WO 2017/092759 Al 8 June 2017 (08.06.2017) P O P C T

Home , Allylglycine, Gabazine

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/092759 Al 8 June 2017 (08.06.2017) P O P C T

(51) International Patent Classification: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, A61K 38/00 (2006.01) C07K 14/705 (2006.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/DK2016/050369 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 16 November 2016 (16.1 1.2016) TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, PA 2015 70783 1 December 2015 (01 .12.2015) DK TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (71) Applicant: UNIVERSITY OF COPENHAGEN DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, [DK/DK]; N0rregade 10, 1165 Copenhagen K (DK). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventors: STR0MGAARD, Kristian; v llehusene 17, GW, KM, ML, MR, NE, SN, TD, TG). st.tv., 4000 Roskilde (DK). MARIC, Hans Michael; Ahle- feldtsgade 29, 2.th., 1359 Copenhagen (DK). Declarations under Rule 4.17 : (74) Agent: H0IBERG P/S; Adelgade 12, 1304 Copenhagen — of inventorship (Rule 4.17(iv)) K (DK). Published: (81) Designated States (unless otherwise indicated, for every — with international search report (Art. 21(3)) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, — with sequence listing part of description (Rule 5.2(a)) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM,

© o (54) Title: GEPHYRIN BINDING PEPTIDES AND USES THEREOF (57) Abstract: The present invention concerns a peptide or peptide analogue that is capable of binding gephyrin with high affinity and specificity. The invention is thus useful for modulating GlyR- or/and GABA AR mediated fast synaptic inhibition. The peptide or peptide analogue may also be used for isolation of post-synaptic density proteins and for an improved and general applicable la beling strategy to visualize inhibitory synapses. Gephyrin binding peptides and uses thereof

Field of invention

The present invention relates to peptides and peptide analogues which bind to gephyrin with high affinity. The invention is thus useful for therapeutic and diagnostic purposes in relation to mental or behavioural disorders and diseases of the nervous system.

Background of invention

Gamma-Aminobutyric acid type A receptors (GABAARs) and glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the human brain. To fulfil this role the receptors are highly concentrated to the synaptic sites. Dysregulation of GABAAR- mediated neurotransmission has been implicated in disorders of the CNS such as epilepsy, anxiety, mood disorders, and neurodevelopmental impairments such as autism, Fragile X syndrome, and schizophrenia (Hines, Davies et al. 2012).

Consequently, GABAAR agonists have been extensively explored and are widely used as clinically relevant drugs such as sedatives, anxiolytics and anticonvulsive drugs, narcotics and anaesthetics, antispasmodics, anti-epileptics, hypnotic and analgesic drugs. More recently, also GABAAR antagonists are being explored for their use in the treatment of Alzheimer's disease and cognitive deficits(Fernandez, Morishita et al. 2007, Ruby, Hwang et al. 2008), as well as neuroprotective agents(Defeudis 2002,

Kiewert, Kumar et al. 2008) and pain(Zhang, L I et al. 2013). All of the aforementioned compounds act on the extracellular receptor domain and thereby also affect the physiological receptor function, potentially causing adverse side effects.

The accumulation of GABAARs subtypes was shown to be dependent on gephyrin, a core component and established marker of the inhibitory post-synaptic density (Craig, Banker et al. 1996; Kim, Schrader et al. 2006; Marie, Kasaragod et al. 2014; Tyagarajan and Fritschy 2014). Thus, the direct binding of gephyrin to the intracellular regions of a subset of GABAARs and GlyRs controls the diffusion dynamics of the receptors and their synaptic clustering (Specht, Izeddin et al. 2013). In addition, gephyrin interacts only with selected subtypes of GlyRs (Kim, Schrader et al. 2006) and

GABAARs (Marie, Kasaragod et al. 2014).

Modulation of inhibitory transmission by targeting gephyrin has been attempted but only partial solutions have been found so far, and none that can be implemented on clinical level. One way that has been pursued is the depletion of gephyrin through genetic manipulation. Notably, both, interference with gephyrin expression by siRNA (Kirsch, Wolters et al. 1993; Yu, Jiang et al. 2007) and knockout approaches (Feng, Tintrup et al.1998; Kneussel, Brandstatter et al. 1999) as well as targeting of expressed gephyrin with intracellular antibodies (Zacchi, Dreosti et al. 2008) or dominant negative gephyrin fragments (Maas, Tagnaouti et al. 2006; Saiyed, Paarmann et al. 2007) resulted in the complete abolishment of gephyrin clusters and hence loss of GABAergic and glycinergic receptor accumulation at synaptic sites. Only very recently, an improved method was developed, which is based on gephyrin targeting intrabodies (Gross, Junge et al. 2013, Gross et al. 2016) and which allows to visualize gephyrin and hence inhibitory post-synaptic sites. The use of any chemical tool such as receptor derived gephyrin binding peptides as intra-cellular modulators of the gephyrin/receptor interaction that would not necessitate the genetic manipulations has never been reported.

X-ray crystallographic studies have identified the structural requirements for gephyrin binding and it was shown that a universal binding site within gephyrin binds linear

peptide motifs within distinct GlyR and GABAAR subunits, with a widely varying affinity (Kim, Schrader et al. 2006; Marie, Mukherjee et al. 201 1 ; Marie, Kasaragod et al. 2014; Brady and Jacob, 2015). Specifically, Marie et al. (2014) describe GlyR-derived peptides that bind gephyrin and are sufficient to effectively compete with other low affinity receptor fragments in vitro, however, their comparable low affinity suggests a limited use when competing with the high-affinity (Specht, Izeddin et al. 2013; Langosch, Hoch and Betz 1992) interaction of the natural full length receptors at post- synaptic sites. There is thus a need for developing compounds capable of binding with high affinity to gephyrin. mary of invention

The present inventors have developed a group of peptides and peptide analogues that bind to gephyrin with exceptionally high affinity.

In one aspect the present invention concerns a peptide or peptide analogue comprising at least 12 amino acid residues having a sequence X SIX2GX 3X 4PX5X6X7X8 (SEQ ID NO: 26), wherein: a . X is selected from phenylalanine (F), tyrosine (Y)

b. X 2 is selected from valine (V), isoleucine (I)

c . X 3 is selected from arginine (R), serine (S) d . X is selected from leucine (L), tyrosine (Y)

e . X 5, X 6 and X 7 are individually selected from arginine (R) and lysine (K)

f. X 8 is selected from arginine (R), lysine (K) and cysteine (C).

In one aspect the invention concerns a peptide or peptide analogue comprising at least 12 proteinogenic or non-proteinogenic amino acid residues comprising or consisting of a sequence (SEQ ID NO: 33), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-pyridinepropionic

acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid

b. X 2 and X 6 are individually selected from serine (S), arginine (R), /V-alpha- methyl-O-f-butyl-L-serine, A/,/\/'-bis-f-butyloxycarbonyl-2-amino-3-guanidino- propionic acid, A/-beta-f-butyloxycarbonyl-L-2,3-diaminopropionic acid

c . X 3 and X are individually selected from valine (V), isoleucine (I), leucine (L), 3- aminopentane-3-carboxylic acid, L-cyclopentylglycine, L-cyclopropylglycine, L- cyclohexylglycine, L-Meucine, L-neopentylglycine, /V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-valine, allylglycine, L-phenylglycine, /V-alpha-methyl-L-leucine, L-cyclohexylalanine

d . X 5 is selected from glycine (G), aminooxyacetic acid, sarcosine

f. X 8, X g and X 0 are individually selected from arginine (R) and lysine (K) g X is selected from arginine (R), lysine (K) and cysteine (C).

In one aspect the invention concerns a biologically active peptide or peptide analogue comprising at least five proteinogenic or non-proteinogenic amino acid residues comprising or consisting of the sequence X G (SEQ ID NO: 37), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-pyridinepropionic

acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid;

b. X 2 is selected from valine (V), isoleucine (I), leucine (L), 3-aminopentane-3- carboxylic acid, L-cyclopentylglycine, L-cyclopropylglycine, L-cyclohexylglycine, L-Meucine, L-neopentylglycine, /V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-

valine, allylglycine, L-phenylglycine, /V-alpha-methyl-L-leucine, L- cyclohexylalanine; wherein the biological activity is capability to act as a ligand of gephyrin.

In one aspect the invention concerns a peptide or peptide analogue as described herein for use as a medicament.

In one aspect the invention concerns a peptide or peptide analogue as described herein for use in a method of prophylaxis and/or treatment of a disorder selected from the group consisting of mental or behavioural disorders and diseases of the nervous system.

In one aspect the present invention concerns a method of preventing and/or treating a mental or behavioural disorder or a disease of the nervous system, comprising administering a peptide or peptide analogue as described herein, or a composition according as described herein to a subject in need thereof. In one aspect the invention concerns a method for modulating GABAAR- and GlyR- mediated fast synaptic inhibition comprising the steps of:

a . providing interconnected neurons wherein GABAAR , GlyR and gephyrin are present, b. providing a peptide or peptide analogue of the present disclosure, c . contacting the peptide or peptide analogue with said neurons, whereby said peptide enters in said neurons, and whereby said peptide is contacted with gephyrin, thereby modulating the binding of gephyrin to GABAAR and/or GlyR and

modulating GABAAR- and GlyR-mediated fast synaptic inhibition.

In one aspect the invention concerns a method for isolation of the inhibitory postsynaptic proteome from brain lysate comprising the steps of: a . providing brain lysate, b. providing a peptide or peptide analogue as disclosed herein, c . immobilizing the peptide or peptide analogue on a resin, thereby obtaining an immobilised composition, d . contacting said brain lysate with said immobilized composition, e . eluting the bound gephyrin and its binding partner, thereby isolating proteins of the inhibitory post-synaptic density thereby obtaining an eluate.

In one aspect the invention concerns a method for exclusive labelling of inhibitory synapses comprising the steps of: a . providing living neurons, wherein inhibitory synapses have formed, b. providing a peptide or peptide analogue comprising a dye conjugate as disclosed herein, and c . visualization of the gephyrin bound peptide by microscopy.

In one aspect the invention concerns a method for improving affinity of a monomeric peptide or peptide analogue for gephyrin, said method comprising the step of linking said monomeric peptide to a second peptide through a linker, thereby obtaining a dimeric peptide or peptide analogue with improved affinity.

In another aspect the invention concerns a method of manufacturing a peptide or peptide analogue as disclosed herein comprising the following steps: a . providing a resin; providing a solution comprising amino acid residues; coupling a first amino acid residue to the resin; coupling a second amino acid residue to the first one, and coupling each following amino acid residues to the previous one to form a peptide comprising at least 12 proteinogenic or non-proteinogenic amino acid residues as disclosed herein; providing cleavage of the so formed peptide from the resin.

Description of the drawings

Figure 1: Principle of action of gephyrin-SBPs. Principle action of the here presented set of compounds. A subset of GABAAR and GlyR subtypes are concentrated at synaptic sites via direct interaction of their large intracellular loops with gephyrin. The peptides exert their activity on GABAergic and glycinergic transmission through their high affinity binding to the receptor binding site of gephyrin which interferes with the recruitment of specific receptor subtypes to synaptic sites.

Figure 2: SPOT membrane layout and representative membrane. Representative SPOT membrane after incubation with the gephyrin-HRP-conjugate and evaluation via chemiluminescence.

Figure 3: Design and evaluation of gephyrin super-binding peptides. Structure-activity relationship study of gephyrin-binding peptides. Raw-heat diagrams of the titration of GephE with monovalent wild type and synthetic peptides. GephE titrated with the native GlyR-derived (GlyR-beta 298-409) peptide 1i (SEQ ID NO: 10) (a) and the improved sequence 1o (SEQ ID NO: 16) (b) as well as the improved and cell membrane penetrating peptide 1r (SEQ ID NO: 19) (c).

Figure 4: Gephyrin dimeric SBPs with picomolar affinity. (a) Raw-heat diagram of the titration of GephE with the dimeric SBP (1y, dimer of SEQ ID NO: 22). (b) Overlay of three independent titration curves. Displacement (squared grey dots) of the dimeric peptidelz (dimer of 1i*, SEQ ID NO: 25) with the improved dimeric peptide 1y (dimer of SEQ ID NO: 22) and the titration of Gephyrin E domain with 1y and 1z alone. The displacement experiment reveals a picomolar gephyrin affinity for 1y.

Figure 5 : Gephyrin SBPs display an unprecedented gephyrin specificity. (a-b) One step affinity purification of native gephyrin from whole-brain lysate using the immobilized peptides (a) Coomassie stained gel reveals that SBP 1o* (SEQ ID NO: 23) binds specifically gephyrin. (b) The immune detection verifies the identity of gephyrin and additionally reveals that the SBP 1o* (SEQ ID NO: 23) removes gephyrin almost quantitatively from the lysate. Immunodetection also reveals that GlyRs were entirely removed from gephyrin. This is in line with effective competitive binding of the SBPs to the receptor binding site of gephyrin.

Figure 6 : Evaluation of gephyrin binding designer peptides in HEK 293 cells. Confocal fluorescence microscopy analysis of HEK 293 cells over-expressing GFP- gephyrin that have been subjected to a) 10 nM of 1v (SEQ ID NO: 19 +TMR) or b) 10 nM of 1w* (SEQ ID NO: 20). GFP-gephyrin shows a punctual distribution in the cytoplasm but not the nucleus of the cells (c) as indicated by the blue 4',6-diamidino-2- phenylindole (DAPI) stain (a-c). Note that 1v (SEQ ID NO: 19 +TMR) but not 1w* (SEQ ID NO: 20) selectively binds gephyrin as indicated by the co-localization. Light transmitted (LT). Scale bars: 10 µηι .

Figure 7 . SBPs target gephyrin at synaptic sites. (a-b) Primary hippocampal neurons were lightly fixed at DIV 14, stained with antibody against endogenous gephyrin and synaptophysin and subjected to 10 nM peptide for 20 minutes. Single plane confocal images show the clustering pattern of gephyrin stained by immunofluorescence or direct peptide binding. Gephyrin clusters marked by antibody and peptide at synaptic sites appear w hite (c) Quantification of the co- localization between the different designed peptides and the conventional immunofluorescence-based gephyrin-staining. The SBP 1v (SEQ ID NO: 19 +TMR) largely co-localizes with the indirect immunofluorescence labelling, while the non- binding point mutant 1w* (SEQ ID NO: 20) does not show a significant co-localization.

Figure 8 . Peptide based super resolution visualization of inhibitory post-synaptic sites in neurons (a-c) SIM imaging of primary hippocampal mouse neurons, stained with antibody against markers of the inhibitory synapse (VGAT and gephyrin) and markers of the excitatory synapse (VGLUT1) or subjected to 2 nM peptide for 13 min. Scale bar: 5 µηι . Note that the peptide based labelling of post-synaptic gephyrin with a) the SBP 1v (SEQ ID NO: 19 +TMR) appears more confined than the corresponding immunogenic labeling of gephyrin (b). c) The SBP 1v (SEQ ID NO: 19 +TMR) appears to bind specifically to inhibitory synapses as displays no co-localization with VGLUT1 at excitatory synapses (d) Consistently the pixel-based co-localization analysis yields significant differences (p<0.05) between experiments with 1v (SEQ ID NO: 19 +TMR) and VGAT and all other experiments (involving VGLUT1 or 1w*) (for VGAT/lw* extracted values are too small to be visible in the graph). Error bars represent the mean standard error from 10 neuronal regions imaged with fluorescence labeled peptides and 10 regions imaged with antibodies.

