatment with selective serotonin reuptake inhibitor (fluoxetine, paroxetinem escitalopram, sertraline), pregabalin, buspirone, duloxetine, imipramine, clomipramine, venlafaxine, moclobemide, propranolol and other beta-adrenoblockers, clonidine, guanfacine, prazosin, or benzodiazepines (alprazolam, chlordiazepoxide, clonazepam, clorazepate, diazepam, lorazepam).
  • compositions of the invention may be characterized by any of the following features or any combinations thereof.
  • the pharmaceutical compositions of the invention may be contained in capsules, tablets, etc. all of which are readily known to the skilled person. Same may be coated e.g. with an EC comprising fatty acids, waxes, shellac, plastics, plant fibers, any of the EC described below, or any combination(s) thereof.
  • the uptake may benefit from varying carriers.
  • Oral administration of protein drugs may be complicated by proteolytic degradation in the gastrointestinal tract.
  • the proteolytic activity is commonly highest in the stomach and duodenum.
  • pepsin cleaves peptide bonds between aromatic amino acids such as phenylalanine, tyrosine and tryptophan.
  • This problem can be circumvented by delivering a peptide / protein solution in acid-resistant (gastro-resistant) capsules.
  • An example are "DRcaps acid resistant hypromellose capsules" (Capsugel).
  • tablets containing the peptide can be coated by "enteric coating” (EC).
  • EC can comprise or consist of fatty acids, waxes, shellac, plastics, and/or plant fibers.
  • peptides or proteins may also be taken up by specific receptor-mediated transport. Therefore, specific binding motifs to receptors present in the intestine can be fused to the peptide of interest. Accordingly, the compounds of the invention may be fused to such binding motifs.
  • Corresponding conjugates are comprised by the compounds of the present invention.
  • Appropriate carrier formulations can be used in addition to EC that improve oral bioavailability of compounds such as peptides or proteins by protecting them from degradation, enhance uptake into the intestinal mucosa, and increase absorption across biological membranes.
  • EC materials are: polyvinyl acetate phthalate (PVAP), cellulose acetate trimelitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), methacrylic acid copolymer, Type C (Eudragit LI 00-55), Methacrylic acid copolymer dispersion (Eudragit L30D-55), methacrylic acid copolymer, Type A, (Eudragit L-100 and Eudragit LI 2,5), cellulose acetate phthalate (CAP) (Aquateric), methacrylic acid copolymer, type B, Eudragit S-100 and Eudragit SI 2,5, shellac (Marcoat 125 &
  • Desired properties for a peptide carrier include the maintenance of the biological activity of the peptide of interest, a high biocompatibility, the ability to incorporate high peptide concentrations, the possibility to target specific cells or tissues, and release kinetics that can be adapted to reach desired pharmacokinetic properties.
  • Examples for corresponding carriers are frequently in the form of nano- or microparticles, such as those with mean diameters ranging from 0.1 to 30 pm:
  • Nanosized polyelectrolyte complexes which may be formed by self-assembly of proteins with natural and synthetic polymers by mixing oppositely charged drug and polymer that will interact by electrostatic-attraction.
  • PECs polyelectrolyte complexes
  • These can for example consist of chitosan, the peptide of interest, and other excipients such as tripolyphosphate (TPP), magnesium sulfate, sodium alginate or cyclodextrin (CD).
  • TPP tripolyphosphate
  • CD cyclodextrin
  • PAAs Poly(amidoamine)s
  • PAAs are synthetic tert-amino polymers obtained by stepwise polyaddition of primary or secondary aliphatic amines to bisacrylamides. These polymers are water-soluble, biodegradable and biocompatible, with lower cytotoxicity profiles than other polycationic vectors.
  • Novel linear PAAs are available containing repetitive disulfide linkages in their backbone (so-called SS-PAAs). These SS-PAAs have the advantage of being relatively stable in the extracellular medium but are fast degraded in the reductive intracellular environment due to reduction of the disulfide bonds.
  • Lipid-based drug delivery systems for example liposomes, mixed micelles, emulsions, micro-manoemulsions, self-emulsifying drug delivery systems (SEDDS), solid lipid nanoparticles (SLN), suspensions, or phospholipid-drug complexes.
  • Liposomes can consist of distearoyl phosphadylcholine. Liposomes can be coated with mucoadhesive polymers like carbopol and chitosan.
  • Acetylsalicylic acid drug/lecithin associates alpha-tocopherol lipid emulsion, amphotericin B cochleate (solid lipid nanoparticles), camptothecin solid lipid nanoparticles, chloroproguanil, hydrochloride-dapsone-artesunate, curcumin, cyclosporin A, lecithin, ethanol, hydroxysafflor yellow
  • a drug-phospholipid complex oil solution phosphatidylcholine-conjugate, lipopolysaccharide
  • gydrogel systems e.g.
  • Nanoparticles and liposomes may also contain an add-on of a cell-penetrating peptide (CPP), such as listed elsewhere in this document.
  • PCP cell-penetrating peptide
  • targeting moieties may be added to nanoparticles or liposomes, for example vitamin B12 or folic acid or transferrin.
  • biodegradable excipients in all those approaches include: amylose a- 1,4 D-glucose, arabinogalactone ⁇ -1,4 & ⁇ -1 ,3 D- galactose, ⁇ -1 ,6 & ⁇ -1 ,3 D-arabinose and D-galactose, Natural pectin, hemicelluloses, chitosan, deacetylated ⁇ -1,4 N-acetyl D- glycosamine, deacetylated chitin, chondroitin sulfate B-1 ,3, D-glucoronic acid & N-acetyl D-glycosamine, mucosopolysaccharides containing sulphate ester group at 4 or 6 position, cyclodextrane a- 1 ,4 D-glucose
  • Specific peptide / protein delivery systems are for example manufactured by Emisphere Technologies, Inc, and known as EligenTM technology based on sodium N-[8-(2- hydroxybenzoyl)aminocaprylate] (SNAC).
  • EligenTM technology based on sodium N-[8-(2- hydroxybenzoyl)aminocaprylate] (SNAC).