Figure 9. Effects of intracellular SBPs on glycinergic transmission. Recordings from neurons in the dorsal medullary reticular nucleus (a) of glycine mediated mlPSCs (b-f) a) approximate localization of the region and structure of biocytin-Alexa 488 stained neuron recorded from in 17-20 days old rat brain, relative to caudal point of cerebellum (drawings from Paxinos and Watson 1996, based on adult rat); b) representative traces showing spontaneous glycine receptor mediated synaptic activity (top trace) and its inhibition by 1 µΜ strychnine (bottom trace). Vertical scale bar as in (d); c) synaptic activity from neurons using pipette solution containing 2 µΜ 1w (SEQ ID NO: 20, top trace) or 2 µΜ 1r (SEQ ID NO: 19, bottom trace); d) Average (thick) of aligned spontaneous glycine mediated mlPSCs (thin) from a neuron using vehicle pipette solution; e-f) summary of mlPSC amplitude (e) and frequency (f) of all neurons showing dose-dependent significant effects of 1r (SEQ ID NO: 19) in comparison to vehicle or 2 µΜ 1w (SEQ ID NO: 20);

Figure 10. Effects of the SBPs on GABA AR localization (a-c) Immunohistochemical analysis of a2 subunit containing GABAAR and SV2 clustering in single hippocampal neurons treated with SBPs. (a) After incubation of DIV12 neurons with 2µΜ 1w (SEQ

ID NO: 20) or 1r (SBP 1r, SEQ ID NO: 19) for 20 h , restricted dendritic areas of single neurons were defined and investigated (b) Upon incubation with 1r but not the negative control peptide 1w frequencies of all (small and large) GABA AR a2 cluster populations appear reduced (c) The ratio of synaptic to extra-synaptic a2 containing

GABA ARs is altered upon incubation with 1r, but not 1w. A large proportion of the GABA ARs appear to be localized at extra-synaptic sites. 96 dendritic regions out of 32 neurons from 3 independent experiments were analyzed per treatment group. Two-way ANOVA was used for statistical analysis (**P < 0.01). Error bars represent mean s.e.m. Scale bars 20 and 2 urn, respectively.

Figure 11. SBP cytotoxicity and effects on excitatory neuronal transmission a) Representative traces of glutamate mediated activation of synaptic glutamate type receptors using vehicle pipette solution (top trace) or containing 2 µΜ 1r (SEQ ID NO: 19, middle trace). Sensitivity to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (bottom trace) indicates that receptors mediating synaptic activity are of AMPA/KA type. Summary of mEPSC amplitude b) and frequency c) analyses, display no effect of 2 µΜ 1r (SEQ ID NO: 19) compared to vehicle on glutamate AMPA/KA type receptors. The total number of neurons included in the analysis were for glycine mlPSCs: SBP 1r 2 µΜ , n = 13, 0.2 µΜ, n = 11, 0.02 µΜ , n = 6 ; Vehicle, n = 14, SBP 1w, n = 10. For AMPA/KA mediated mEPSCs n = 6 for both SBP 1r 2 µΜ and vehicle and glutamate mediated mEPSCs. (d-e) Comparison of the cytotoxicity of 1r and Glutamate (Glu) when subjected at varying concentrations to primary hippocampal neurons (DIV12). (d) Real time viability assay. The number of viable cells is determined based on their metabolic potential to generate a chemiluminescence substrate. Numbers are given as % viability vehicle = 100 % . Incubation of the peptide 1r at concentrations between 20 nM and 2 µΜ shows no significant decrease in cell viability. In presence of 20 µΜ of 1r the assay detects a 10-15 % reduction of cells viability as compared to control (vehicle) treatment (e) Toxcicity in % in relation to the toxicity of 1 % Triton X-100. Measurements are based on the quantification of the release of lactatedehydrogenase (LDH). SBP 1r shows no significant cytotoxicity at

concentrations between 20 nM and 2 µΜ. At 20µΜ an increase ( 1 %) in toxicity can be detected after 24 and 48 hrs. ANOVA and the one sample t-test were used for statistical analyzes (*P<0.05, ***P<0.001; n=3-4). Error bars represent meanis.e.m.

Figure 12. The SBPs inhibit gephyrins receptor binding activity (a-c) ITC competition experiments based on the titration of one ligand to a stoichiometric (1:1) complex of the E domain of gephyrin (GephE) and a putative competitor ligand. (a) Raw heat signatures of the titrations of 1x to GephE (top) and the titration (middle) of 1x to

GephE preincubated with the SBP 1r ( 1 :1). The overlay of the heat release per mole of

both titrations is shown in the bottom diagram (b) Heat signatures of the titrations of 1i to GephE (top), and GephE + 1r ( 1 :1) (middle). The bottom diagram shows the overlay of the integrated heat signatures of both titrations (c) Raw heat diagrams of the titration of 1r to GephE alone or in complex with either 1x or 1i. Overlaid molar heat signatures of all three 1r titrations are shown at the bottom. Note that the pre- incubation of gephyrin with 1r renders the protein inactive towards the GABAAR alpha3 (residues 368-379) fragment 1x (a) as well as the GlyR beta (residues 398-41 1) fragment 1i (b). Vice versa, 1r binds sufficiently tight to uncouple a preformed GABAAR or GlyR gephyrin complex (c).

Detailed description of the invention

Definition of abbreviations and terms:

The term "amide bond" refers to a bond that is formed by a reaction between a carboxylic acid and an amine (and concomitant elimination of water). Where the reaction is between two amino acid residues, the bond formed as a result of the reaction is known as a peptide linkage (peptide bond).

Proteinogenic "amino acids" are named herein using its 1-letter code according to the recommendations from lUPAC, see for example http://www. chem.qmw.ac.uk/iupac. If nothing else is specified an amino acid may be of D or L-form.

The term "biological activity" as used herein refers to the capability of a chemical moiety to act as ligand of gephyrin. In particular, the term is used to refer to the capability of the peptide or peptide analogue disclosed in the present invention to specifically bind to gephyrin. Preferably human gephyrin in case of therapethic applications and mouse and rat gephyrin for visualization and isolation.

The term "BMPEG", bis-maleimide polyethylene glycol (PEG)-linkage, here refers to a structural unit composed of 2 maleimide moieties conjugated to the 2 terminals of PEG BMPEG was used in one embodiment of the present invention to link 2 peptides together. Each maleimide moiety was linked through the nitrogen atom to an ethylene glycol moiety and through a carbon atom (one involved in a double C-C bond) to the sulfur atom of a terminal cysteine of one of the peptides. The term "CNS" refers to central nervous system.

The term "CPP", cell penetrating peptide, refers to a peptide characterized by the ability to cross the plasma membrane of mammalian cells, and thereby may give rise to the intracellular delivery of cargo molecules, such as peptides, proteins, oligonucleotides to which it is linked.

The term "dimer", or "dimeric peptide", here refers to a first peptide or peptide analogue linked to a second peptide or peptide analogue through a linker. It can be homogeneous, where both peptides have the same amino acid sequence, or heterogeneous, where the 2 peptides differs for length and/or amino acid sequence.

The term "ethylene glycol moiety", also called oxyethylene, here refers to the structural unit that constitutes a PEG.

The term "fast synaptic inhibition", refers to the fast potsynaptic action (miliseconds) of

GABAARs and GlyRs when the corresponding presynaptic terminal receives an action

potential ; herein also referred to as GlyR-mediated and GABAAR-mediated fast synaptic inhibition.

The terms "fluorophore" and "fluorescent moiety" as understood herein refer to any substance that can re-emit light upon excitation. Fluorescence is generated when the fluorophore, lying in its ground state, absorbs light energy at a short wavelength, creating an excited electronic singlet state, and emits light energy at a longer wavelength, creating a relaxed singlet state. The fluorophore then returns to its ground state.

The term "GABAAR", refers to gamma-aminobutyric acid type A receptor. These ionotropic receptors are the major mediators of fast synaptic inhibition in the human brain, together with GlyRs.

The term "GlyR", refers to the glycine receptor. These receptors are the major

mediators of fast synaptic inhibition in the human brain, together with GABAARs. The beta subunit, GlyR-beta, comprises a fragment that engages into direct gephyrin interactions. This fragment displays highest affinity among all known natural gephyrin binding ligands.

The term "IPSCs", inhibitory postsynaptic currents, refers to currents comprised in an inhibitory post-synaptic potential which is a potential that prevents post-synaptic neurons from generating an action potential. Common neurotransmitters involved in IPSPs are GABA and glycine.

The term "K " refers to a dissociation constant and is a measure of the affinity of a molecule for another molecule. The lower the K , the higher the affinity of a peptide for its binding site.

The term "linker" as used herein refers to a chemical moiety that binds to a first compound and to a second compound thereby linking the first compound to the second. In some embodiments of the present invention, 2 peptides and linked together via a linker thus forming a dimer. The linker may link together more than 2 compounds; in some embodiments the linker links together 2 peptides and an additional moiety e.g. a detectable moiety and/or a CPP.

The term "maleimide moiety", also called 2,5-pyrroledione, refers to a structural unit having the chemical formula C4O2N H3.

The term "non-proteinogenic amino acids" also referred to as non-coded, non-standard, unnatural or non-natural amino acids are amino acids, as used herein refers to amino acids which are not encoded by the genetic code. A non-exhaustive list of non- proteinogenic amino acids include alpha-amino-n-butyric acid, norvaline, norleucine, isoleucine, alloisoleucine, alpha-amino-n-heptanoic acid, pipecolic acid alpha, beta- diaminopropionic acid, alpha,gamma-diaminobutyric acid, ornithine, allothreonine, homocysteine, homoserine, beta-alanine, beta-amino-/V-butyric acid, beta- aminoisobutyric acid, gamma-aminobutyric acid, alpha-aminoisobutyric acid, isovaline, sarcosine, /V-ethyl glycine, -propyl glycine, /V-isopropyl glycine, /V-methyl alanine, N- ethyl alanine, /V-methyl beta-alanine, /V-ethyl beta-alanine, isoserine, alpha-hydroxy- gamma-aminobutyric acid, 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4-methylenedioxyphenyl)propionic acid, 3,3- diphenylpropionic acid, 1-naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-pyridinepropionic acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4-

tetrahydroisoquinoline-3-carboxylic acid, /V-alpha-methyl-O-f-butyl-L-serine, Λ ,Λ/'-bis-f- butyloxycarbonyl-2-amino-3-guanidino-propionic acid, /V-beta-f-butyloxycarbonyl-L-2,3- diaminopropionic acid, 3-aminopentane-3-carboxylic acid, L-Meucine, aminooxyacetic

acid, L-cyclopentylglycine, L-cyclopropylglycine, L-cyclohexylglycine, L-

neopentylglycine, /V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-valine, allylglycine, L- phenylglycine, /V-alpha-methyl-L-leucine and L-cyclohexylalanine.

The term "Λ/PEG", is a linker derived from the classical PEG, but where one or more of the backbone oxygen atoms is replaced with a nitrogen atom.

The term "PEG", polyethylene glycol, refers to a polymer of ethylene glycol having

n+ 20n + and the repeating structure:

The term "peptide", as used herein is a sequence of at least 2 amino acid residues linked via amide bonds.

The term "peptide analogue" as used herein refers to a compound comprising a peptide, wherein the peptide may be modified with moieties that do not necessarily consist of amino acid residues. A fluorescently tagged peptide for example is a peptide analogue.

The term "SBPs" or super binding peptides, as used herein refers to a peptide having a K below 1 µΜ for gephyrin.

I. Peptide or peptide analogue The invention is as defined in the claims.

In one aspect, the present invention relates to a peptide or peptide analogue comprising at least 12 amino acid residues comprising or consisting of a sequence

XiSIX 2GX3X PX5 6 7 8 (SEQ ID NO: 26), wherein: a . X is selected from phenylalanine (F), tyrosine (Y)

b. X2 is selected from valine (V), isoleucine (I)

c . X3 is selected from arginine (R), serine (S)

d . X4 is selected from leucine (L), tyrosine (Y)

e . X5, X6 and X7 are individually selected from arginine (R) and lysine (K)

f. X8 is selected from arginine (R), lysine (K) and cysteine (C).

Another aspect of the present invention relates also to a peptide or peptide analogue comprising 12 proteinogenic or non-proteinogenic amino acid residues having a

sequence (SEQ ID NO: 33), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-pyridinepropionic

acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid

b. X2 and X6 are individually selected from serine (S), arginine (R), /V-alpha- methyl-O-f-butyl-L-serine, A/,/\/'-bis-f-butyloxycarbonyl-2-amino-3-guanidino- propionic acid, A/-beta-f-butyloxycarbonyl-L-2,3-diaminopropionic acid

c . X3 and X are individually selected from valine (V), isoleucine (I), leucine (L), 3- aminopentane-3-carboxylic acid, L-cyclopentylglycine, L-cyclopropylglycine, L- cyclohexylglycine, L-Meucine, L-neopentylglycine, /V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-valine, allylglycine, L-phenylglycine, /V-alpha-methyl-L-leucine, L-cyclohexylalanine

d . X5 is selected from glycine (G), aminooxyacetic acid, sarcosine

e . X7 is selected from valine (V), isoleucine (I), leucine (L), tyrosine (Y), 3- aminopentane-3-carboxylic acid, L-cyclopentylglycine, L-cyclopropylglycine, L- cyclohexylglycine, L-Meucine, L-neopentylglycine, /V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-valine, allylglycine, L-phenylglycine, /V-alpha-methyl-L-leucine, L-cyclohexylalanine f. X , X g and X are individually selected from arginine (R) and lysine (K) 8 0 g . X is selected from arginine (R), lysine (K) and cysteine (C). Another aspect of the present invention relates also to a peptide or peptide analogue comprising at least five proteinogenic or non-proteinogenic amino acid residues comprising or consisting of the sequence X G (SEQ ID NO: 37), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-pyridinepropionic

acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid;

b. X2 is selected from valine (V), isoleucine (I), leucine (L), 3-aminopentane-3- carboxylic acid, L-cyclopentylglycine, L-cyclopropylglycine, L-cyclohexylglycine, L-Meucine, L-neopentylglycine, /V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-

valine, allylglycine, L-phenylglycine, /V-alpha-methyl-L-leucine, L- cyclohexylalanine; wherein the biological activity is capability to act as a ligand of gephyrin.

According to one embodiment of the present invention, the N-terminal end is a characterizing feature of the peptide or peptide analogue as described herein. In some embodiments, the sequence of the five N-terminal amino acid residues is X G (SEQ ID NO: 37). In other embodiments, the sequence of the eight N-terminal amino

acid residues is X SIX2GX3X4P (SEQ ID NO: 28) . In further embodiments, the sequence

of the twelve N-terminal amino acid residues is X SIX2GX3X PX5X6X7X8 (SEQ ID NO: 26). In even further embodiments, the sequence of the twelve N-terminal amino acid

residues is (SEQ ID NO: 33).

In some embodiments, the peptide or peptide analogue has the sequence

(SEQ ID NO: 27), wherein X9, X 0 , X and X 2 are individually selected from R and K .

Said peptide or peptide analogue may comprise between 5 and 25 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 24 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 23 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 22 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 2 1 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 20 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 19 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 18 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 17 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 16 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 15 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 14 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 13 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 12 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 11 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 10 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 9 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 8 amino acid residues. Said peptide or peptide analogue may comprise between 5 and 7 amino acid residues. The peptide or peptide analogue may comprise no more than 25 amino acid residues. Thus in some embodiments, the peptide or peptide analogue

consists of 12 amino acid residues having a sequence X SIX2GX3X4PX5X6X7X8 (SEQ ID NO: 26), wherein: a . X is selected from F, Y

b. X2 is selected from V, I

c . X3 is selected from R , S d . X is selected from L , Y

e . X5, X6 and X7 are individually selected from R and K

f. X8 is selected from R , K and C.

In other embodiments, the peptide or peptide analogue consists of 13 amino acid residues comprising the 5 amino acid sequence SIX (SEQ ID NO: 37) or the 12

amino acid sequence X SIX2GX3X PX5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid Χ Χ Χ Χ β θ ιο ιι sequence 2 3 5 7 8 (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 14 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X 1SIX2GX3X PX5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence Χ Χ Χ Χ Χ Χ Χ Ρ Χ Χ Χ οΧ 1 2 3 5 6 7 8 9 (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 15 amino acid residues comprising the 5 amino acid

sequence X SIX2G (SEQ ID NO: 37) or the 12 amino acid sequence X1SIX2GX3X4P 5 6 7 8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 16 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 17 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X1SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 18 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X1SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 19 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X1SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 20 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X1SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 2 1 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X SIX2GX3X PX5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 22 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X1SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 23 amino acid residues comprising the 5 amino acid sequence X IX (SEQ ID NO: 37) or the 12 amino acid sequence

X1SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 24 amino acid residues comprising the 5 amino acid sequence X G (SEQ ID NO: 37) or the 12 amino acid sequence

X 1SIX2GX3X4P 5 6 7 8 (SEQ ID NO: 26) or the 12 amino acid sequence (SEQ ID NO: 33). In other embodiments, the peptide or peptide analogue consists of 25 amino acid residues comprising the 5 amino acid sequence X G (SEQ ID NO: 37) or the 12 amino acid sequence

X 1SIX2GX3X PX 5X6X7X8 (SEQ ID NO: 26) or the 12 amino acid sequence

In some embodiments, some amino acid residues comprised in the peptide or peptide analogue may be substituted by unnatural or non-proteinogenic amino acid residues. This substitution may result in increased affinity to the gephyrin binding site.The resulting peptide or peptide analogue will have a sequence comprising or consisting of a sequence (SEQ ID NO: 33) as disclosed herein. In other embodiments, the resulting peptide or peptide analogue will have a sequence comprising or consisting of a sequence X G (SEQ ID NO: 37) as disclosed herein. In some embodiment only one residue is a non-proteinogenic amino acid. In other embodiments the peptide or peptide analogue comprises 2 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 3 non- proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 4 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 4 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 5 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 6 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 7 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 8 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 9 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 10 non-proteinogenic amino acid residues. In other embodiments the peptide or peptide analogue comprises 11 non-proteinogenic amino acid residues.

According to one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue comprised in the composition described in the present invention may be further modified, for example they may be conjugated to a moiety that adds further features. Said conjugated moiety may allow easy detection of the peptide or peptide analogue and of the protein binding thereto. Said conjugated moiety may also facilitate penetration of the peptide or peptide analogue through a membrane. Examples of moieties that can be conjugated to the monomeric or dimeric peptide or peptide analogue comprised in the compositions described herein are found in the section below "Conjugated moiety". A non-limiting list of examples of conjugated moieties comprises a cell penetrating peptide (CPP), an albumin binding domain (ABM) and a detectable moiety.

In some embodiments, the peptide or peptide analogue comprised in the composition may be conjugated to a CPP. The CPP comprises at least 4 and at the most 13 amino acid residues selected from arginine and/or lysine. In one embodiment, the CPP comprises 5 arginine residues. In another embodiment, the 5 arginine residues are conjugated to the C-terminus of the peptide or peptide analogue. The CPP may be conjugated to the peptide or peptide analogue via an amide bond. Accordingly, in some embodiments, the peptide or peptide analogue comprises or consists of the sequence

ID NO: X·,, Xs, Xe, X SIX2GX3X 4P 5 6 8 9 o 2 (SEQ 27), wherein X2, X3, X4, X and X are as defined above and X , X , and X are individually selected from R 8 9 0 X 2 and K . In other embodiments the peptide or peptide analogue comprises or consists of the sequence (SEQ ID NO: 35), wherein X as X are defined above and X 2 , X13, X i 4 and X 5 are individually selected from R and K . In a further embodiment the peptide or peptide analogue comprises or consists of

the sequence X^IX^XsX^sXe iSEQ ID NO: 38), wherein X X2 are as defined above

and X3, X , X5 and X6 are individually selected from R and K . In a particular embodiment, the peptide or peptide analogue consists of the sequence FSIVGRYPRRRRRRRRR (SEQ ID NO: 19, 1r).

In one embodiment of the present invention, the sequence of the peptide or peptide analogue comprised in the composition may further comprise a terminal cysteine residue, whereby said cysteine facilitates the attachment of conjugated moieties to said peptide or peptide analogue. In a preferred embodiment, the cysteine is at the C- terminal end of said peptide or peptide analogue. Accordingly, in some embodiments,

the peptide or peptide analogue comprises the sequence X SIX2GX3X P X 5X6X7X8C (SEQ ID NO: 29). In other embodiments the peptide or peptide analogue comprises the sequence (SEQ ID NO: 34). In further embodiments, the peptide or peptide analogue comprises or consists of the sequence X SIX2GX 3X 4P 5 6 7 8 9 0 2C (SEQ ID NO: 30). In other embodiments, the peptide or peptide analogue comprises or consists of the sequence

X1X2X 3X4X5X 6X7PX 8X 9X 0X X 2X 3X 4X 5C (SEQ ID NO: 36). In other embodiments the peptide or peptide analogue comprises the sequence X GC (SEQ ID NO: 39). In even further embodiments the peptide or peptide analogue comprises the sequence

X 1SIX2G X 3X4X5X 6C (SEQ ID NO: 40). Accordingly, the peptide or peptide analogue may consist of 5 to 25 amino acids.

According to some embodiments, the peptide or peptide analogue may further comprise a detectable moiety allowing in vitro and/or in vivo detection. Said detectable moiety is in some embodiments selected from a group comprising fluorophores, chromophores and radioactive compounds as described in detail in the section "Conjugated moieties" below. In one embodiment, said detectable moiety is a

fluorophore. In another embodiment, said detectable moiety is the fluorophore 5/6 carboxy-tetramethyl rhodamine (TMR). In an even further embodiment said detectable moiety is a dye that can be used for visualization in super-resolution microscopy, for example Alexa Fluor ® 488, Alexa Fluor ® 532, Alexa Fluor ® 647, ATTO 488 or ATTO 532. The conjugated detectable moiety may allow visualization of the gephyrin bound peptide by microscopy, for example conventional fluorescence microscopy or super- resolution microscopy.

The peptide or peptide analogue may be in a monomeric or multimeric form. In some embodiments, the peptide or peptide analogue is in a monomeric form. In other embodiments, the peptide or peptide analogue is in a dimeric form, as detailed in the section below "Dimeric peptide".

In some embodiments of the present invention, the monomeric or dimeric peptide or

peptide analogue may be useful for modulating GABA AR and GlyR mediated fast synaptic inhibition, and can thus be used for preventing and/or treating a disorder selected from the group consisting of mental or behavioral disorders and diseases of the nervous system. The peptide or peptide analogue comprised in the composition may bind the protein gephyrin. Throughout the present disclosure, the term 'modulating' as used herein may be a positive modulation, i.e. the compositions induce fast synaptic inhibition, or a negative modulation, i.e. the compositions inhibit fast synaptic inhibition. The peptide or peptide analogue comprised in the present compositions preferably has high affinity to gephyrin. Gephyrin is a protein that interacts with the GABA A and Gly receptors, thereby modulating fast synaptic inhibition mediated by said receptors. In one embodiment, the peptide or peptide analogue binds to gephyrin and inhibits the binding of gephyrin to a specific subset of receptor subunits. Specifically the peptide or peptide analogue interferes with gephyrin binding of receptor subtypes that contain the β GlyR-beta subunit or the GABA AR-a1-3 or GABA AR 1-2 subunit. Thereby the peptide or peptide analogue modulates GABA AR and GlyR mediated fast synaptic inhibition.

Thereby inducing or enhancing GABA AR and GlyR mediated fast synaptic inhibition.

One embodiment of the present invention refers to a peptide or peptide analogue having nanomolar affinity for gephyrin. In particular, it has a K for gephyrin below than 1 µΜ, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM. Methods for determining the dissociation constant K of a peptide or peptide analogue for gephyrin are known to the skilled person. An example of such methods is isothermal titration calorimetry (ITC).

The peptide or peptide analogue comprised in the composition may be a competitive inhibitor. In one embodiment, said peptide or peptide analogue is a competitive inhibitor of gephyrin thereby accelerating the exchange rate of desensitized receptors at post synaptic sites and hence increasing the frequency and amplitude of fast synaptic inhibition. In another embodiment, said peptide or peptide analogue is a competitive inhibitor of gephyrin thereby uncoupling receptors from synaptic sites and thus decreasing frequency and amplitude of fast synaptic inhibition.

Dimeric peptide

In some embodiments, the peptide or peptide analogue is in a dimeric form. The dimeric peptide or peptide analogue comprises a first peptide or peptide analogue (P^ and a second peptide or peptide analogue (P2) linked together via a linker (L) as in the generic structure: L

The dimeric peptide or peptide analogue comprises a first peptide or peptide analogue and a second peptide or peptide analogue linked together by a linker, wherein said first and/or second peptide or peptide analogue comprise at least twelve amino acid residues comprising or consisting of a sequence X SIX2GX 3X 4PX5X6X7X8 (SEQ ID NO: 26), wherein: a . X is selected from phenylalanine (F), tyrosine (Y)

b. X 2 is selected from valine (V), isoleucine (I)

c . X 3 is selected from arginine (R), serine (S) d . X is selected from leucine (L), tyrosine (Y)

e . X 5,X 6 and X 7 are individually selected from arginine (R) and lysine (K)

f. X 8 is selected from arginine (R), lysine (K) and cysteine (C).

In some embodiments, the dimeric peptide or peptide analogue comprises a first peptide or peptide analogue and a second peptide or peptide analogue linked together by a linker, wherein said first and/or second peptide or peptide analogue comprise at least twelve proteinogenic or non-proteinogenic amino acid residues comprising or consisting of a sequence (SEQ ID NO: 33), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-pyridinepropionic

acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid

d . X5 is selected from glycine (G), aminooxyacetic acid, sarcosine

In even further embodiments, the dimeric peptide or peptide analogue comprises a first peptide or peptide analogue and a second peptide or peptide analogue linked together by a linker, wherein said first and/or second peptide or peptide analogue comprise at least five proteinogenic or non-proteinogenic amino acid residues comprising or consisting of the sequence X G (SEQ ID NO: 37), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-pyridinepropionic

acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid;

b. X2 is selected from valine (V), isoleucine (I), leucine (L), 3-aminopentane-3- carboxylic acid, L-cyclopentylglycine, L-cyclopropylglycine, L-cyclohexylglycine, L-Meucine, L-neopentylglycine, /V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-

valine, allylglycine, L-phenylglycine, /V-alpha-methyl-L-leucine, L- cyclohexylalanine;

The dimeric peptide may comprise two identical peptides or peptide analogues as described herein linked together via a linker. The dimeric peptide may therefore be a homodimer. In some embodiments, the dimeric peptide or peptide analogue may be a heterodimer.

The dimeric peptide comprised in said composition may comprise 2 identical monomeric peptides or peptide analogues. The two peptides or peptide analog comprised in the dimeric peptide are linked together via a linker. Said linker may comprise an alkane chain, diaminoacetic acid (DA), maleimide, ethylene glycol, PEG and combinations thereof. Said linker may also comprise L-alanine, 3-amino; 2,4- diamino-butanoic acid; L-ornithine or L-lysine. Another example of said linker is maleimide-PEG. A further example is a DA-PEG based linker. Said linker may also comprise Λ/PEG.

See further details in the section below "Linker".

The dimeric peptide or peptide analogue may have the following structure:

N-ter(P )C-ter-Linker-C-ter(P 2)-N-ter, wherein P and P2 are the first and the second peptides comprised in the dimer and can have identical or different sequences as disclosed herein. Each one of the two peptides or peptide analogues comprised in dimeric peptide has a sequence comprising between 5 and 25 proteinogenic or non-proteinogenic amino acid residues. The

peptides P and P2 comprised in the dimeric peptide are as the peptide or peptide analogue described in the section above "Peptide or peptide analogue". In one

embodiment P and P2 have sequence FSIVGRYPRRRC (SEQ ID NO: 22). In another

embodiment P and P2 have sequence FSIVGRYPRRRR (SEQ ID NO: 16). In a further

embodiment P . and P2 have sequence FSIVGRYPRRRRC (SEQ ID NO: 23). In an

even further embodiment P and P2 have sequence FSIVGRYPRRRRRRRRR (SEQ ID NO: 19).

Each of the first and/or second peptide or peptide analogue may further comprise a terminal cysteine residue. Said cysteine residue may facilitate the attachment of conjugated moieties to said peptides or peptide analogues. In a preferred embodiment, the cysteine is at the C-terminal end of said peptides or peptide analogues. In another embodiment, the linker linking the two peptides or peptide analogues to form a dimeric peptide is conjugated to the cysteine situated at the C-terminal end of each of the first and/or second peptides or peptide analogues.

In some embodiments, the conjugated moiety is a peptide such as a cell penetrating peptide (CPP). The CPP may comprise at least 4 amino acid residues selected from arginine and/or lysine. In one embodiment, the CPP comprises 5 arginine residues. In another embodiment, the CPP comprises at the most 13 amino acid residues. The CPP may be linked to the first and/or second peptide or peptide analogue via an amide bond. The CPP may also be linked to the linker. In some embodiments, the CPP further comprises a terminal cysteine at the C-terminus. In other embodiments, the linker linking the two peptides or peptide analogues to form a dimeric peptide is conjugated to the cysteine situated at the C-terminal end of each of the first and/or second peptides or peptide analogues. In further embodiments, a detectable moiety is conjugated to the cysteine situated at the C-terminus of the CPP.

The dimeric peptide comprised in the composition may be conjugated to a detectable moiety and may thereby be detectable in vitro and/or in vivo. Said detectable moiety is selected from a group comprising fluorophores, chromophores and radioactive compounds as described in detail in the section below "Conjugated moiety". In one embodiment, said detectable moiety is a fluorophore. In another embodiment, said detectable moiety is the fluorophore 5/6 carboxy-tetramethyl rhodamine (TMR). In a further embodiment, said detectable moiety is a fluorophore that can be used in super- resolution microscopy. Examples of such dyes are Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488 and ATTO 532. The dimeric peptide conjugated to a detectable molecule may bind to other compounds in vitro and/or in vivo, thereby allowing detection of said compounds. The detectable moiety may be conjugated to the N-terminus of one or both the peptides comprised in the dimer. The detectable moiety may also be conjugated to the linker via the CPP. In some embodiments, the

detectable moiety is conjugated to a peptide via a C-terminal cysteine residue. In further embodiments, said detectable moiety is conjugated to the CPP attached to the linker via a C-terminal cysteine residue.

In one embodiment of the present invention, at least one of the peptides or peptide analogues comprised in the dimeric peptide has the sequence FSIVGRYPRRRRC (SEQ ID NO: 23). In a further embodiment, at least one of the peptides or peptide analogues comprised in the dimeric peptide has the sequence FSIVGRYPRRRC (SEQ

ID NO: 22). In another embodiment the dimeric peptide is BMPEG(FSIVGRYPRRRC) 2.

In one embodiment of the present invention, each of the peptides comprised in the dimeric peptide or peptide analogue bind to a gephyrin E domain moiety. In one embodiment of the present invention, the dimeric peptide has higher affinity to gephyrin than the corresponding monomeric peptides or peptide analogues. Said dimeric peptide may have picomolar affinity for gephyrin. In particular, the K for gephyrin may be below 1 nM, such as below 900 pM, such as below 800 pM, such as below 700 pM, such as below 600 pM, such as below 500 pM, such as below 400 pM, such as below 300 pM, such as below 200 pM, such as below 100 pM.

In one embodiment of the present invention, the dimeric peptide has nanomolar affinity for gephyrin. In particular, it may have a K for gephyrin below 100 µΜ , such as below 90 µΜ, such as below 80 µΜ , such as below 70 µΜ, such as below 60 µΜ, such as below 50 µΜ, such as below 40 µΜ, such as below 30 µΜ, such as below 20 µΜ, such as below 10 µΜ.

The dimeric peptide or peptide analogue comprised in the composition may be a competitive inhibitor of gephyrin. In one embodiment, said dimeric peptide or peptide analogue is an inhibitor of gephyrin thereby accelerating the exchange rate of desensitized receptors at post-synaptic sites and hence increasing the frequency and amplitude of fast synaptic inhibition. In another embodiment, said dimeric peptide or peptide analogue is an inhibitor of gephyrin thereby uncoupling receptors from synaptic sites and thus decreasing frequency and amplitude of fast synaptic inhibition. In one embodiment of the present invention the dimeric peptide is a heterodimer. One of the peptides comprised in the heterodimer may have high affinity to the domain E of gephyrin. One of the peptides comprised in the heterodimer may have high affinity to a different binding site of gephyrin. One of the peptides comprised in the heterodimer may also have high affinity to a moiety that is not gephyrin.