  • Other suitable proprietary systems include: CLEC, Bio-Oral, Oral-Lyn, HIM2, Oradel, Macrulin, Orasome. All of the above described carriers, coatings, excipients, systems, etc., and any combination(s) thereof, are envisaged as embodiments for the pharmaceutical compositions and (medical) methods and uses described herein.
  • the compounds of the invention may e.g. be administered orally such as in the form of pills, tablets, such as lacquered or sugar-coated tablets, granules, gelatin capsules having a soft or hard shell, solutions such as aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.
  • Administration may also be carried out parenterally, for example via injection or infusion, and includes subcutaneous, intramuscular and intravenous injection or infusion.
  • Other suitable administration modes are, for example, percutaneous, topical, rectal, via inhalation, via microcapsules, via implants or via rods.
  • the compounds of he invention such as those contained in a pharmaceutical composition of the invention, may e.g. be administered via a route including, but not limited to a route selected from parenteral (s.c, i.v.), oral, buccal, transdermal, pulmonary, intranasal, intraocular, rectal and vaginal routes.
  • parenteral s.c, i.v.
  • oral, buccal, transdermal, pulmonary, intranasal, intraocular, rectal and vaginal routes are the most promising alternatives to parenteral administration. Accordingly, oral, nasal and parenteral administration is particularly preferred.
  • Example 1 Particular Materials and Methods Animal experiments. Male Wistar rats (250g bodyweight) were used. They were housed at constant temperature (23°) and relative humidity (60%) with a fixed 12 h light / dark cycle and ad libitum access of food and water. AAV vectors are injected bilaterally at 4 sites per adult hippocampus. For each hippocampus a total volume of 4 ⁇ was injected of a stock solution of 7 x 10 9 number of particles per millilitre. Fear conditioning was performed using a specialized apparatus (Med Associates, St Albans, VT, USA). Open field, Morris water maze, and 8-arm maze were monitored by proprietary camera setup and software (SYGNIS tracker). All animal experiments were conducted in a blinded and randomized fashion. Animal experiments were approved by the responsible authorities (Reg michsprasidium Düsseldorf, Germany).
  • Plasmids/Constructs The expression plasmids (pExp_NterFlag_PK ! ⁇ _hu; pExp_Ntermyc_KIBRA_hu; pExp_nV5_KIBRA; pExp_NterFlag_KIBRA_hu; ⁇ ⁇ ⁇ _ ⁇ 5_ ⁇ _ ⁇ ; pExp_NterFlag_PK ⁇ _hu) were constructed by PCR with Phusion DNA polymerase (New England Biolabs). We modified the respective PCR products with attBl/2 recombination sites compatible for the Gateway recombination cloning system (Invitrogen).
  • the amplified ORFs were recombined with a gateway donor vector to obtain a Gateway-compatible ENTRY clone.
  • Another recombination of the ENTRY clones with the respective DEST vectors revealed the expression plasmids (CMV promoter). All Gateway related reagents and plasmids were from Invitrogen and Gateway cloning procedures were performed according to protocols provided by the manufacturer.
  • Vectors intended for small hairpin RNA (shRNA) expression were based on the AAV2 ITR-flanked shRNA expression cassette pAM U6-pl-CBA-hrGFP-WPRE-BGHpA described earlier ⁇ Szumlinski, et al. 2005).
  • shRNA small hairpin RNA
  • a target sequence at position 1276 of the Kibra ORF (GAT CCG TTG AAG TTA AAC AGC AAG ATT CAA GAG ATC TTG CTG TTT AAC TTC AAC CTT TTT TGG AAA) (SEQ ID NO: 51) was used.
  • HEK293 cells were used for the production of pseudotyped chimeric AAV 1/2 vectors (containing a 1 : 1 ratio of capsid proteins serotype 1 and 2) as described previously ⁇ Szumlinski, et al. 2005).
  • the genomic titre of the viral solutions was determined by real-time PCR (light cycler, Roche diagnostics).
  • vectors intended for small hairpin RNA (shRNA) expression were based on the AAV2 ITR-flanked shRNA expression cassette pAM/U6-pl-CBA- hrGFP-WPRE-BGHpA described earlier (Franich, et al.
  • RNA polymerase III compatible human U6 promoter For knock-down of Kibra transcript levels, a target sequence at position 1276 of the Kibra ORF (GAT CCG TTG AAG TTA AAC AGC AAG ATT CAA GAG ATC TTG CTG TTT AAC TTC AAC CTT TTT TGG AAA) (SEQ ID NO: 51) was identified with Invitrogen's BLOCK-iTTM RNAi Designer web tool, and complementary DNA oligonucleotides encoding a shRNA directed against this target sequence were generated using Ambion ' s pSilencerTM Expression Vectors Insert Design Tool.
  • sequence GTG AAG CCA CAG ATG was used as described previously by Zeng & Cullen (Zeng et al., 2004). Annealed oligos were then BamHI x Hindlll subcloned into the polylinker site. The resulting vector was termed AAV-(rat)-KIBRA RNAi.
  • AAV-eGFP is used as control vector. Generation of AAV eGFP was performed by subcloning the coding sequence of eGFP into the AAV2 backbone plasmid containing the chicken ⁇ -actin promoter and an IRES-eGFP sequence, flanked by AAV2 ITR sequences.
  • HEK293 cells were used for the production of pseudotyped chimeric AAV 1/2 vectors (containing a 1 : 1 ratio of capsid proteins serotype 1 and 2) as described previously ⁇ Klugmann, et al. 2005).
  • Cultured cells (80% confluent) propagated in complete DMEM were transfected with the AAV construct and helper plasmids (pH21, pRVl and pFA6) using calcium phosphate.
  • cells were harvested in PBS, centrifuged, and pellets from 5 plates were pooled in 25 mL of a buffer consisting of 150 mM NaCl, 20 mM Tris pH8, 1.25 mL of 10% sodium deoxycholate and 50 U/mL of benzonase. After an incubation of 1 hour at 37°C, 25 mL of 150 mL NaCl and 1.25 mL of 10% sodium deoxycholate were added and the solution was centrifuged.