Linker

One embodiment of the present invention relates to a dimeric peptide or peptide analogue comprising a first peptide or peptide analogue (P^ and a second peptide or peptide analogue (P2) connected via a linker (L). Said linker may be covalently bound to the first peptide or peptide analogue and to the second peptide or peptide analogue simultaneously. Said linker may be bound to the C-terminus of each peptide. In one embodiment, the linker is bound to a cysteine situated at the C-terminal end of each peptide. The binding may be via the sulfur atom comprised in the cysteine. The said linker may be attached to the C-terminal end of each peptide, for example via an amide bond with the terminal amino acid.

The linker keeps the peptide or peptide analogues comprised in the dimer at a certain distance from each other, so that they can interact with two binding sites simultaneously, hence enhancing the affinity of the molecule. The two binding sites may be comprised in the same moiety. The two binding sites may be two gephyrin E domains. The linker may keep the two peptides or peptide analogues comprised in the dimer at a distance between 4 and 340 Angstrom (A). The distance may be at least 4 A . The distance may also be at least 10 A The distance may also be at least 20 A . The distance may also be at least 30 A . The distance may also be at least 40 A . The distance may be at least 50 A . The distance may also be at least 60 A . The distance may also be at least 70 A . The distance may also be at least 80 A . The distance may be at least 90 A . The distance may also be at least 100 A . The distance may also be at least 150 A . The distance may also be at least 200 A . The distance may also be at least 250 A . The distance may also be at least 300 A . The distance may also be e.g. 340 A .

In one embodiment of the present invention the linker comprises 2 maleimide moieties. In a further embodiment the linker comprises at least 2 maleimide moieties and at least one PEG molecule comprising at least 2 ethylene glycol moieties. The linker may be bound to the 2 peptides or peptide analogues comprised in the dimeric peptide via the maleimide moieties. In a preferred embodiment, each maleimide moiety may bind a terminal cysteine of each of the peptides or peptide analogues. Each maleimide moiety may also be bound to PEG and thereby the PEG molecule may be comprised within the 2 maleimide moieties. The linker may be a bis-maleimide PEG-linkage (BMPEG). In a further embodiment the linker may be 1,8-bis(maleimido)diethylene glycol. A general structure of the linker is shown below: where P and P2 are the first and the second peptides comprised in the dimer and can have identical or different sequences as disclosed herein.

The dimer may have any of the above structures, where P and P2 are the first and the second peptides comprised in the dimer and can have identical or different sequences as disclosed herein and wherein PEG and Λ/PEG may comprise 2 to 40 ethylene glycol moieties. PEG and Λ/PEG may also comprise 2 to 5 ethylene glycol moieties. PEG and Λ/PEG may also comprise 6 to 10 ethylene glycol moieties. PEG and Λ/PEG may also comprise 11 to 15 ethylene glycol moieties. PEG and Λ/PEG may also comprise 21 to 25 ethylene glycol moieties. PEG and Λ/PEG may also comprise 26 to 30 ethylene glycol moieties. PEG and Λ/PEG may also comprise 3 1 to 35 ethylene glycol moieties. PEG and Λ/PEG may also comprise 36 to 40 ethylene glycol moieties. In a preferred embodiment the linker comprises a PEG moiety composed of 4 ethylene glycol moieties.

In one embodiment P and P2 have sequence FSIVGRYPRRRR (SEQ ID NO: 16). In

another embodiment P and P2 have sequence FSIVGRYPRRRRC (SEQ ID NO: 23).

In a further embodiment P . and P2 have sequence FSIVGRYPRRRC (SEQ ID NO: 22).

In an even further embodiment P and P2 have sequence FSIVGRYPRRRRRRRRR (SEQ ID NO: 19).

In one embodiment, the peptide or peptide analogue is dimeric and is

BMPEG(FSIVGRYPRRRC) 2.

In some embodiments of the present invention, the linker may comprise an alkane

chain, diaminoacetic acid (DA), maleimide or PEG. Said linker may also comprise L- alanine, 3-amino; 2,4-diamino-butanoic acid; L-ornithine or L-lysine. An example of said 2

linker is maleimide-PEG. A further example of the linker is a DA-PEG based linker. An even further example is a linker comprising DA and an alkane chain. In some embodiments the linker comprises 2 alkane chains. The linker may also comprise 3 alkane chains. Each alkane chain may comprise 1 carbon atom. The alkane chain may between 1 and 4 carbon atoms. Said linker may also comprise Λ/PEG. Furthermore, the linker may comprise between 1 and 3 alkane chains and at least 1 PEG moiety. In may also comprise 2 PEG moieties. Each PEG moiety may comprise between 1 and 3 ethylene glycol units. Each PEG moiety may comprise between 1 and 40 ethylene glycol units. In some embodiments, the PEG comprises 1 ethylene glycol unit. The PEG may also comprise 2 ethylene glycol units. The PEG may also comprise 5 ethylene glycol units. The PEG may also comprise 7 ethylene glycol units. The PEG may also comprise 9 ethylene glycol units. The PEG may also comprise 12 ethylene glycol units. The PEG may also comprise 20 ethylene glycol units. The PEG may also comprise 36 ethylene glycol units. Here below are provided some non-limiting examples (a-e) of the general structure that the linker may have: In the examples above, where and P2 are the first and the second peptides comprised in the dimerand can have identical or different sequences as disclosed herein; Y represent a conjugated moiety and can comprise an additional linker, a CPP, a ABM and/or a detectable moiety. The moiety Y may have the general structure: wherein Z can be a CPP, a ABM and/or a detectable moiety. In some embodiments, Z is a CPP. In other embodiments, Z is an ABM. In further embodiments, Z is a detectable moiety. In even further embodiments, Z is a CPP conjugated to a detectable moiety. The CPP may be conjugated to a detectable moiety (Z) via an amide bond. In some embodiments, the CPP further comprises a cysteine residue at the C-terminus. Said linker may further comprise a detectable moiety conjugated to the C-terminal end of the CPP. The detectable moiety may be conjugated to the C-terminal cysteine residue. The detectable moiety may also be conjugated to the C-terminal end of one or both peptides comprised in the dimer. The detectable moiety may also be conjugated to the N-terminal end of one or both peptides comprised in the dimer. The first and/or the second peptide comprised in the dimer may have sequence FSIVGRYPRRRR (SEQ ID NO: 16). In another embodiment they have sequence FSIVGRYPRRRRC (SEQ ID NO: 23). In a further embodiment they have sequence FSIVGRYPRRRC (SEQ ID NO: 22). In an even further embodiment they have sequence FSIVGRYPRRRRRRRRR (SEQ ID NO: 19).

Conjugated moiety

The peptide or peptide analogue as well as the dimeric peptide comprised in the composition disclosing the present invention may be further conjugated to an additional moiety (Z). Said moiety may be conjugated to a terminal end of the peptide or peptide analogue. In a dimeric peptide, said moiety may be conjugated to a terminal end of one of the peptides as in the generic structure below: L

In a dimeric peptide the moiety may be conjugated to the linker as in the generic structure below:

In one embodiment, said conjugated moiety is a peptide, which may be linked via an amide bond to the monomeric peptide or peptide analogue. Said conjugated peptide may be conjugated to the linker in the dimeric peptide or peptide analogue via an amide bond. Said conjugated peptide may be a cell penetrating peptide (CPP) facilitating penetration of the composition through e.g. a cellular membrane. Said conjugated peptide may comprise at least 4 amino acid residues selected from a group comprising arginine and lysine. In another embodiment of the present invention, said conjugated peptide comprises 5 arginine residues. In a further embodiment, said conjugated peptide comprises at the most 13 amino acid residues. Accordingly, in some embodiments, the peptide or peptide analogue comprises or consists of the

sequence X SIX 2GX 3X 4P 5 6 7 8 9 o 2(SEQ ID NO: 27). In other embodiments, the peptide or peptide analogue comprises or consists of the sequence

X1X2X3X4X5X6X7PX8X9X10X1 X 2X 3X 4X 5 (SEQ ID NO: 35). In a particular embodiment, the peptide or peptide analogue consists of the sequence FSIVGRYPRRRRRRRRR (SEQ ID NO: 19).

In other embodiments, said conjugated moiety is an albumin binding moiety (ABM) herein defined as any suitable chemical group binding albumin. A non-limiting example of a suitable ABM is a fatty acid, but other chemical groups may be used. In some embodiments the ABM is conjugated to the linker in the dimeric peptide or peptide analogue. In one embodiment, said conjugated moiety is a detectable moiety (J), wherein said moiety may be detected using a technology based on fluorescence or on radioactivity or UV spectrometry. Said detectable moiety may be conjugated to an N-terminus of the monomeric or dimeric peptide or peptide analogue. Said detectable moiety (J) may also be conjugated to the C-terminus of a CPP attached to the linker of the dimeric peptide or peptide analogue, such as at a C-terminal cysteine residue attached to the CPP as in the generic structure below:

The monomeric or dimeric peptide or peptide analogue as described herein may be conjugated to a detectable moiety, for example to a fluorophore. Suitable fluorophores are known to the skilled person and include Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488 and ATTO 532 and 5/6 carboxy-tetramethyl rhodamine (TMR), 6-carboxyfluorescein (6-FAM), Alexa Fluor® 350, DY-415, ATTO 425, ATTO 465, Bodipy® FL, fluorescein isothiocyanate, Oregon Green® 488, Oregon Green® 514, Rhodamine Green™, 5'-Tetrachloro-Fluorescein, ATTO 520, 6-carboxy-4',5'-dichloro- 2',7'-dimethoxyfluoresceine, Yakima Yellow™ dyes, Bodipy® 530/550, hexachloro- fluorescein, Alexa Fluor® 555, DY-549, Bodipy® TMR-X, cyanine phosphoramidites (cyanine 3 , cyanine 3.5, cyanine 5 , cyanine 5.5), ATTO 550, Rhodamine Red™, ATTO 565, Carboxy-X-Rhodamine, Texas Red (Sulforhodamine 101 acid chloride), LightCycler® Red 610, ATTO 594, DY-480-XL, DY-610, ATTO 610, LightCycler® Red 640, Bodipy 630/650, ATTO 633, Bodipy 650/665, ATTO 647N, DY-649, LightCycler® Red 670, ATTO 680, LightCycler® Red 705, DY-682, ATTO 700, ATTO 740, DY-782, IRD 700 and IRD 800, CAL Fluor® Gold 540 nm, CAL Fluor® Gold 522 nm, CAL Fluor® Gold 544 nm , CAL Fluor® Orange 560 nm, CAL Fluor® Orange 538 nm, CAL Fluor® Orange 559 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 569 nm, CAL Fluor® Red 591 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 635 nm, Quasar® 570 nm, Quasar® 548 nm, Quasar® 566 nm (Cy 3), Quasar® 670 nm, Quasar® 647 nm, Quasar® 670 nm (Cy 5), Quasar® 705 nm, Quasar® 690 nm, Quasar® 705 nm (Cy 5.5), Pulsar® 650 Dyes, SuperRox® Dyes. In some embodiments the fluorophore is TMR. Said fluorophore may be detected via conventional fluorescence microscopy. In other embodiments the detectable moiety is a dye that can be used in super-resolution microscopy, for example Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488 and ATTO 532. Said fluorophores may be detected via super-resolution microscopy.

In one embodiment, the presence of said detectable moiety allows detection of the monomeric or dimeric peptide or peptide analogue comprised in the composition under in vitro and/or in vivo conditions. Accordingly, in a further embodiment, said detectable moiety allows indirect detection of any compound binding to the peptide or peptide analogue in vitro and or in vivo. Said other compounds may be proteins that interact with the peptide or peptide analogue. In one embodiment, said other compounds may be synaptic proteins. In a further embodiment, the detectable moiety may allow detection of gephyrin.

The monomeric or dimeric peptides or peptide analogues may also be conjugated to a polymer. Said polymer may be a resin, wherein said resin may be contacted with a mixture comprising proteins. In one embodiment of the present invention, said resin is in the form of beads. The peptides or peptide analogues conjugated to the resin may bind to a protein comprised in said mixture, thereby allowing their isolation. Said monomeric or dimeric peptides or peptide analogues may be conjugated to the resin via a terminal amino acid residue. In one embodiment of the present invention, the monomeric peptide or peptide analogue is conjugated to said resin via a terminal cysteine residue. In another embodiment of the present invention, the dimeric peptide or peptide analogue is conjugated to the resin via an amino acid residue situated at the C-terminus of the CPP, wherein said CPP is conjugated to the linker. Said residue may be a cysteine. Further, the monomeric or dimeric peptide or peptide analogue may be conjugated to the resin covalently, e.g. via thiol-based chemistry.

II. Pharmaceutical composition

Whilst it is possible for the compounds or salts of the present invention to be administered as the raw peptide or peptide analogue, it is preferred to present them in the form of a pharmaceutical formulation. Accordingly, the present invention further provides a pharmaceutical formulation, which comprises a compound of the present invention or a pharmaceutically acceptable salt or ester thereof, as herein defined, and a pharmaceutically acceptable carrier therefor. The pharmaceutical formulations may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.

The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more excipients which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

The compounds of the present invention may be formulated for parenteral administration and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers, optionally with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.

Pharmaceutically acceptable salts of the instant compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases.

Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent.

The compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount.

Pharmaceutical formulations for oral administration

The compounds of the present invention may be formulated in a wide variety of formulations for oral administration. Solid form preparations may include powders, tablets, drops, capsules, cachets, lozenges, and dispersible granules. Other forms suitable for oral administration may include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations, such as solutions, suspensions, and emulsions.

In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.

Drops according to the present invention may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

Pharmaceutical formulations for parenteral administration

Injections and infusions The compounds of the present invention may be formulated in a wide variety of formulations for parenteral administration.

For injections and infusions the formulations may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules, vials, pre-filled syringes, infusion bags, or can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.

The formulations for injection will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution.

Nasal, pulmonary and bronchial administration Formulations for use in nasal, pulmonary and/or bronchial administration are normally administered as aerosols in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages, bronchial tract or the lung. The term "aerosol particle" is used herein to describe the liquid or solid particle suitable for nasal, bronchial or pulmonary administration, i.e., that will reach the mucous membranes.

Typically aerosols are administered by use of a mechanical devices designed for pulmonary and/or bronchial delivery, including but not limited to nebulizers, metered dose inhalers, and powder inhalers. With regard to construction of the delivery device, any form of aerosolization known in the art, including but not limited to spray bottles, nebulization, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used.

Liquid Aerosol Formulations in general contain a compound of the present invention in a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like.

Formulations for dispensing from a powder inhaler device will normally comprise a finely divided dry powder containing pharmaceutical composition of the present invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device. Dry powder formulations for inhalation may also be formulated using powder- filled capsules, in particularly capsules the material of which is selected from among the synthetic plastics. The formulation is formulated to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy and known to the person skilled in the art. The propellant may be any propellant generally used in the art. Specific non-limiting examples of such useful propellants are a chlorofluorocarbon, a hydrofluorocarbon, a hydrochlorofluorocarbon, or a hydrocarbon.

The formulations of the present embodiment may also include other agents useful for pH maintenance, solution stabilization, or for the regulation of osmotic pressure.

Enteric coating

When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.

One aspect of the present invention relates to composition comprising the peptide or peptide analogue as defined herein.

In one embodiment, the composition is a pharmaceutical composition.

The compound as defined herein can be in the form of a pharmaceutically acceptable salt or prodrug of said compound. In one embodiment of the present invention the compound as defined in any one of the general formulas (I), (II), (III), (IV), (V) and (VI) can be formulated as a pharmaceutically acceptable addition salt or hydrate of said compound, such as but not limited to K+, Na+, as well as non-salt e.g. H+.

III. Method for preventing and/or treating mental or behavioral disorders and diseases of the nervous system

Many mental and behavioral disorders as well as in diseases of the nervous system are thought to be based on the pathological alternation of the exact balance of neuronal inhibition and excitation and it was shown that therapeutic benefit can be

created by directly acting on the proteins that control this balance. GABAARs and GlyRs are the major mediators of fast synaptic inhibition and a number of clinically

relevant drugs are targeting the extra-cellular regions of GABAARs. However, this conventional approach is limited by the side effects that result from the altered receptor functions as well as the conserved structural features of the targeted extracellular binding sites which limit the overall potential to design subtype selective inhibitors.