  • a buffer consisting of 150 mM NaCl, 20 mM Tris pH8, 1.25 mL of 10% sodium deoxycholate and 50 U/mL of benzonase.
  • the supernatant was collected and filtered with 450 mM NaCl, 20 mM Tris pH 8 through a high affinity heparin column (1 mL HiTrap Heparin, Sigma) previously equilibrated with buffer (150 mM NaCl, 20 mM Tris pH 8), at a speed of 1 mL/min as described.
  • the genomic titer of the viral solutions was determined by real-time PCR (light cycler, Roche diagnostics, Mannheim, Germany).
  • AAV vectors were injected bilaterally into the adult hippocampus at 4 sites per hemisphere. For each hemisphere a total volume of 4 ⁇ was injected of a virus solution containing 7 x 10 9 virus particles /ml.
  • the coordinates used for each hemisphere were 1) AP 5.8 mm from the lambda; ML 1.0 mm from the sagittal suture and DV 4.2 mm from the bregma, 2) AP 4.4 mm from the lambda; ML 2.9 mm from the sagittal suture and DV 3.9 mm from the bregma, 3) AP 3.8 mm from the lambda; ML 4.0 mm from the sagittal suture and DV 3.8 mm from the bregma, 4) AP 3.2 mm from the lambda; ML 4.6 mm from the sagittal suture and DV 6.2 mm from the bregma.
  • Vector delivery was performed with a microprocessor controlled mini pump (World Precision Instruments, Sarasota, FA, USA) with 34xG beveled needles (World Instruments) in a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA).
  • a microprocessor controlled mini pump World Precision Instruments, Sarasota, FA, USA
  • 34xG beveled needles World Instruments
  • a stereotaxic frame Kopf Instruments, Tujunga, CA, USA.
  • the open field consists of an opaque PVC box (90x90x90 cm 3 ). To evaluate locomotor activity the arena was divided into 4x4 equal squares and the 4 inner squares were defined as the center. For testing the rat is placed in the lower right corner of the box facing the wall. Exploration is analyzed automatically for 5 min using SYGNIS Tracker software. Time and pathlength in the center and in the periphery is calculated.
  • Contextual fear conditioning The rat is placed in a fear conditioning chamber (Med Associates, St Albans, VT, USA) and freezing behavior is monitored. In the acquisition phase at day 1 the animal is placed in the chamber for 8 min. After 3 min of exploration the animal is exposed to 4 tone - shock pairs. The conditioned stimulus represented by the tone (1000 Hz; 90 dB, 20 s) was terminated by the unconditioned stimulus represented by a footshock (1.2 mA, 1 s). Exposures are repeated for 4 times with an 1 min interval. To test the contextual fear memory the rat is placed in the chamber in the absence of any stimuli one day later, and freezing behavior is measured for 8 min.
  • a fear conditioning chamber Med Associates, St Albans, VT, USA
  • the rat is placed into a distinct context by changing the internal shape, texture, and smell of the chamber on day 3, and freezing is monitored during a 5 min presentation of the tone. Freezing response is defined as a complete lack of movement and is analyzed by a dedicated software package (Med Associates).
  • the water maze is a circular water tank (diameter 1.70 m, depth 0.60 m) partially filled with water.
  • Non toxic colour is added to the water preventing the animals from seeing a submerged transparent plexiglas platform that is placed slightly below the water. The platform is left in this position for the duration of the experiment.
  • External visual cues are placed in the surrounding environment of the water maze. These cues are visible from the pool and serve as spatial orientation for the rats.
  • a camera placed over the center of the pool connected to a video recorder tracks the behavior of the animals. To ensure that the rats learned the spatial task efficiently they first performed a non-spatial pretraining where the platform is made visible by placing cues directly over the platform.
  • rats During non-spatial training cues positioned outside the maze are hidden by surrounding the pool with black curtains.
  • rats have 4 trials per day, one trial has a maximal time of 60 s.
  • Animals were released from the SW, S, SO and O points of the pool in a randomized but balanced manner such that the start point frequency and the distance to the platform were distributed equally across the test. After climbing onto the platform, rats are allowed to remain on the platform for 30 s before being returned to the cage for a 30 s inter-trial interval. In cases where the rat was unable to find the platform, it was guided by the experimenter to the target and was allowed to remain there for 30 s.
  • Latency and pathlength to find the platform are calculated using proprietary software. Additional parameters measuring the proximity (time and distance in the area of the platform) are calculated.
  • Radial 8-arm maze The maze consisted of 8 equally spaced arms radiating from a central platform, which the rat has to enter in order to retrieve a food pellet placed in 4 of the arms.
  • the central platform was 25 cm in diameter, each arm measured 50 cm x 10 cm.
  • Walls consisted of clear plexiglas walls with 20 cm height.
  • the rat is randomly placed in one of the unbaited arms (random start arm) and is allowed to enter the baited arms to pick up the pellet.
  • the animal will acquire a working memory relating to arms that were baited as well as arms already entered. In addition, the animal will learn to avoid unbaited arms during the testing phase (reference memory).
  • Testing time was 300 s with 3 trials per day over 3 weeks (15 days). Animals were food deprived for the test until a 10% drop in intial bdy weight was reached, and were kept at that weight during the trial. Correct choices and errors were recorded using proprietary analysis software.
  • COS 1 cells were cultured in Dulbecco's modified eagle medium (DMEM) high glucose supplemented with 10% fetal calf serum (FCS). Cells were transfected with appropriate plasmids as indicated in the result section.
  • DMEM Dulbecco's modified eagle medium
  • FCS fetal calf serum
  • COS-1 cells were harvested and resuspended in electroporation (EP)-buffer (50 mM K 2 HP0 4 , 20 mM CH 3 COOK, 20 mM KOH, pH 7.4) at a densitiy of 3.5xl0 6 cells per transfection.
  • EP electroporation
  • Cells were plated at a density of 10000 cells per well of a 24-well-plate on glass cover slips coated with poly-L-lysine. Following an incubation of three hours, the medium was replaced by neurobasalmedium containing lxB27 supplement (Invitrogen, Düsseldorf, Germany) mixed with 50% (final concentration) conditioned medium. For viral infection, cells were cultured for 14 DIV and then virus was given to the culture. After another seven days, primary neurons were harvested for RNA preparation or protein extraction.