The here presented invention opens a novel pharmacological pathway to modulate synaptic inhibition by acting on the diffusion dynamics of the receptors. This is achieved by their unprecedented high affinity and specificity towards the intra-cellular GlyR and GABAAR binding site of the post-synaptic sub-membranous scaffold protein gephyrin. Our data presented herein below not only demonstrate exceptional thermodynamic binding properties towards the gephyrin protein in vitro but also towards the native protein at post-synaptic sites within neurons. Finally, electrophysiological testing validated the functionality of the invention as acute modulators of fast synaptic inhibition in living neurons within mouse derived brain slices.

One aspect of the present invention relates to a composition comprising the monomeric or dimeric peptide or peptide analogue as disclosed herein for use as medicament.

Another aspect of the present invention provides a method for preventing or treating mental or behavioral disorders and diseases of the nervous system, said method comprising providing a composition as described herein and administering it to a subject in need thereof. The subject in need thereof may be a subject suffering from or suspected to suffer from mental or behavioral disorders. The subject in need thereof may be a subject suffering from or suspected to suffer from a disease of the nervous system. In some embodiments, the method comprises the step of administering a therapeutically effective amount of the composition. The method may comprise the step of administering a therapeutically effective amount of the peptide or peptide analogue described herein.

The present compositions may modulate GABAAR and GlyR mediated fast synaptic inhibition. In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue comprised in the composition modulate GABAAR and GlyR mediated fast synaptic inhibition in a subject suffering from a mental or behavioral disorder such as a disease of the nervous system, wherein said disorder and said disease comprise dysregulation of GABAAR-mediated neurotransmission. In a preferred embodiment, the monomeric or dimeric peptide or peptide analogue comprised in the composition modulates GABAergic transmission.

Examples of such mental or behavioral disorders comprise anxiety disorders, autistic disorders, mood and affective disorders, mental and behavioural disorders due to psychoactive substance use, schizophrenia, schizotypal disorders, delusional disorders, organic mental disorders, neurotic disorders, stress-related disorders, somatoform disorders, anxiety disorders, behavioural syndromes associated with physiological disturbances and physical factors, disorders of adult personality and behaviour, mental retardation, disorders of psychological development, depression and behavioural and emotional disorders with onset usually occurring in childhood and adolescence. .

Examples of such diseases of the nervous system comprise epilepsy, Alzheimer's disease, hyperekplexia, chorein deficiency, ischemic brain damage, inflammatory diseases of CNS, systemic atrophiesprimarily affecting CNS, extrapyramidal and movement disorders, Parkinson's disease, degenerative diseases of CNS, demyelinating diseases of CNS, episodic and paroxysmal disorders, insomnia, nerve-, nerve root and plexus disorders, polyneuropathies, disorders of the peripheral nervous system, diseases of myoneural junction and muscle, cerebral palsy and paralytic syndromes. The subject to which the compositions or peptides are administered may be a mammal, such as, but not limited to, a human or a domestic mammalian animal, for example a bull, a sheep, a pig, a horse, a dog or a cat. In other embodiments, the subject is a poultry bird, such as a chicken, a goose, a duck or a turkey.

The compositions used for treatment or prophylaxis of a mental or behavioral disorder such as a disease of the nervous system may be as described herein elsewhere. In particular, the composition may comprise the peptides or peptide analogues described herein above.

In some embodiments, the method comprises administering, to a subject in need thereof, a composition comprising a monomeric or dimeric peptide or peptide analogue comprising a first peptide or peptide analogue and a second peptide or peptide analogue linked together by a linker, wherein said first and/or second peptide or peptide analogue comprise at least 12 amino acid residues having a sequence

X SIX2GX3X4PX5X6X7X8 (SEQ ID NO: 26) as disclosed herein.

In other embodiments, the method comprises administering, to a subject in need thereof, a composition comprising a monomeric or dimeric peptide or peptide analogue comprising a first peptide or peptide analogue and a second peptide or peptide analogue linked together by a linker, wherein said first and/or second peptide or peptide analogue comprise at least 12 proteinogenic or non-proteinogenic amino acid residues having a sequence (SEQ ID NO: 33) as disclosed herein.

In further embodiments, the method comprises administering, to a subject in need thereof, a composition comprising a monomeric or dimeric peptide or peptide analogue comprising a first peptide or peptide analogue and a second peptide or peptide analogue linked together by a linker, wherein said first and/or second peptide or peptide analogue comprise at least five proteinogenic or non-proteinogenic amino acid residues comprising or consisting of the sequence X G (SEQ ID NO: 37) as disclosed herein.

In particular embodiments, the peptide is a dimer and has the sequence

BMPEG(FSIVGRYPRRRC) 2.

In other embodiments, the peptide is a monomer and has the sequence FSIVGRYPRRRR (SEQ ID NO: 16). In further embodiments, the peptide is a monomer and has the sequence FSIVGRYPRRRRC (SEQ ID NO: 23).

In other embodiments, the peptide is a monomer and has the sequence FSIVGRYPRRRRRRRRR (SEQ ID NO: 19). IV. Method to modulate GABAAR and GlyR mediated fast synaptic inhibition

One aspect of the present invention relates to a composition that can be used to modulate GABAAR and GlyR mediated fast synaptic inhibition. Said composition may comprise a peptide or peptide analogue such as a monomeric or dimeric peptide as described herein elsewhere, which can interact with gephyrin. The interaction between the peptide or peptide analogue with gephyrin directly competes with gephyrin mediated GlyR and GABAAR recruitment to post-synaptic sites and hence affects the frequency and the amplitude of IPSCs by acting on the diffusion dynamics of the selected receptor subtypes within the post-synaptic membrane. The invention therefore acts on the synaptic recruitment of a specific subset of GABAAR and GlyR subtypes. The peptide or peptide analogue as described herein can act as direct competitor to specific GABAAR and GlyR subtypes thereby accelerating the exchange rate of the respective desensitized receptor subtypes at post-synaptic sites. In other embodiments, the peptide or peptide analogue can act as direct competitor to specific

GABAAR and GlyR subtypes thereby uncoupling the respective receptor subtypes from synaptic sites and thus decreasing the frequency and amplitude of their fast synaptic inhibition.

The peptide or peptide analogue as described herein enters the neurons by diffusion and interacts with gephyrin. In a preferred embodiment, the peptide or peptide analogue is contacted with gephyrin within seconds after it has entered the neurons.

In one embodiment the method is a method to enhance GABAAR and GlyR mediated fast synaptic inhibition, wherein said method comprises contacting a composition comprising gephyrin binding peptides or peptide analogues with neurons in vitro or in vivo and wherein said peptides or peptide analogues are direct competitors of the gephyrin/GABA AR and gephyrin/GlyR interaction. In a preferred embodiment, the neurons are acute brain slices.

The peptide or peptide analogue comprised in the composition may be administered at nanomolar concentrations. The dimeric peptide comprised in the composition may be administered at picomolar concentrations. In one embodiment of the present invention, the method according reduces the synaptic GABAAR and/or GlyR concentration, thereby inhibiting GABAAR- and GlyR- mediated fast synaptic inhibition. In another embodiment, the method enhances the synaptic GABAAR and/or GlyR concentration, thereby inducing GABAAR- and GlyR- mediated fast synaptic inhibition.

In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue provided by the method does not alter the properties of GABAAR and GlyR. In another embodiment, the monomeric or dimeric peptide or peptide analogue provided by the method alters the gephyrin receptor clustering activity. In another embodiment, the monomeric or dimeric peptide or peptide analogue provided by the method alters GABAAR and/or GlyR clustering at post-synaptic sites. In one embodiment, the monomeric or dimeric peptide or peptide analogue provided by the method alters GABAAR and/or GlyR clustering at extra-synaptic sites.

In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue provided by the method alters gephyrin clustering. In another embodiment, the monomeric or dimeric peptide or peptide analogue provided by the method alters GABAAR and/or GlyR diffusion properties within the post-synaptic neuronal membrane. In one embodiment, the monomeric or dimeric peptide or peptide analogue provided by the method alters fast synaptic transmission by altering the exchange rate of desensitized GABAARs and/or GlyRs. In another embodiment, the monomeric or dimeric peptide or peptide analogue provided by the method alters the turnover rate of GABAARs and/or GlyRs.

In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue provided by the method alters the turnover of gephyrin.

In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue provided by the method alters the neuronal balance of fast synaptic inhibition and excitation.

In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue provided by the method alters the diffusion properties and/or exchange rates and and/or turnover rates of a specific subset of GABAARs and/or GlyRs subtypes.

In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue provided by the method specifically alters the diffusion properties

and/or exchange rates and and/or turnover rates of a specific subset of GABAARs and/or GlyRs subtypes.

In one embodiment of the present invention, the monomeric or dimeric peptide or peptide analogue provided by the method specifically alters the diffusion properties

and/or exchange rates and and/or turnover rates of GABAARs and/or GlyRs subtypes and/or gephyrin within a specific subset of neuronal cells and/or specific brain regions.

V. Method for isolation of postsynaptic proteome

One aspect of the present invention relates also to methods to isolate inhibitory postsynaptic proteome. In particular, the present compositions comprise peptides and peptide analogues that are capable of binding synaptic proteins. Said peptides and peptide analogues may be conjugated to a resin, wherein said resin is contacted with a mixture comprising proteins and thereby allow said peptides and peptide analogues to bind selectively to synaptic proteins. In one embodiment, the mixture comprising synaptic proteins is or is derived from a brain lysate. An example of said synaptic proteins is gephyrin. The resin can be selected from a group comprising iodoacetyl- activated beads. In one embodiment the peptide or peptide analogue is covalently linked to the resin. In a further embodiment, the covalent linkage between the resin and the peptide or peptide analogue is based on thiol-selective chemistry.

The synaptic proteins bound to the peptides or peptide analogues such as to the dimeric peptides may subsequently be eluted and the supernatant may be collected. Said supernatant may be analyzed, for example using electrophoresis and /or chromatography. The person skilled in the art knows suitable methods for analyzing the supernatant and optionally separating the different components comprised in the supernatant. Methods for further characterizing said components are also known in the art. The peptide or peptide analogue may be in a monomeric form or in a dimeric form as disclosed herein. The monomeric or dimeric peptide or peptide analogue has a structure as described in the sections "Peptide or peptide analogue" and/or "Dimeric peptide".

VI. Method for labelling of inhibitory synapses

An aspect of the present disclosure also provides a method for labeling of inhibitory synapses in living interconnected neurons in the form of primary hippocampal neurons, dissociated cultures and/or brain slices by contacting said neurons with a monomeric or dimeric peptide or peptide analogue as defined above conjugated with a detectable moiety. In a preferred embodiment, the living interconnected neurons are primary hippocampal neurons at day fourteen (Days in Vitro 14, DIV14, primary hippocampal neurons).

The living interconnected neurons may be fixated and permeabilized before being contacted with the peptide or peptide analogue of the invention.

In some embodiments of the present invention, the monomeric or dimeric peptide or peptide analogue as disclosed herein is conjugated to a detectable moiety. In other embodiments, said conjugated monomeric or dimeric peptide or peptide analogue is contacted with interconnected neurons and therefore binds to gephyrin. Microscopy can be used to visualize said gephyrin bound monomeric or dimeric peptide or peptide analogue, thanks to the presence of a conjugated moiety. In some embodiments, said conjugated moiety is a fluorophore such as 5/6 carboxy-tetramethyl rhodamine (TMR). Conventional fluorescence microscopy techniques can be used to visualize such fluorophores. In other embodiments, said conjugated moiety is a dye that can be used in super-resolution microscopy for example Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488 and ATTO 532. Super-resolution microscopy techniques can be used to visualize such dyes and their conjugates at the nanoscale dimension. Examples of super-resolution microscopy techniques are structured illumination microscopy, stimulated-emission-depletion fluorescence microscopy, photoactivation localization microscopy and stochastic optical reconstruction microscopy. In some embodiments of the present invention, the living neurons are contacted with the peptide or peptide analogue conjugated to a detectable moiety at picomolar or nanomolar concentrations. In a preferred embodiment, the concentration of the peptide or peptide analogue conjugated to a detectable moiety is between 0.05 and 50 nM.

The peptide or peptide analogue may be in a monomeric form or in a dimeric form as disclosed herein. The monomeric or dimeric peptide or peptide analogue has a structure as described in the sections "Peptide or peptide analogue" and/or "Dimeric peptide".

VII. Method for improving affinity of a peptide for gephyrin

One aspect of the present disclosure also provides a method for increasing the affinity of a peptide, in particular of a monomeric peptide, to gephyrin. This can be achieved by dimerizing said monomeric peptide.

The peptide or peptide analogue may have a structure and a sequence as described in the section "Peptide or peptide analogue".

In one embodiment of the present invention, the affinity of a first peptide for gephyrin is increased by linking said peptide to a second peptide via a linker. The linker may be as described herein above in the section "Linker". Linkers suitable for linking two peptides together are known in the art.

The affinity may increase at least 10-fold compared to the affinity of the monomeric peptide or peptide analogue for gephyrin, such as at least 25-fold, such as at least 50- fold, such as at least 100-fold, such as at least 250-fold, such as at least 500-fold, such as at least 750-fold, such as at least 1000-fold compared to the affinity of the monomeric peptide or peptide analogue.

Thus in some embodiments, the peptide or peptide analogue in a dimeric form is method has a K for gephyrin of less than 1 nM, such as below 900 pM, such as below 800 pM, such as below 700 pM, such as below 600 pM, such as below 500 pM, such as below 400 pM, such as below 300 pM, such as below 200 pM, such as below 100 pM. In some embodiments, the first peptide and the second peptide are identical. In other embodiments, the first peptide and the second peptide are different. The dimeric peptide may thus be a homodimer or a heterodimer. In some embodiments, the first peptide and the second peptide have different sequence of identical lengths. In other embodiments, the first peptide and the second peptide have identical sequences. In other embodiments, the first peptide and the second peptide have different sequences of different lengths.

In particular embodiments, the peptide has sequence BMPEG(FSIVGRYPRRRRC) 2.

In particular embodiments, the peptide has sequence BMPEG(FSIVGRYPRRRC) 2.

VIII. Method for manufacturing a peptide having high affinity for gephyrin

In one aspect, this invention relates to a method of manufacturing a peptide or peptide

analogue comprising or consisting of the sequence X SIX2GX 3X 4PX5X6X7X8 (SEQ ID NO: 26). The method can also be used for manufacturing a peptide or peptide analogue comprising or consisting of the sequence (SEQ ID NO: 33). Further, the method can be used for manufacturing a peptide or peptide analogue comprising or consisting of the sequence X G (SEQ ID NO: 37).

In another aspect the invention concerns a method of manufacturing a peptide or peptide analogue as disclosed herein comprising the following steps: a . providing a resin; b. providing a solution comprising amino acid residues;

c . coupling a first amino acid residue to the resin; d . coupling a second amino acid residue to the first one, and coupling each following amino acid residues to the previous one to form a peptide comprising at least 5 , e.g. at least 6 , such as at least 7 , e.g. at least 8 , such

as at lerast 9 , e.g. at least 10, such as at least 11 , e.g. at least 12 proteinogenic or non-proteinogenic amino acid residues as disclosed herein; e . providing cleavage of the so formed peptide from the resin. The method for manufacturing a peptide or peptide analogue of the present invention typically comprises the following steps: a . providing a resin; b. providing a solution comprising amino acid residues; c . coupling a first amino acid residue to the resin; d . coupling the other amino acid residues to the first one in a sequence as described herein; e . providing cleavage of the so formed peptide from the resin.

The peptides of the invention can be manufactured according to methods known by those of skill in the art.