  • lysate was spun down at 13000 rpm at 4 °C for lO min and the protein concentration of the supernate was determined (BCA-Test, Pierce). 1/5 volume of 5x sample-buffer (with ⁇ -Mercaptoethanol) was added and samples were denatured at 95 °C for 5 min. 50 ⁇ g of protein was run on a SDS-PAGE and proteins were transferred to nitrocellulose membranes using an iBlotTM Dry Blotting System (Invitrogen, Düsseldorf, Germany).
  • the blots were incubated with the secondary antibody (anti -rabbit- or anti-mouse-HRP-conjugated- antiserum) for 1 h at room temperature. Signals were detected using the supersignal chemiluminescence system (Pierce, Rockford, IL, USA) and exposed to CL-Xposure film (Pierce). For quantification of scanned autoradiographs Windows ImageJ (NIH, Bethesda, MD,USA) was used.
  • COS cells were washed twice with ice-cold lxPBS, and protein extraction was carried out by lysis of cells with lysis-buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.5% NP40, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 2 mM orthovanadate, protease inhibitor cocktail) at 4 °C. After incubation for 20 min at 4°C, the lysate was spun down at 13500 rpm at 4 °C for 10 min. One part of the supernatant was kept for direct Western analysis (lysate).
  • lysis-buffer 50 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.5% NP40, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 2 mM orthovanadate, protease inhibitor cocktail
  • the other part was incubated with prepared anti-flag beads (ANTI-FLAG M2 Affinity Gel, Sigma, Taufkirchen, Germany) at 4 °C for 2 h. After precipitation of the anti-flag-antigen-complex, the beads were washed 3x with TBS (50 mM Tris/HCl, 150 mM NaCl). To release the flag-bound- complex from the beads, lx SDS loading buffer (without reducing reagents) was added and incubated at 95°C for 5min. After spinning down the beads, supernatant (IP) was analyzed by performing SDS-PAGE and transferring proteins onto nitrocellulose membranes (iBlotTM Dry Blotting System, Invitrogen, Carlsbad, CA, USA).
  • TBS 50 mM Tris/HCl, 150 mM NaCl
  • Blots were blocked with 5% milk powder and incubated overnight at 4 °C with the primary antibody (anti-flag M2 Monoclonal Antibody, 1 : 10000 (Sigma, Taufkirchen, Germany) and anti-V5 Antibody, 1 :5000 (Sigma, Taufmün, Germany), respectively). After washing, the blots were incubated with the secondary antibody (anti-mouse antiserum HRP-coupled, 1 :8000 (Dianova, Hamburg, Germany) for lh at room temperature. Signals were detected using the supersignal chemiluminescence system (Pierce, Rockford, IL, USA) and exposed to CL-Xposure film (Pierce, Rockford, IL, USA)
  • Quantitative PCR Quantitative PCR. KIBRA-overexpressing primary neuronal cells were harvested 5 d after transduction of KIBRA by AAV for RNA preparation using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturers recommendations. cDNA was synthesized from 2 ⁇ g total RNA using oligo-dT primers and superscript II reverse transcriptase (Invitrogen, Düsseldorf, Germany) according to standard protocols. Quantitative RT-PCR was performed using the Lightcycler system (Roche Diagnostics, Mannheim, Germany) with SYBR-Green staining of double-stranded DNA.
  • the following primer pairs were used: KIBRA sp-1 2982mm “GAA GGA GCT GAA GGA GCA TTT” (SEQ ID NO: 52), KIBRA asp-1 3219mm “CCT GAA AGA CTG CAC TTC TGG” (SEQ ID NO: 53); PKC zeta 5'fwd “CGC TCAC CCTC AAG TGG GTG GAC AG” (SEQ ID NO: 54), PKC zeta and PKM zeta 3 'rev "GGC TTG GAA GAG GTG GCC GTT GG” (SEQ ID NO: 55); PKM zeta 5 ' fwd "CCA CCC GGG CCT GGA GAC ATG” (SEQ ID NO: 56).
  • Cycling conditions were as follows: lOmin at 95°C; 5s at 95°C, 10s at 60°C, 30s at 72°C, and 10s at 84 for 45 cycles for KIBRA, and lOmin at 95°C; 5s at 95°C, 10s at 67°C, 30s at 72°C, and 10s at 83 for 45 cycles for PKC/M zeta. Melting curves were determined using the following parameters: 95 °C cooling to 50 °C; ramping to 99 °C at 0.2 °C/ sec. Specificity of product was ensured by melting point analysis and agarose gel electrophoresis.
  • cDNA content of samples was normalized to the expression level of Cyclophilin (primers: intocyc5" ACC CCA CCG TGT TCT TCG AC (SEQ ID NO: 57); consequentacyc300" CAT TTG CCA TGG ACA AGA TG (SEQ ID NO: 58)) .
  • Relative regulation levels were derived after normalization to native cortical neurons.
  • Proteasome activity assay For assaying the influence of KIBRA on proteasome activity in COS1 cells, a 20S Proteasome activity assay (#APT280, Millipore, Germany) was used.
  • a labeled substrate (LLVY-7-Amino-4-methylcoumarin (AMC)) is cleaved and fluorescence of the free AMC fluorophore can be quantified using a 380/ 460 nm filter set.
  • COS cells were transfected with pExp_nV5_KIBRA_hu, pExp_NterFlag_PKMzeta_hu or with the respective V5-/Flag- control-plasmids. 2d after transfection, cells were harvested for the assay using a lysis-buffer containing 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl and 1% Triton X-100.
  • Cell extraction and proteasome activity assay were performed according to the manufacturer's instructions, including a proteasome positive control and Lactacystin as proteasome inhibitor. Fluorescence data was collected by using a BMG FLUOstar plate reader (BMG, Ortenberg, Germany) using 340 nm excitation and 450 nm emission filters. Kinase assays. CREB phosphorylation assays were performed using the SignaTECT Protein Kinase C Assay System from Promega and 33 P -ATP (10 ⁇ / ⁇ 1) provided by Perkin Elmer following the manufacturer's instructions.