Examples

Several experimental tests were performed to demonstrate the present invention and are described below. They refer to:

1. expression of gephyrin in E . coli and subsequent purification; 2 . profiling of the gephyrin/receptor interaction site by assessing the binding of 240 peptides derived from GlyR-beta (all the 20 different amino acids were tested at 12 of 15 positions of the native peptide); 3 . synthesis of the gephyrin binding peptides; 4 . quantification of the affinity of the synthesised peptides for gephyrin using isothermal titration calorimetry (ITC); 5 . production of dimeric peptides and quantification of their affinity for gephyrin using ITC; 6 . use of the synthesized peptides for selective isolation of gephyrin from brain lysate; 7 . assessment of the ability of the synthesized peptides to bind gephyrin in HEK 293 cells; 8 . assessment of the ability of the synthesized peptides to bind gephyrin at synaptic sites in neurons; 9 . assessment of the ability of the synthesised peptides to define the localization of gephyrin within neurons in the nanometer range; 10. assessment of the ability of the synthesized peptides to act on the amplitude and frequency of GlyR dependent IPSCs in brain slices; 11. a list of the sequences comprised in the present invention; 12. assessment of the ability of the synthesized peptides to act on the clustering

of GABAA receptors in living neurons; 13. assessment of the cytotoxicicity of the synthesized peptides; 14. assessment of the ability of the synthesised peptides to prevent gephyrin/receptor binding as well as to dissociate the gephyrin/receptor complex.

Example 1: Expression and purification of gephyrin

Full-length gephyrin (P2 splice variant residues 1-736) was expressed in E . coli and purified in a three-step purification. Concisely, the protein was initially purified via His- tag and subjected to size-exclusion chromatography (SEC) column (HiLoad 16/600 Superdex 200pg, GE Healthcare) on an AEKTA system (GE Healthcare) followed by a final ion-exchange chromatography, which allowed to separate higher oligomers of gephyrin from the primary trimeric homogenous gephyrin protein.

For the microarray experiments trimeric gephyrin was pooled as visualized via native PAGE. The E domain for gephyrin (GephE) (gephyrin P2 splice variant residues 318-

736) was expressed in E . Coli and purified in a two-step purification as described earlier. Concisely, the protein was purified using via Intein-tag (Chitin beads, New England Biolabs) and after self-cleavage the protein could be obtained by size- exclusion chromatography (SEC) column (HiLoad 16/600 Superdex 200pg, GE Healthcare) on an AKTA explorer system (GE Healthcare).

Conclusion : The E domain of gephyrin and full-length gephyrin (P2 splice variant residues 318-736 and 1-736) were successfully expressed in E.coli and successively purified. Example 2 : Systematic profiling of the gephyrin/receptor interaction site.

Specific sequence requirements of each amino acid for gephyrin binding were assessed using a 15-mer peptide sequence corresponding to GlyR beta (residues

397-41 1) known to bind gephyrin with an affinity of 13-17 µΜ (Marie H . M . 2014; Marie,

Kasaragod et al. 2014) as a starting point. Then, position - 1 to 10 (SEQ ID NO: 10) was probed with the 20 proteinogenic amino acids, resulting in 240 peptide variants using a

Celluspot µ ΓΓ format (µβΡΟΤ ) . To evaluate affinity in a semi-quantitative fashion, a sensitive self-reporting gephyrin-affinity-probe was generated by reacting recombinant gephyrin E domain with native HRP that has been activated by oxidation of its sugar moieties to aldehydes. After extensive washing bound GephE-HRP-conjugate was detected by chemiluminescence using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and the chemiluminescent bio-imaging system MicroChemi (DNR Bio-imaging Systems). The resulting dot-blots were analyzed using the array analyze software (Active Motif) which defines the error range of the each data set by comparing the intensities of each peptide duplicate on the analyzed array. Fig. 2 shows a representative membrane after incubation and evaluation. A clear correlation between position and nature of the amino acid was found: in positions 1-4, the native amino acid was in all cases clearly superior to any of other amino acids. In positions 5 , 7 and 8 , the native amino acid was also among the best peptide binders, but notably for the remaining five positions, -1, 0 , 6 , 9 and 10, there was marked increase in affinity by replacing the native amino acid with either Tyr (position - 1 , 0 and 6) or Arg (position 9 and 10).

Conclusion : The amino acids residues that play a key role for binding of a GlyR-beta derived peptide to gephyrin (E domain) were identified. These residues are: the native amino acids in positions 1-5, 7 and 8 ; tyrosine (Y) or arginine (R) in positions - 1 , 0 , 6 , 9 and 10. Example 3 : Peptide synthesis.

Based on the results of the profiling analysis, peptides composed of 8 to 14 amino acids were designed and tested. First, 8-mer peptides were prepared, which are known to comprise the minimal active native GlyR beta sequence (residues 398-405), 1a (SEQ ID NO: 2 , FSIVGSLP, K = 13 µΜ) and showed that affinity could be improved 3- fold by a Leu-to-Tyr mutation in position 6 (SEQ ID NO: 5 , FSIVGSYP,1d). Next, 12- mer peptides were designed and synthesized, using the native GlyR sequence,

FSIVGSLPRDFE (SEQ ID NO: 10, 1i, K = 8.3 µΜ) as a starting point. Introduction of 5 affinity-enhancing mutations, lead to peptide 1o (SEQ ID NO: 16, FSIVGRYPRRRR), which is the first nanomolar, monomeric gephyrin inhibitor with a K of 140 nM.

The peptides were manually synthesized by Fmoc-based solid-phase peptide synthesis (SPPS) at a 0.25 mmol scale on a MiniBlock (Mettler-Toledo, OH, USA) using a 2- chlorotrityl chloride resin (200-400 mesh, 1% DVB). After swelling the resin in dry Dichloromethane (DCM) for 15-30 min, the first amino acid was loaded by dissolving (4 equiv.) in dry DCM (1ml/g resin) and adding the solution to the resin with diisopropylethylamine (DIPEA) (8 equiv.), followed by agitation for 1 hr and washing of the resin with DCM. Capping of the resin was performed with DCM/MeOH/DIPEA

(17:2:1 , 3 ml_) for 3 1 min followed by washing with DCM and Dimethylformamide (DMF). Fmoc deprotection was carried out with 20% piperidine in DMF (2 2 min) washing with DMF in-between and after. Coupling steps were carried out using Λ/-[1 Η- benzotriazol-1-yl)(dimethylamino)methylene]-/\/-methyl-methanaminium hexafluorophosphate /V-oxide (HBTU) and DIPEA (resin/amino acid/HBTU/DIPEA, 1:4:4:8) or /V-[(dimethylamino)-1 H-1 ,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-/V- methylmethanaminium hexafluorophosphate /V-oxide (HATU) and 2,4,6- trimethylpyridine with a resin/linker/HATU/2,4,6-trimethylpyridine ratio of 1:2:2:3, in dry

DMF (3 ml_) for 30 min. Finally, the Fmoc group was removed from the terminal residue. Cleavage from the solid support was conducted by treatment of the resin for 2

hrwith a cleavage mixture containing TFA/Triisopropylsilane (TIPS) / H20 / Ethanedithiol / Thioanisol (90:2.5:2.5:2,5:2,5, 3 ml_). After evaporation in vacuum, cold ether precipitation and reverse phase HPLC purification, the peptides were lyophilised and characterized by LC-MS. All prepared peptides with exclusion of peptide 1m were water soluble at neutral pH in the millimolar range.

Conclusion : Super-binding peptides (SBPs) were synthesised, in particular the peptide having sequence FSIVGRYPRRRR (SEQ ID NO: 16, 1o) and nanomolar binding affinity to gephyrin (E domain).

Example 4 : The peptides have very high affinity to gephyrin.

Affinity of the synthetized peptides to gephyrin was measured with isothermal titration calorimetry (ITC) (Fig.3) . The affinity constants (K ) of each peptide are listed in Table 1.

Table 1. Affinity of the synthesized peptides to gephyrin. Selected peptides, were subsequently, labeled with the fluorophore TMR at the N- terminus. The peptides were N-terminally deprotected and on-resin coupled with the TMR fluorophore. The fluorescently-labeled version of 1o, 1t (TMR-FSIVGRYPRRRR), still showed nanomolar inhibition of gephyrin (K = 300 nM). The µβΡΟΤ array revealed the possibility to introduce arginine residues in the C-terminal gephyrin binding region while at the same time maintaining or even enhancing the overall peptide affinity. Thus, five additional arginine residues were added in the C-terminus of both 1o and 1p, resulting in 1r (SEQ ID NO: 19, FSIVGRYPRRRRRRRRR) and 1w* (SEQ ID NO: 20 +TMR, TMR-FSIVGRYPRRRRRRRRR) as potential cell-penetrating versions of the high-affinity peptide binders. Notably both 1o and 1r are nanomolar inhibitors of gephyrin with K values of 500 and 300 nM, respectively (Tab. 2).

Table 2. Affinity of the TMR-labeled synthesized peptides to gephyrin.

Conclusion : Some of the synthesized SBPs have significantly higher affinity for gephyrin compared to the wild type GlyR-beta-derived fragment (397-41 1). The high affinity was maintained even when a fluorescent group was conjugated to the peptides.

Example 5 : Dimeric SBPs have picomolar affinity for gephyrin.

Using the optimized monomeric peptide 1o* (SEQ ID NO: 23, FSIVGRYPRRRRC), a dimeric ligand, 1y* (BMPEG(FSIVGRYPRRRC) 2) , is prepared using a short polyethylene glycol (PEG) linker. The dimeric ligand 1y was tested in ITC and showed exceptional high affinity, which required a displacement measurement against another dimeric high-affinity gephyrin-binding peptide (1z, (BMPEG(FSIVGSLPRDFEC) 2 Fig. 4). This revealed that the affinity to gephyrin was in the picomolar range, with a K value of 200 ± 10 pM (Tab. 3) , thus having 1600-fold enhanced affinity compared to c oc

monomeric peptide 1i, and 40-fold more potent than a previously optimized dimeric ligand, 1z.

Table 3. Affinity of two dimeric peptides to gephyrin.

Conclusion : The affinity of a SBPs increased when 2 identical SBPs were conjugated, using a BMPEG linker, to form a dimer. The dimer displays an enhanced picomolar affinity towards gephyrin (E domain).

Example 6 : Use of SBP for quantitative and selective isolation of native gephyrin from brain lysate.

After cervical dislocation, whole brains from 54 weeks old C57BI/6J male mice were

removed from the scull and rapidly homogenized in 1 ml_ lysate buffer (20 mM Hepes, 100 mM KCH3COOH, 40 mM KCI, 5 mM EGTA, 5 mM MgCI2, 5 mM DTT, 1 mM PMSF, 1% Triton X , protease inhibitor Roche complete, pH 7.2) per 200 mg using a pistol homogenizer (8 strokes at 900 rpm). The homogenate was centrifuged at 10,000 x g for 15 min. Subsequently, the supernatant was removed and aliquots were flash- frozen in liquid nitrogen and stored at -80 °C. The peptide 1o was synthesized with an additional C-terminal Cys (1o*, SEQ ID NO: 23, FSIVGRYPRRRRC) and coupled to UltraLinklodoacetyl Gel (Thermo Scientific) according to the protocol of the manufacturer. The peptides were dissolved in coupling buffer (50 mM Tris, 5mM EDTA, pH 8.5) at a concentration of 1 mM and incubated for 2 hrs at RT with UltraLink beads, which had been washed and equilibrated with coupling buffer before. After removing excess peptides, the UltraLink beads were subjected to 1 mM cysteine for 2 hrs to quench possible unreacted iodoacetyl groups. The resin was washed three times and equilibrated with 1 M NaCI and stored at 4 °C. The resin with the immobilized peptides was incubated with whole brain lysate for 1 hr at 4 °C. After three washing steps with lysate buffer, the beads were boiled with Laemmli buffer containing 10% SDS. The supernatant was applied to gel electrophoresis and analyzed by western blotting. As a control, resin-bound 1p (SEQ ID NO: 17, FSIVRGYPRRRR) comprising a 'swapped', non-binding peptide moiety was employed. The cross-linked SBP 1o* but not 1p (where the amino acids in positions 4 and 5 were swapped) displayed an unmatched ability to recover native gephyrin from mouse brain in a remarkable selective manner (Fig. 5a). Western blotting additionally revealed that gephyrin was quantitatively removed by the resin-bound SBP from the whole brain homogenate (Fig. 5a) and immune-detection verified the absence of GlyRs at the gephyrin-peptide complexes (Fig 5b). The absence of GlyRs strongly suggest that the gephyrin SBPs compete effectively with bound GlyRs by targeting the same binding site on gephyrin, albeit more efficiently than GlyRs.

Conclusion : The SBPs display an extraordinary specific binding towards gephyrin which can be used to isolate gephyrin even from highly complex mixtures such as brain lysate in single step. Additionally, the affinity of the SBPs is sufficient to compete successfully with the binding of native gephyrin ligands such as the pentameric full-length GlyR.

Example 7 : Gephyrin SBPs target gephyrin in HEK cells.

A transfection-assay in HEK 293 cells, as originally used to identify gephyrin/receptor interactions, was used (Kneussel, Hermann et al. 1999), where co-expression of the GlyR-beta subunitwith gephyrin causes retention of the receptor on intracellular gephyrin aggregates (Kirsch, Kuhse et al. 1995; Meyer, Kirsch et al. 1995). The GFP- gephyrin fusion construct and its human embryonic kidney (HEK) 293 cells transfection and expression were prepared according to methods known in the art, for example (Fuhrmann, Kins et al. 2002). HEK 293 cells were cultured on 14 mm glass coverslips pre-coated with poly-L-ornithine (1.5 µg/ml) and transfected by calcium phosphate co- precipitation. Coverslips with the primary hippocampal or HEK 293 cells were washed with PBS and incubated with methanol at -20 °C for 10 minutes. After washing with PBS the fixed cells were blocked with 1% (w/v) bovine serum albumin (Applichem, Darmstadt, Germany). The GFP-gephyrin overexpressing HEK 293 cells were subjected to 20 nM of the fluorescent peptides and analyzed via confocal fluorescence microscopy (Fig. 6). To facilitate fluorescence detection of gephyrin, N-terminal GFP- tagged gephyrin was overexpressed so to produce intracellular aggregates, whereas overexpression of GFP alone was diffusely distributed throughout the cytoplasm of HEK 293 cells (Fig. 6a). GFP and the TMR-labeled peptides were directly visualized by auto-fluorescence.

TMR-labeled peptide derived from the GlyR, 1s (TMR- FSIVGSLPRDFE, SEQ ID NO: 10 +TMR), which displays micromolar affinity towards gephyrin, was compared with a control, TMR-labeled peptides 1u (TMR- FSIVRGYPRRRR, SEQ ID NO: 17 +TMR) /1w* (TMR- FSIVRGYPRRRRRRRRR, SEQ ID NO: 20 +TMR) where positions 6 and 7 were swapped to yield a non-binding peptide and two SBPs, 1t (TMR- FSIVGRYPRRRR, SEQ ID NO: 16 +TMR) and 1v (TMR-FSIVGRYPRRRRRRRRR, SEQ ID NO: 19 +TMR).

The analysis (Fig 6b) revealed that the fluorescently-labeled gephyrin SBPs 1t and 1v but not the non-binding peptide 1u, target gephyrin in HEK 293 cells. Furthermore, the native GlyR-beta subunit sequence 1s did not effectively target gephyrin, thus indicating that enhanced affinity of the SBPs is required for targeting gephyrin in a cellular context with externally applied peptides. This is also mirrored in the quantification of the co-localization for the different peptides with gephyrin, where 81- 87% of gephyrin was found to co-localize with the SBPs while the GlyR-derived peptides and the swapped negative control displayed less than 1% co-localization (Fig. 6b). Co-localization indices were determined using the MetaMorph Imaging Software, (Universal Imaging, West Chester, PA). For the quantification of co-localization in primary hippocampal cells the neuron nucleus was excluded. Co-localization was determined for 86 to 144 HEK 293 cells, the results were averaged and the root means square derivation was calculated.

Conclusion : Externally applied SBPs (1t and 1v) can target gephyrin and thus co-localize with the protein in HEK 293 cells. SBPs can therefore be used for detection of gephyrin instead of antibodies. Because of their small size, they allow detection of gephyrin at higher resolution than antibodies. In addition, they can be produced at lower costs and the staining protocol is simplified as it does not need fixation and permeabilization steps. Gephyrin acts as universal marker for the inhibitory synapse and hence its visualization is of general interest. Example 8 : SBPs target gephyrin at synaptic sites within neurons.

Immunohistochemical staining of endogenous gephyrin (rabbit polyclonal antibody, Alexis Biochemicals) and synaptophysin (guinea pig polyclonal antibody, Synaptic Systems) within primary neurons was compared with the staining resulting from a simple incubation with the gephyrin SBPs at nanomolar concentrations under identical conditions.

Primary cultures of hippocampal neurons were prepared from early postnatal mice or rats at primary culture and passage 1 and maintained as previously described (Fuhrmann, Kins et al. 2002). In brief, hippocampi were obtained from embryonic day 18 (E18) rat embryos (Wistar) and incubated with 0.5 mg/ml papain and 10 µg/ml DNAse I in PBS containing 10 mM glucose for 15 min at 37°C. After washing once in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal calf serum (Invitrogen), 25 µg/ml pyruvate, and 2 mM glutamine, cells were dissociated by trituration and seeded in DMEM containing supplements at a density of 30,000 cells per well onto 14 mm glass coverslips coated with poly-L-ornithine (1.5 µg/ml). After 3 hr, the medium was replaced with Neurobasal medium containing 2 mM glutamine, 25 µg/ml pyruvate, and 2% (v/v) B27 supplement (Invitrogen). All media contained 50 lU/ml penicillin and 50 µg/ml streptomycin (Invitrogen). At 3 d in vitro (DIV), 3 µΜ 1- beta-D-Darabinofuranosylcytosine was added to suppress astrocyte growth. Every week, one-third of the culture medium was exchanged for fresh medium.

For the quantification of co-localization in primary hippocampal cells the neuron nucleus was excluded. Co-localization was determined for 24 to 28 neurons, the results were averaged and the root means square derivation was calculated. The designed SBPs 1t (TMR-FSIVGRYPRRRR, SEQ ID NO: 16 +TMR) and 1v (TMR- FSIVGRYPRRRRRRRRR, SEQ ID NO: 19 +TMR) effectively stained synaptic gephyrin, in contrast to the corresponding native GlyR peptide sequence 1s (TMR- FSIVGSLPRDFE, SEQ ID NO: 10 +TMR) or the swapped variant 1w* (TMR- FSIVRGYPRRRRRRRRR, SEQ ID NO: 16 +TMR) (Fig. 7a). The indirect antibody- based staining of gephyrin resulted in a largely overlapping labeling compared to our direct fluorescent gephyrin SBP labeling (Fig. 7a). Specifically, it was found that gephyrin co-localizes with the SBP 1t 49 ± 18 % and with SBP 1v 69 ± 17 % , while the GlyR derived fragment 1s and the point mutated negative control 1w* show less than 7 ± 3 % co-localization with gephyrin (Fig.7b). Additionally, the SBP marked gephyrin clusters appear primarily synaptic as they co-localize with the synaptic marker synaptophysin (Fig. 7). Co-localization indices were determined using the MetaMorph Imaging Software, (Universal Imaging, West Chester, PA).

Conclusion : Fluorescently labeled SBPs can successfully stain endogenous gephyrin in neurons (immuonohistochemical staining was used as control). The results also indicate that gephyrin-SBPs were localized as synaptic site (immunohistochemical staining of synaptophysin was used as control). Gephyrin acts as an universal marker for the inhibitory synapses. In contrast to conventional immunological approaches to visualize inhibitory synapses the SBPs can be produced at lower costs and do not necessitate fixation or permeabilization steps and hence can be applied in live imaging.

Example 9 : SBPs can be used in super-resolution imaging techniques to visualize inhibitory post-synaptic sites.

Based on the smaller size of the SBPs compared to antibodies and because of their stoichiometric binding, the SBPs can be used in higher resolution imaging techniques such as structured illumination microscopy (SIM).

Primary cultures of hippocampal neurons were prepared as described in example 8 . PFA fixed cells (DIV14 or DIV21 neurons) were permeabilized with 0.1% Triton X-100 for 10 min. After washing with PBS cells were blocked with 5% bovine serum albumin and 40 nM swapped variant 1w* (TMR- FSIVRGYPRRRRRRRRR, SEQ ID NO: 20 +TMR), followed by incubation with primary antibody (overnight at 4 °C) and secondary antibody (2 h at room temperature) or gephyrin peptide 1v (TMR- FSIVGRYPRRRRRRRRR, SEQ ID NO: 19 +TMR) (2 nM for 13 min at room temperature) in blocking buffer.

Manufacturer and dilution of the used antibodies: Primary Antibodies directed against: phosphorylated gephyrin (mouse monoclonal antibody, clone mAb7a, Synaptic Systems (SySy), dilution 1:200, VGAT (guinea pig polyclonal antibody, SySy, Cat. No. 131 004, dilution 1:2000), VGLUT1 (guinea pig polyclonal antibody, SySy, Cat. No. 135 303, dilution 1:5000). Alexa Fluor 647 conjugated anti-guinea pig IgG (Life Technologies, dilution 1:200), Alexa Fluor 647 conjugated anti-rabbit IgG (Life Technologies, dilution 1:200), Alexa Fluor 532 conjugated goat anti-mouse (Life Technologies, dilution 1:200).

We compared the 1v (TMR-FSIVGRYPRRRRRRRRR, SEQ ID NO: 19 +TMR) labeling, with a conventional antibody-based labeling of gephyrin (Fig. 8a) and other established markers for GABAergic (Fig. 8b) and glutamatergic synapses (Fig. 8c) in hippocampal mouse neurons. The co-localization of 1v (TMR-FSIVGRYPRRRRRRRRR, SEQ ID NO: 19 +TMR) with VGAT is clearly visible in DIV21 neurons (Fig. 8b), whereas there is no co-localization of 1v (TMR-FSIVGRYPRRRRRRRRR, SEQ ID NO: 19 +TMR) and VGLUT1 (Fig. 8c), and neither for the control TMR-2j with VGAT (Fig. 8d).

Conclusion : As SIM provides a lateral resolution beyond the size of typical inhibitory synapses, the spatial difference between post- and presynaptic localizations for peptide and VGAT is clearly visible in all SIM images and allows only limited overlap of pre- and postsynaptic markers. The SBPs display a more confined labeling of gephyrin than the conventional immunogenic approach and most likely are better representations of the molecular clusters whose size is below the SIM resolution limit.

Example 10: SBPs inhibit GlyR-dependent IPSCs.

Coronal slices were prepared from medulla oblongata (ventral for caudal half-part of cerebellum and rostral for spinal cord), and cells were recorded from an area corresponding to the gigantocellular reticular nucleus (assessed by relative position to cerebellum). Neurons recorded therefrom were indeed large with soma ranges of 12-15 urn, and capacitance from 14-18 pS. Neurons were recorded in artificial cerebrospinal fluid (ACSF) containing 2 mM kynurenic acid, 10 uM SR95531 (gabazine), 1 uM

CGP54626 (GABA B antagonist), 1 uM TTX, Ca/Mg ratio 0.5/3.5 and at 34 °C. Intracellular solution was high chloride, CsCI based. Glycine mediated spontaneous inhibitory postsynaptic currents (IPSCs) were characterized by high amplitude and very fast decay as well as sensitivity to bath applied strychnine. Typical waveforms of the averaged, non-contaminated IPSC were determined as well as channel conductance and average number of channels per release site (Fig. 9). Values for conductance fall in an interval of most probable conductance (range 85 - 111 pS), dependent on subunit stoichiometry. Recordings began immediately after establishment of the whole cell configuration with 60 s average amplitude of non-contaminated IPSCs in a 600 s long recording. Inclusion in the postsynaptic patch pipette of 0.2 or 2 µΜ SBP 1v (TMR- FSIVGRYPRRRRRRRRR, SEQ ID NO: 19 +TMR), acutely resulted in inhibited GlyR- mediated IPSCs (Fig. 9). The measurements visualized a dose-dependent effect of the intracellular gephyrin-interacting SBPs on the amplitude and also frequency of strychnine sensitive IPSCs when administered in the intracellular solution. Notably, there was no significant IPSC alternation in controls without ligand. At lower

concentrations of SBP v (0.02 µΜ) , the GlyR IPSC inhibition was lost (Fig. 9).

Conclusion : The SBPs specifically act on the frequency and amplitude of inhibitory IPSCs such as glycinergic transmission but do not alter AMPAR related currents. The SBPs show functionality at doses of 0.2 or 2 µΜ.

Example 1 1 : The SBPs to act on GABAA receptor clustering in living neurons

To analyze synaptic GABAAR a2 levels, separate signals in co-localization with the presynaptic marker protein SV2 (magenta signals) were determined using the

"threshold image" function and quantified. Non-synaptic GABAAR a2 levels were defined as non-co-localizing clusters with SV2. Receptor cluster number and sizes were assessed by Metamorph-based analysis using the "Integrated Morphometry Analysis" tool, calculating the number, area, perimeter and radius of single objects. (Fig 10). In total 96 dendritic regions out of 32 neurons from 3 independent experiments were analyzed per treatment group. Statistical analysis was performed with Microsoft Excel or SPSS 18.0 (SPSS Inc.). Statistical significance was assessed with one or two way ANOVA. All values from quantitative data represent the mean s.e.m. from n independent experiments.

DIV12 neurons ere incubated with 2µΜ 1r (FSIVGRYPRRRRRRRRR, SEQ ID NO: 19) or the non-binding control peptide 1w (FSIVRGYPRRRRRRRRR, SEQ ID NO: 20) for

20 h . Subsequently, restricted dendritic areas of single neurons were defined and investigated. Upon incubation with 2µΜ 1r but not the negative control peptide 1w (FSIVRGYPRRRRRRRRR, SEQ ID NO: 20) frequencies of all (small and large)

GABAAR a2 cluster populations were reduced. (Fig. 10a-b) The ratio of synaptic to α extra-synaptic 2 containing GABAARs is altered upon incubation with 1r, but not the negative control 1w (Fig. 10c).

Conclusion :

Exposure to the SBPs redistributes the a2 subunit containing GABAARs and, at the same time, changes their aggregation behaviour. Exposure to SBPs results in a reduced clustering of the receptors and that the receptors are shifted from synaptic to non-synaptic sites.

Example 12: Assessment of the SBP toxicology

Glycine mediated spontaneous inhibitory postsynaptic currents (IPSCs) were characterized in the same fashion as described in example 10. Application of the SBPs at micromolar concentrations, and hence 1000 fold concentrated compared to the visualization approaches, did not interfere with healthy neuronal function such as glutamergic transmission. (Fig. 11a-b). Neuronal viability was analysed in the presence of the SBPs by monitoring the cell membrane integrity by the release of lactate dehydrogenase (LDH) of dying neurons. When applied at 20 µΜ for up to 48 hours the SBPs induce less than 1.5% toxicity (relative to 1 % TX-100 application) (Fig. 11c). Moreover, application of the SBPs at concentrations as high as 20 µΜ leads to a limited decrease (5-10%) in cell viability as compared to control (vehicle only) treatment. (Fig 11d)

Conclusion :

The cell viability and toxicology assays and the assessment of the AMPAR mediated currents in presence of SBPs indicate that the SBPs show no cytotoxic effects at pharmacological relevant concentrations.

Example 13: The SBPs directly compete with GABAARs and GlyRs for gephyrin binding.

The affinity of the SBPs, GABAARs and GlyRs to gephyrin was measured with isothermal titration calorimetry (ITC) in presence and absence of stoichiometric amounts of the SBPs, GlyRs or GABAARs. We found that stoichiometric pre- equilibration of gephyrin with 1r (FSIVGRYPRRRRRRRRR, SEQ ID NO: 19) abolishes gephyrin binding to the native receptor subunit fragments comprising the highest β reported gephyrin affinities, namely the GlyR and GABAAR a3 subunits (Fig. 12 a-b).

The SBPs also uncouple preformed gephyrin/GlyR or gephyrin/GABA AR complexes 5 (Fig. 12c).

Conclusion : The competition experiments demonstrate that the SBPs occupy an overlapping binding site with the receptor fragments and that the enhanced affinity of the SBPs

0 outcompetes GABAAR and GlyR derived fragments, respectively. The ITC studies demonstrate that the SBPs dissociate the preformed receptor/gephyrin complexes by

direct competition and that GABAARs can be uncoupled at lower SPB doses than GlyRs due to their reduced gephyrin binding affinity.

5 Example 14: Sequences

SEQ ID NAME ORGANISM SEQUENCE Notes NO: SEQ ID GlyR-beta (397- SPOT DFSIVGSLPRDFELS NO: 1 4 1 1) SEQ ID GlyR-beta (398- 1a FSIVGSLP NO: 2 405) SEQ ID 1b Artificial FSIVRSLP NO: 3 SEQ ID 1c Artificial FSIVGRLP NO: 4 SEQ ID 1d Artificial FSIVGSYP NO: 5 SEQ ID 1e Artificial FSIVGRYY NO: 6 SEQ ID 1f Artificial YSIVGSLP NO: 7 SEQ ID 1g Artificial YFSIVGSYP NO: 8 SEQ ID 1h Artificial YYSIVGSYP NO: 9 SEQ ID GlyR-beta (398- 1i FSIVGSLPRDFE +TMR(1s) NO: 10 409) SEQ ID Artificial FSIVGSLYRDFE 1j NO: 11 SEQ ID 1k Artificial FSIVGSLPRRRR NO: 12 SEQ ID 1 1 Artificial FSIVGSYYRRRR NO: 13 SEQ ID GlyR-beta (397- 1m DFSIVGSLPRDFE NO: 14 409) SEQ ID 1n Artificial YYSIVGRYYRRRR NO: 15 SEQ ID +T R (1t) 1o Artificial FSIVGRYPRRRR NO: 16 SEQ ID p Artificial FSIVRGYPRRRR +T R(1u) NO: 17 SEQ ID GlyR-beta (398- q FSIVGSLPRDFELS NO: 18 4 1 1) SEQ ID r Artificial FSIVGRYPRRRRRRRRR +T R(1v) NO: 19 SEQ ID w Artificial FSIVRGYPRRRRRRRRR + T R (1w*) NO: 20 SEQ ID GABA R-alpha3 x A FNIVGTTYPINL NO: 2 1 (368-379) SEQ ID 2 peptides having this sequence in y Artificial FSIVGRYPRRRC NO: 22 the dimer with B PEG ( 1 y*) SEQ ID o* Artificial FSIVGRYPRRRRC +Dimer with DA/DA-PEG(2a-c) NO: 23 SEQ ID * Artificial FSIVRGYPRRRRC NO: 24 SEQ ID GlyR-beta (398- i* FSIVGSLPRDFEC + in dimer with BMPEG(1 z) NO: 25 409)

is selected from F, Y ; X2 is selected from V , I; X is selected SEQ ID from R , S ; is selected from L , Y ; Artificial X S IX2 X3X PX5X X X NO: 26 X5, X and X are individually selected from R and K ; X is selected from R , K and C.

is selected from F, Y ; X2 is selected from V , I; X is selected from R , S ; is selected from L , Y ; SEQ ID X1SIX2GX3X4PX5X6X7X8X9X1 0X 11X 2 Artificial X , X and X are individually NO: 27 5 selected from R and K ; X is

selected from R , K and C ; X -X 2 are individually selected from R and K . is selected from F, Y ; X is SEQ ID 2 Artificial X1SIX2GX3X4P selected from V , I; X is selected NO: 28 from R , S ; is selected from L , Y ;

is selected from F, Y ; X2 is selected from V , I; X is selected SEQ ID from R , S ; is selected from L , Y ; Artificial X SIX2GX3X4PX5X6X7X8C NO: 29 X5, X and X are individually selected from R and K ; X is selected from R , K and C.