  • FLAG-tagged PKC zeta was harvested as described under "Co-immunoprecipitation" in lysis-buffer containing 1% NP40. Harvested protein samples were quantified by Western blotting, and equal amounts were applied in the phosphorylation assay. Bead suspensions were diluted 1 :2 in 0.1 mg/ml BSA / 0.05% Triton.
  • PKM zeta is identical to the C-terminal half of the atypical Protein kinase C ⁇ (PKC zeta) lacking its N-terminal auto-inhibitory domain and generated by an independent promoter within the gene (Hernandez et al, 2003).
  • PKM zeta mRNA is stored locally in dendrites and translated after sufficient synaptic stimulation (Osten et al, 1996; Muslimov et al, 2004).
  • PKM zeta is constitutively active after phosphorylation by PDKl (Kelly et al, 2007).
  • the present inventors initially confirmed interaction of PKM zeta and PKC zeta with KIBRA in pulldown assays from transfected COS cells (Fig. la). The binding was very robust and reproducible under multiple lysis conditions and detergents (not shown). This interaction was also relevant in a cellular context as co- expression of a myristoylated PKC zeta redirected a KIBRA-GFP fusion protein completely towards the cell membrane (Fig. lb). The present inventors have previously demonstrated that PKM zeta can phosphorylate - at least in vitro - two serine residues near the KIBRA C-terminus (But her et al, 2004). Mutating those residues to alanine or glutamate had no influence on the interaction observed (Fig. 6a).
  • Example 3 mRNA levels of PKM zeta were monitored to determine whether this enhancement in levels was caused by influences of KIBRA on the mRNA level, for example on mRNA stability. Endogenous PKC zeta or PKM zeta mRNA levels were not influenced by increased presence of KIBRA in primary cortical neurons (Fig. 2a) or in other cells (not shown). The present inventors therefore asked whether KIBRA presence led to a stabilization of PKM zeta on the protein level. Expression of PKM zeta in the presence of the protein translation inhibitor cycloheximide (CHX) in COS cells led to a rapid decrease of the kinase levels within 48 h (Fig.
  • CHX protein translation inhibitor cycloheximide
  • PKC zeta The constitutive kinase activity of PKM zeta is thought to be crucial in its role in maintaining long-term memory storage (Sacktor 2008). The present inventors therefore asked whether binding to KIBRA altered PKC/ PKM zeta kinase activity.
  • PKC zeta was immunoprecipitated from COS cells in absence or presence of KIBRA, and phosphorylation of a CREB target peptide was determined by a radioactive filter assay in comparison to in vitro synthesized and purified PKC zeta. Equal amounts of PKC zeta determined by Western blot were entered into the assay. Presence of KIBRA did not decrease the kinase activity of PKC zeta (Fig. 3a).
  • PDK1 previous activation by PDK1 may be important for KIBRA to bind and protect PKM zeta from degradation, and that the bound kinase retains its activity towards other proteins.
  • PDK1 itself is not contained in the complex, fitting to the assumption of a prior phosphorylation event and subsequent KIBRA binding (Fig. 6b).
  • KIBRA KIBRA itself is highly stable. Indeed, KIBRA amounts remain stable after 48 h CHX exposure of transfected cells (Fig. 6c).
  • the binding motif(s) in KIBRA responsible for binding were determined. By deletion series, it was determined that a region in the C-terminal third was important for binding (data not shown). Fine deletion mapping indicated amino acids 946 - 985 as important (Fig. 4a). EGFP-fusion of different stretches within that region indicated a 20 aa motif (PPFVRNSLERRSVRMKRPSS (aa 956 - 975), SEQ ID NO: 22) is sufficient for PKM zeta binding (Fig. 4b).
  • Example 6 In another experiment, consequences of reducing KIBRA expression in the rat hippocampus by AAV -mediated siRNA delivery were studied. siRNA-treatment produced an overall reduction of KIBRA expression by almost 50% on the protein level (Fig. 7). The initial open field test revealed no significant differences between siRNA-treated and control animals in parameters such as overall distance travelled (Fig. 5a), mean velocity, or time or distance spent in center (Fig. 5b). In the Morris water maze, the acquisition phase over 16 trials using random choices of 4 start positions yielded similar performances of both groups with a slight disadvantage for the siRNA group that resulted in the same run time reached at trial 16 (Fig. 5c).
  • KIBRA downregulation influenced working memory performance equally over the duration of the trial, while the slope behavior of reference memory performance was changed significantly by treatment, indicating that the speed of reference memory acquisition was altered.
  • KIBRA downregulation impairs spatial learning and memory performance in two paradigms.
  • Inhibiting PKM zeta activity is unambiguously linked to a decrease in the ability to maintain long-term memories, while a simple increase in PKM zeta levels enhances the capabilities for memory storage (Shema et al, 2007; Serrano et ai, 2008; Shema et ah, 2011).
  • Hippocampal PKM zeta levels in rats where KIBRA levels have been downregulated by siRNA were determined.
  • PKM zeta levels were decreased by over 50% in the hippocampi of those animals (Fig. 5g).
  • the present inventors consider the mechanism observed in cell culture to obviously also be active in vivo, and to be sufficient to explain the influence of altered KIBRA levels on learning and memory performance.
  • Example 7 Moreover, it was shown that KIBRA overexpressed by AAV in primary neurons localizes to different spines in different amounts, suggesting preferential localization to spines in a certain, yet unknown state of plasticity (cf.. Fig. 8).
  • Example 8 To monitor stabilization of PKM zeta by KIBRA, KIBRA peptides, peptide derivatives or peptide conjugates, e.g. SH-SY5Y cells (or other immortalized cell lines such as COS- or HEK293 -cells) can be transfected with a plasmid containing the ORF of PKM zeta. In such assays, cells may for example be co-transfected with a plasmid harbouring the ORF of KIBRA or a fragment etc. thereof. To determine the impact on degradation of the PKM zeta protein, cells can be treated Id after transfection with cycloheximide (CHX, ⁇ ) for e.g. 24 h and 48 h to inhibit de novo protein synthesis.
  • CHX, ⁇ cycloheximide
  • cells may be transfected with a PKM zeta-reporter construct only.
  • Id after transfection KIBRA peptides or the like are transferred into the cells.
  • De novo protein synthesis is stopped on day 2 after transfection using e.g. cycloheximide.
  • a reporter such as luciferase
  • a luciferase assay using a plate reader will be employed.
  • an ELISA can be performed using an antibody targeted against the tag for capturing PKM zeta. Detection of the kinase may be done using an anti-PKC/M zeta antibody. Read-outs can be statistically analyzed, and hits identified.
  • Optical densities were measured at 450 nm using a Fluostar plate reader. Background levels of untransfected cells were subtracted from read out values and PKM zeta stability was calculated comparing the OD450 nm at 0 h, 24 h and 48 h after CHX treatment.
  • Example 9 On the basis of the findings of the present inventions, it may be screened for molecules (such as small molecules or oligopeptides) that have the ability to bind to PKM zeta at the position where binding of a peptide of the invention interferes with degradation of the kinase. Such screen can yield molecules with similar or higher affinity to PKM zeta than native KIBRA or a peptide of the invention. These molecules can be further derivatized and optimized for medical uses.
  • molecules such as small molecules or oligopeptides
  • human KIBRA or peptide fragments such as a 20mer containing amino acids 956-975 of KIBRA, and PKM zeta may be expressed in SH-SY5Y-, COS- or HEK293- cells (or other immortalized cell lines, or primary cells such as neurons) with different labels (e.g. EGFP or luciferase or tag sequences such as flag-, myc- or V5-tag).
  • labels e.g. EGFP or luciferase or tag sequences such as flag-, myc- or V5-tag.
  • a small molecule library may be screened for binders that displace the native KIBRA or KIBRA peptide, such as the 20mer.
  • a flag-tagged PKM zeta construct may be overexpressed in a cell system (like e.g. COS, HEK, SHSY5Y), immunoprecipitated using flag-beads (e.g. agarose beads) under stringent conditions, released from the beads, and bound to ELISA plates harbouring an immobilized anti- FLAG -antibody.
  • flag-beads e.g. agarose beads
  • PKM zeta generated in a cell-free system may be used (e.g. from a reticulocyte or wheat germ lysate translation system).
  • a biotinylated KIBRA or KIBRA peptide may then be added to the system, and will bind after incubation of a short time period (e.g. 1 h) to PKM zeta.
  • the test compound or e.g. small molecule library
  • concentrations of typically 1 or 10 ⁇ are then added in concentrations of typically 1 or 10 ⁇ .
  • the ELISA plates can be washed under relatively mild conditions, and the label (here: biotin moiety) of KIBRA or the KIBRA peptide is detected e.g. using Streptavidin coupled to a fluorophore.
  • the fluorophore intensity may be quantitated in an adequate reader, and wells will be identified with a significantly diminished fluorophore intensity, indicating displacement of the KIBRA or KIBRA peptide by the respective test compound (such as a small molecule).
  • a test compound such as a small molecule
  • a peptide library or the like may be used.
  • flag-tagged PKM zeta and a KIBRA peptide luciferase fusion protein may be expressed. These two proteins will form a complex within the cells, and this complex will be isolated by immunoprecipitation using flag-beads. After this step, the above procedure can be used without the need for adding KIBRA peptide.
  • the readout system may be more versatile in this approach, as either luciferase, or a protein-encoded fluorophore such as EGFP or YGFP may be used. If the test compound, such as a small molecule, carries a tag itself, this may be measured along the lines to KIBRA-detection, as well. An increase of the signal will give information if the molecule itself binds PKM zeta.
  • a phage display library may be employed (e.g. available form New England Biolabs, Kit Components: Ph.D.TM-12 Phage Display Peptide Library, E. coli K12 ER2738, Biotin, Streptavidin, lyophilized).
  • a common vector used in such assay is the bacteriophage Ml 3.
  • the phage library displays random peptide sequences of seven or 12 amino acids, where also longer sequences may be used.
  • the randomized peptide sequences in the NEB libraries are expressed at the N-terminus of the minor coat protein pill, resulting in a valency of five copies of the displayed peptide per virion.
  • PKM zeta may again be immobilized in wells, and the phages may be added to the well. Phages with appropriate sequences would bind, and remain bound after the washing step. Bound phages are then eluted, a suitable bacterial host (such as Escherichia coli bacterial cells, including e.g. as TGI , SS320, ER2738, or XLl-Blue) is infected, and phages are amplified. After e.g. three washing and enrichment steps (often called "panning"), the sequence preference can be determined when sequencing the DNA of all remaining phages.
  • a suitable bacterial host such as Escherichia coli bacterial cells, including e.g. as TGI , SS320, ER2738, or XLl-Blue
  • a modified assay may test for displacement of KIBRA or of a KIBRA peptide of the invention, to avoid the use of binders not related to the KIBRA-interacting site on PKM zeta.
  • phage libraries can be employed that present antibody structures on their surface in order to identify antibodies that could displace the KIBRA peptide.
  • optimized peptides for medical use may be derived from the most preferred sequence identified.
  • Example 11 Alternatively, e.g. for assays involving interaction of KIBRA (or of a peptide, peptide derivative of peptide conjugate of the invention) with PKM zeta, such assays may also be performed with sections of PKM zeta including the KIBRA binding site. That is, specificity of such assays might be further improved if only the part of PKM zeta is used therein that binds KIBRA or the like. With such modified assays, further compounds being able to interact with PKM zeta and/or PKC zeta may be identified. To this end, several approaches may be employed. PKM zeta can be divided in smaller protein stretches by using PCR-based subcloning.
  • PKM zeta bound to e.g. the 20mer KIBRA peptide may be determined. This may routinely be done using X-ray crystallography or nuclear magnetic resonance (NMR). Protein nuclear magnetic resonance is usually performed highly purified protein in aqueous solution. The protein concentration may e.g. be in the range 0.1 - 3 mM, in a total buffer volume of 300 to 600 ⁇ .
  • the NMR sample is usually prepared in a thin walled glass tube.
  • the pure protein may be produced in an expression system such as mammalian cells (e.g.
  • the protein may be produced in a cell-free system, such as reticulocyte lysate or wheat germ extract.
  • the protein may commonly be labelled with carbon- 13 and nitrogen- 15. NMR spectra can then be used to determine the 3D-structure of the proteins examined, including e.g. the KIBRA binding site of PKM zeta.
  • the determination of the binding pocket in PKM zeta for the KIBRA peptide is considered to allow modeling of test compounds (such as small molecule structures) into the binding pocket that allow to mimic the protective function of the KIBRA peptide. That way, further compounds being able to interact with PKM zeta and/or PKC zeta may be identified.
  • Example 13 Neuronal Kibra knock-out results in anxiety symptoms.
  • KIBRA knock-out mice were generated by targeting exon 15 of the KIBRA locus for conditional excision by the Cre-lox system. These mice were crossed to transgenic mice expressing Cre recombinase under control of the Nestin promoter ("Nes-Cre mice"), thus producing knock-outs of the KIBRA allele in many neurons. These mice were subjected to testing in an open field and a fear conditioning experiment. For the open field experiment, animals were tested for 5 min in a 90 x 90 cm open box. All animals were placed in the lower right corner facing the wall, and exploration was analyzed automatically using the SYGNIS tracking software.
  • the open field was divided in 16 equal squares, with the inner 4 squares defined as the centre. Mice with the KIBRA knock-out spent less time and had lower pathlengths in the center of the open field, an indication of anxiety (cf. Fig. lla,b).
  • a sound and a mild shock are paired, such that the animals rapidly learn to associate the sound with a foot shock occurence.
  • KIBRA knock-outs demonstrated a higher basic level of anxiety / fear (cf. Fig. 12c).
  • KIBRA histone deacetylase
  • a stronger basic fear and anxiety level is present in the absence of KIBRA.
  • treatment approaches that either upregulate KIBRA levels such as shown in the present invention, or mediate the key effects of KIBRA (i.e. peptides from the KIBRA sequence protecting PKM zeta) are suitable for treating anxiety disorders.
  • Example 14 In addition, experiments in context with the present invention illustrate that stimulation with H-DAC inhibitors elevates the amount of KIBRA transcripts in cells.
  • a qCR Experiment measured the amount of KIBRA transcript in SHSY5Y cells treated with the HDAC-inhibitor sodium butyrate at a concentration of 5 mM in comparison to untreated cells (cf. Fig. 13a). Cells were seeded in 6-well plates at a density of 0,5 x 10 6 cells / well and stimulated for 48h and cyclophiline served for normalization.
  • KIBRA binds PKM zeta via the 20mer motif KIB 956"975 and residue Arg965 is important for this process as alanine substitution mutant KIBRA R965A completely abolishes the interaction. Binding of KIBRA to PKM zeta counteracts proteasomal degradation of the kinase, thus facilitating stabilization of PKM zeta protein levels. The present inventors therefore asked whether the KIB 956"975 motif is sufficient to elicit this effect.
  • PKM zeta binding would also counteract kinase degradation
  • the inventors next analyzed dissipation of PKM zeta protein levels induced by treatment of cells with the translation inhibitor cycloheximide (CHX) in the presence of CPP-tagged dimeric repeats of KIB 956"975 or KIB 956"975 R965A.
  • CHX translation inhibitor cycloheximide
  • PKM zeta protein levels were stabilized by KIB 956"975 compared to KIB 956"975 R965A using either antp or TAT as CPP (Fig. 21).
  • Example 16 Since PKM zeta kinase activity is thought to be crucial for memory consolidation and long-term memory storage (Sacktor 2008, Shema 2011) the present inventors asked whether stabilization of brain PKM zeta protein levels would translate into enhanced memory performance.
  • the coding sequences for the PKM zeta stabilizing fragment KIB 956"975 or the non-PKM zeta stabilizing double mutant fragment KIB 56"975 R965A/R969A were fused in-frame downstream to a CBA promoter-driven ORF of the EGFP reporter gene, and the expression cassette packed to adeno-associated virus (AAV) vectors.
  • the present inventors asked to which extend the interaction of WWC2 (SEQ ID NO 66) and WWC3 (SEQ ID NO 62) binding motifs compare to the binding of the 20mere KIBRA motif (SEQ ID NO 22) to PKM zeta, and if said WWC2 or WWC3 interaction confers enhanced stability to PKM zeta.
  • WWC2 and WWC3 20mere with PKMz biotinylated Peptides were sythesized. These peptides were tested for their ability to co-immunoprecipitate PKM zeta protein as well as for their capacity to displace KIBRA from the preformed KIBRA-PKM zeta complex.
  • COS cells were transiently transfected with a plasmid that harbours a flag-PKM zeta ORF and lysed 48hrs after transfection. Lysates were incubated with different concentrations of biotinylated peptides (WWC1 , 2, and 3 20meres, 1 ⁇ -10 ⁇ final concentration) and precipitated using Streptavidine-beads. The 2nd supernatant of those precipitations were probed in Western Blot using an anti-flag-antibody to detect coprecipitated PKM zeta.
  • biotinylated peptides WWC1 , 2, and 3 20meres, 1 ⁇ -10 ⁇ final concentration
  • the present invention provides the following items 1 to 15:
  • a peptide comprising the sequence FVRNSLERRSVRM, wherein said peptide has a sequence selected from the group consisting of:
  • FVRNSLERRSVRM (SEQ ID NO: 4);
  • PPFVRNSLERRSVRM (SEQ ID NO: 6);
  • FVRNSLERRSVRMKR (SEQ ID NO: 12);
  • FVRNSLERRSVRMKRPSS (SEQ ID NO: 20); PPFVRNSLERRSVRMKRPSS (SEQ ID NO: 22);
  • FVRNSLERRSVRMKRPSSVKS (SEQ ID NO: 28);
  • peptide, peptide derivative or peptide conjugate for use of item 1 , wherein the peptide derivative or peptide conjugate is selected from any of the below A, B, C, D and E:
  • v) having a sequence that is at least 80 % identical to a sequence as defined in item 1 and comprising the amino acid residue R at the position corresponding to position 965 of the full-length sequence of human KIBRA (SEQ ID NO: 2) and/or
  • ii) being a cyclic derivative of a peptide as defined in item 1 or of a peptide derivative according to A);
  • v a sequence interacting with the postsynaptic density, particularly with a PDZ motif.
  • peptide, peptide derivative or peptide conjugate for use of item 1, wherein the peptide derivative or peptide conjugate is selected from full-length KIBRA and a derivative or conjugate thereof, particularly a conjugate comprising a moiety as defined in item 2, E) i) -v ),
  • KIBRA has the sequence of SEQ ID NO: 2.
  • nucleic acid for use in a method of treating an anxiety disorder, wherein the nucleic acid a) encodes a peptide as defined in item 1 ; a peptide derivative as defined in item 2 or 3); or a peptide conjugate as defined in item 2 or 3,
  • nucleic acid having a sequence selected from the group consisting of:
  • iii an isolated nucleic acid having a sequence of 39 to 600 nucleotides that comprises a sequence as defined in i);
  • nucleic acid having a sequence of 39 to 1500 nucleotides that comprises a sequence as defined in i) and encodes a peptide or polypeptide that is capable of binding to PKM zeta;
  • c) is capable of hybridizing under stringent conditions to a nucleic acid according to any one of a) and/or b).
  • a recombinant vector for use in a method of treating an anxiety disorder wherein said vector comprises a nucleic acid as defined in item 5,
  • said vector is derived from a viral vector, especially wherein said viral vector originates from a virus selected from the group consisting of retroviruses, lentiviruses, foamyviruses, adenoviruses and adeno-associated viruses, in particular from adeno-associated viruses.
  • a host cell for use in a method of treating an anxiety disorder comprising a nucleic acid as defined in item 5 and/or a vector as defined in item 6.
  • GAD generalized anxiety disorder
  • MCI mild cognitive impairment
  • xi a method for improving or treating a brain trauma
  • xiii) a method for treating an anxiety disorder.
  • HDAC inhibitor for use according to item 9, wherein the HDAC inhibitor is selected from the group consisting of Suberoylanilide Hydroxamic Acid (SAHA), Entinostat (MS-275), Tacedinaline (CI-994), BML-210, M344, Mocetinostat (MGCD0103), Belinostat (PXD101), Panobinostat (LBH-589), and sodium butyrate, preferably wherein the HDAC inhibitor is selected from the group consisting of Suberoylanilide Hydroxamic Acid (SAHA) and sodium butyrate.
  • SAHA Suberoylanilide Hydroxamic Acid
  • MS-275 Entinostat
  • Tacedinaline CI-994
  • BML-210 BML-210
  • M344 Mocetinostat
  • MGCD0103 Mocetinostat
  • PXD101 Belinostat
  • Panobinostat LH-589
  • sodium butyrate preferably wherein the HDAC inhibitor is selected from the group consisting of Suberoylanilide
  • a proteasome inhibitor particularly selected from the group consisting of Bortezomib (Velcade), Carfilzomib, Marizomib (NPI-0052; salinosporamide A), PS-519, CEP- 18
  • MCI mild cognitive impairment
  • xi a method for improving or treating a brain trauma
  • xiii) a method for treating an anxiety disorder.
  • proteasome inhibitor for use according to item 12, wherein the proteasome inhibitor is selected from the group of: Bortezomib (Velcade), Carfilzomib, Marizomib (NPI-0052; salinosporamide A), PS-519, CEP- 18770, MLN-9708, ONX 0912, Clioquinol (5-chloro-7-iodo-quinolin-8-ol), EGCG ((-)- epigallocatechin-3-gallate), PR-924, ISPS-101, 5AHQ (5-amino-8- hydroxyquinoline), chloroquine, PR957, MG132, Epoxomycin, Lactacystin, preferably wherein the proteasome inhibitor is selected from the group consisting of MG132, and Epoxomycin.
  • the proteasome inhibitor is selected from the group of: Bortezomib (Velcade), Carfilzomib, Marizomib (NPI-0052;
  • SAHA Suberoylanilide Hydroxamic Acid
  • Velcade Bortezomib
  • Carfilzomib Marizomib
  • GAD generalized anxiety disorder

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Abstract

Cette invention concerne, entre autres, un peptide comprenant la séquence FVRNSLERRSVRM, ledit peptide présentant une séquence sélectionnée dans le groupe comprenant les SEQ ID NO 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, ou présentant une séquence de 13 à 40 acides aminés consécutifs de SEQ ID NO: 42 ou un dérivé peptidique correspondant ou un conjugué peptidique du peptide ou dérivé peptidique, pour une utilisation dans le cadre d'une méthode pour traiter des troubles anxieux.
PCT/EP2012/003974 2011-09-21 2012-09-21 Utilisation de peptides kibra, d'un inhibiteur de hdac ou d'un inhibiteur du protéasome pour améliorer des troubles anxieux Ceased WO2013041238A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015124540A1 (fr) * 2014-02-19 2015-08-27 F. Hoffmann-La Roche Ag Navette de la barrière hémato-encéphalique

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WO2007120955A2 (fr) * 2006-01-27 2007-10-25 Translational Genomics Research Institute Gènes agissant sur les performances de la mémoire humaine
US20080051360A1 (en) * 2006-08-09 2008-02-28 Reed Thomas D Pkc ligands and polynucleotides encoding pkc ligands
WO2008019395A2 (fr) * 2006-08-10 2008-02-14 Translational Genomics Research Institute Composés pour améliorer l'apprentissage et la mémoire

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015124540A1 (fr) * 2014-02-19 2015-08-27 F. Hoffmann-La Roche Ag Navette de la barrière hémato-encéphalique
US9993564B2 (en) 2014-02-19 2018-06-12 Hoffmann-La Roche Inc. Blood brain barrier shuttle

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