X is selected from F, Y ; X2 is selected from V , I; X is selected from R , S ; is selected from L , Y ; SEQ ID X SIX2GX3X4PX5X6X7X8X9X10X1 1X12C Artificial X , X and X are individually NO: 30 5 selected from R and K ; X is

selected from R , K and C ; X -X 2 are individually selected from R and K . SEQ ID Artificial RRRRR CPP NO: 3 1 SEQ ID Artificial RRRRRC CPP+Cysteine NO: 32 X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(?-butyl)hydrocinnamic acid, 4- (trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4- SEQ ID Artificial X X2X3X4X5X6X7PX8X9X1 0X1 1 tetrahydro-2-naphthoic acid, 4- NO: 33 pyridinepropionic acid, 1-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, d-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid

X2 and X are individually selected from serine (S), arginine (R), N - alpha-methyl-O-i-butyl-L-serine, /V,/V'-bis-?-butyloxycarbonyl-2-amino- 3-guanidino-propionic acid, /V-beta-f- butyloxycarbonyl - L-2,3- diaminopropionic acid X and are individually selected from valine (V), isoleucine (I), leucine (L), 3-aminopentane-3- carboxylic acid, L-cyclopentylglycine, L-cyclopropylglycine, L- cyclohexylglycine, L-Meucine, L- neopentylglycine, /V-alpha-methyl -L- isoleucine, /V-alpha-methyl - L-valine, allylglycine, L-phenylglycine, N- alpha-methyl - L-leucine, L- cyclohexylalanine

X 5 is selected from glycine (G), aminooxyacetic acid, sarcosine X is selected from valine (V), isoleucine (I), leucine (L), tyrosine (Y), 3-aminopentane-3-carboxylic acid, L-cyclopentylglycine, L- cyclopropylglycine, L- cyclohexylglycine, L-Meucine, L- neopentylglycine, /V-alpha-methyl -L- isoleucine, /V-alpha-methyl - L-valine, allylglycine, L-phenylglycine, N- alpha-methyl - L-leucine, L- cyclohexylalanine X , X and X are individually selected from arginine (R) and lysine (K) X11 is selected from arginine (R), lysine (K) and cysteine (C).

SEQ ID X1X2X3X4X5X6X7PX8X9X10X11 are Artificial NO: 34 X X 2X 3 X 5X6 9 0X defined as for SEQ ID NO: 33. X1X2X3X4X5X6X7PX8X9X10X11 are SEQ ID defined as for SEQ ID NO: 33. Artificial X X 2X 3X4X 5X X X 8X 9X 0X11 X 2X NO: 35 X12X13X14X15 are individually selected from R and K . X1X2X3X4X5X6X7PX8X9X10X11 are SEQ ID defined as for SEQ ID NO: 33. Artificial X X 2X 3X4X 5X X X 8X 9X 0X X 2X 3X 14X NO: 36 X12X13X14X15 are individually selected from R and K . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(?-butyl)hydrocinnamic acid, 4- (trifluoromethyl)hydrocinnamic acid, 3-(3,4- methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1- naphthaleneacetic acid, 1,2,3,4- tetrahydro-2-naphthoic acid, 4- pyridinepropionic acid, L-1 ,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, D-1 ,2,3,4- SEQ ID Artificial X1SIX2G tetrahydroisoquinoline-3-carboxylic NO: 37 acid;

X 2 is selected from valine (V), isoleucine (I), leucine (L), 3- aminopentane-3-carboxylic acid, L- cyclopentylglycine, L- cyclopropylglycine, L- cyclohexylglycine, L-Meucine, L- neopentylglycine, /V-alpha-methyl-L- isoleucine, /V-alpha-methyl-L-valine, allylglycine, L-phenylglycine, N- alpha-methyl-L-leucine, L- cyclohexylalanine Brady, M . L . and T. C. Jacob (2015). "Synaptic localization of alpha 5 GABA (A) receptors via gephyrin interaction regulates dendritic outgrowth and spine maturation." Developmental Neurobiology 75(1 1): 1241-51 . Craig, A . M., G . Banker, et al. (1996). "Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons." The Journal of neuroscience: the official journal of the Society for Neuroscience 16(10): 3166-3177. Defeudis, F. V. Bilobalide and neuroprotection. Pharmacol. Res. 46, 565-568 (2002).

Feng G., H . Tintrup, J. Kirsch, M.C. Nichol, J. Kuhse, H . Betz and J.R. Sanes (1998). "Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity." Science 282: 1321-4. Fuhrmann, J. C , S . Kins, et al. (2002). "Gephyrin interacts with Dynein light chains 1 and 2 , components of motor protein complexes." The Journal of neuroscience: the official journal of the Society for Neuroscience 22(13): 5393- 5402. Fernandez, F. et al. Pharmacotherapy for cognitive impairment in a mouse model of Down syndrome. Nat Neurosci 10, 4 11-413, doi:10.1038/nn1860 (2007). Gross, G . G . et al. Recombinant probes for visualizing endogenous synaptic proteins in living neurons. Neuron 78, 971-985, doi:10.1016/j.neuron.2013.04.017 (2013). Gross, G . G . et al. An E3-ligase-based method for ablating inhibitory synapses, Nat. Methods, 13, 673-678 (2016). 9 . Hines, R . M., P. A . Davies, et al. (2012). "Functional regulation of GABAA receptors in nervous system pathologies." Current opinion in neurobiology 22(3): 552-558. 10. Kiewert, C. et al. Role of glycine receptors and glycine release for the neuroprotective activity of bilobalide. Brain Res. 1201 , 143-150 (2008).

11. Kim, E . Y., N . Schrader, et al. (2006). "Deciphering the structural framework of glycine receptor anchoring by gephyrin." The EM BO journal 25(6): 1385-1395. 12. Kirsch, J., J. Kuhse, et al. (1995). "Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells." Molecular and cellular neurosciences 6(5): 450-461 .

13. Kirsch J, I. Wolters, A . Triller and H . Betz (1993). "Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons." Nature 366: 745-748.

14. Kneussel, M . , J.H. Brandstatter, B. Laube, S . Stahl, U. Muller and H . Betz (1999). "Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice." J. Neurosci. 19: 9289-97. 15. Kneussel, M., A . Hermann, et al. (1999). "Hydrophobic interactions mediate binding of the glycine receptor beta-subunit to gephyrin." Journal of neurochemistry 72(3): 1323-1326.

16. Langosch D., W. Hoch and H . Betz (1992). "The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase." FEBS L 298: 113-1 17.

17. Maas C , N . Tagnaouti, S . Loebrich, B. Behrend, C . Lappe-Siefke and M . Kneussel (2006). "Neuronal cotransport of glycine receptor and the scaffold protein gephyrin." The Journal of Cell Biology 172(3): 441-451 .

18. Marie H . M . , K . V . B., Haugaard-Kedstrom L . , Torben J. H . , Kneussel M . ,

Schindelin H . , Stromgaard K . (2015). "Design and synthesis of high-affinity dimeric inhibitors targeting the interactions between gephyrin and inhibitory neurotransmitter receptors." Angewandte Chemie International Edition 54(2): 490-4.

19. Marie, H . M., J. Mukherjee, et al. (201 1). "Gephyrin-mediated gamma- aminobutyric acid type A and glycine receptor clustering relies on a common binding site." The Journal of biological chemistry 286(49): 42105-421 14.

20. Marie, H . M., V. B. Kasaragod, et al. (2014). "Modulation of gephyrin-glycine receptor affinity by multivalency." ACS Chemical Biology 9(1 1): 2554-62. Marie, H . M., V. B. Kasaragod, et al. (2014). "Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin." Nature communications 22(5): 5767. Meyer, G., J. Kirsch, et al. (1995). "Identification of a gephyrin binding motif on the glycine receptor beta subunit." Neuron 15(3): 563-572. Ruby, N . F. et al. Hippocampal-dependent learning requires a functional circadian system. Proc. Natl. Acad. Sci. USA 105, 15593-15598 (2008).

Saiyed T., I. Paarmann, B. Schmitt.S. Haeger, M . Sola, G . Schmalzing, W.

Weissenhorn and H . Betz (2007) . "Molecular basis of gephyrin clustering at inhibitory synapses: role of G- and E-domain interactions." J Biol Chem 282: 5625-5632.

Specht C.G., I. Izeddin, P.C. Rodriguez, M . El Beheiry, P. Rostaing, X . Darzacq, M . Dahan and A . Triller (2013). "Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites." Neuron 79(2): 308-321. Troy, D. B. and Beringer P. (2005). "Remington: The Science and Practice of Pharmacy." Lippincott, Williams & Wilkins.

Tyagarajan, S . K . and J. M . Fritschy (2014). "Gephyrin: a master regulator of neuronal function?" Nature reviews. Neuroscience 15(3): 141-156.

Yu W, M . Jiang, CP. Miralles, R.-W. Li, G . Chen and A.L. De Bias (2007). "Gephyrin clustering is required for the stability of GABAergic synapses." Mol Cell Neurosci. 36(4): 484-500.

Zacchi P., E . Dreosti, M . Visintin, M . Moretto-Zita, I. Marchionni, I. Cannistraci,

Z . Kasap, H . Betz, A . Cattaneo and E . Cherubini (2008). "Gephyrin selective intrabodies as a new strategy for studying inhibitory receptor clustering." Journal of Molecular Neuroscience 34(2): 141-148.Zhang, X., LI, S., BAO, L , Ye, Y. & Yao, S . Substances for treatment or relief of pain, US patent 201 50374705 A 1 (2013) Claims

A peptide or peptide analogue (P^ comprising at least 12 amino acid residues

of the sequence X SIX2GX3X4P 5 6 7 8 (SEQ ID NO: 26), wherein: a . X is selected from phenylalanine (F), tyrosine (Y)

b. X2 is selected from valine (V), isoleucine (I)

c . X3 is selected from arginine (R), serine (S) d . X is selected from leucine (L), tyrosine (Y)

e . X5, X6 and X7 are individually selected from arginine (R) and lysine (K)

f. X8 is selected from arginine (R), lysine (K) and cysteine (C).

A peptide or peptide analogue (P^ comprising at least 12 proteinogenic or non-proteinogenic amino acid residues comprising or consisting of the sequence (SEQ ID NO: 33), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4-methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1-naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4- pyridinepropionic acid, l-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, d-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

b. X 2 and X 6 are individually selected from serine (S), arginine (R), N- alpha-methyl-O-f-butyl-L-serine, /V,/V'-bis-f-butyloxycarbonyl-2-amino-3-

guanidino-propionic acid, /V-beta-f-butyloxycarbonyl-L-2,3- diaminopropionic acid

/V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-valine, allylglycine, L-

phenylglycine, /V-alpha-methyl-L-leucine, L-cyclohexylalanine

d . X 5 is selected from glycine (G), aminooxyacetic acid, sarcosine

/V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-valine, allylglycine, L-

phenylglycine, /V-alpha-methyl-L-leucine, L-cyclohexylalanine f. X , X g and X are individually selected from arginine (R) and lysine (K) 8 0 g . X is selected from arginine (R), lysine (K) and cysteine (C).

A biologically active peptide or peptide analogue (P^ comprising at least five proteinogenic or non-proteinogenic amino acid residues comprising or consisting of the sequence X G (SEQ ID NO: 37), wherein: a . X is selected from phenylalanine (F), tyrosine (Y), 3-phenylpropanoic acid, alpha-(f-butyl)hydrocinnamic acid, 4-(trifluoromethyl)hydrocinnamic acid, 3-(3,4-methylenedioxyphenyl)propionic acid, 3,3-diphenylpropionic acid, 1-naphthaleneacetic acid, 1,2,3,4-tetrahydro-2-naphthoic acid, 4-

pyridinepropionic acid, L-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic

acid, D-1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

b. X2 is selected from valine (V), isoleucine (I), leucine (L), 3- aminopentane-3-carboxylic acid, L-cyclopentylglycine, L- cyclopropylglycine, L-cyclohexylglycine, L-Meucine, L-neopentylglycine,

/V-alpha-methyl-L-isoleucine, /V-alpha-methyl-L-valine, allylglycine, L- phenylglycine, /V-alpha-methyl-L-leucine, L-cyclohexylalanine; wherein the biological activity is capability to act as a ligand of gephyrin.

The peptide or peptide analogue according to any one of the preceding claims, wherein said at least five amino acid residues constitute the five N-terminal residues of the peptide or peptide analogue.

The peptide or peptide analogue according to any one of the preceding claims, wherein the sequence of the eight N-terminal amino acids is

X 1SIX2GX3X4P (SEQ ID NO: 28).

The peptide or peptide analogue according to any one of the preceding claims, wherein said at least twelve amino acid residues constitute the twelve N-terminal residues of the peptide or peptide analogue.

The peptide or peptide analogue according to any one of the preceding claims, wherein the sequence of the twelve N-terminal amino acids is

X SIX2GX3X4PX5X6X7X8 (SEQ ID NO: 26) . The peptide or peptide analogue according to any one of the preceding

claims wherein P comprises or consists of a sequence X SIX GX X4P 5 6 8 9 o 2 (SEQ ID NO: 27), wherein X , X , X 2 3 9 0 K . and X 2 are individually selected from R and

The peptide or peptide analogue according to any one of the preceding

claims, wherein P comprises between 12 and 25 amino acid residues.

10. The peptide or peptide analogue according to any one of the preceding

claims, wherein P comprises between at least 13 amino acid residues, such as at least 14 amino acid residues, e.g. at least 15 amino acid residues, such as at least 16 amino acid residues, e.g. at least 17 amino acid residues, such as at least 18 amino acid residues, e.g. at least 19 amino acid residues, such as at least 20 amino acid residues, e.g. at least 2 1 amino acid residues, such as at least 22 amino acid residues, e.g. at least 23 amino acid residues, such as at least 24 amino acid residues, e.g. 25 amino acid residues.

11. The peptide or peptide analogue according to any one of the preceding claims, further comprising a conjugated moiety.

12. The peptide or peptide analogue according to any one of the preceding claims, wherein the conjugated moiety is selected from the group consisting of a Cell Penetrating Peptide (CPP), an Albumin Binding Moiety (ABM), a detectable moiety (J) and a linker (L).

13. The peptide or peptide analogue according to any one of the preceding claims, wherein the conjugated moiety is a CPP.

14. The peptide or peptide analogue according to any one of the preceding claims, wherein the CPP comprises at least four amino acid residues

individually selected from R and K .

15. The peptide or peptide analogue according to any one of the preceding claims, wherein the CPP comprises five arginine residues.

16. The peptide or peptide analogue according to any one of the preceding claims, further comprising a C-terminal cysteine residue. 17 . The peptide or peptide analogue according to any one of the preceding claims, wherein said peptide or peptide analogue has the generic structure of Formula (I):

L-Y Formula (I)

thus generating a dimeric peptide comprising a first peptide or peptide

analogue (P^ and a second peptide or peptide analogue (P2) linked together via the linker (L), wherein (L) is optionally substituted with a moiety Y.

18 . The peptide or peptide analogue according to any one of the preceding

claims, wherein P is identical to P2.

19 . The peptide or peptide analogue according to any one of the preceding

claims, wherein the number of amino acid residues of P is identical to the

number of amino acids of P2.

20. The peptide or peptide analogue according to any one of the preceding claims, wherein the dimer is a homodimer.

21. The peptide or peptide analogue according to any one of the preceding claims, wherein the dimer is a heterodimer.

22. The peptide or peptide analogue according to any one of the preceding claims, wherein (L) comprises or consists of an alkane chain, diaminoacetic acid, maleimide, ethylene glycol, PEG, Λ/PEG or any combination thereof.

23. The peptide or peptide analogue according to any one of the preceding claims, wherein (L) comprises at least 2 maleimide moieties and at least 2 ethylene glycol moieties. 73 74 25. The peptide or peptide analogue according to claim 24, wherein (Y) has the structure of formula (II):

Formula (II)

wherein (Z) is a CPP and/or a detectable moiety (J).

26. The peptide or peptide analogue according to any one of the preceding claims, wherein (J) is conjugated to the C-terminus of a CPP conjugated to the linker thus having the generic structure of formula (III):

CPP — J Formula (III)

27. The peptide or peptide analogue according to any one of the preceding claims, further comprising a moiety (Z) conjugated to the C-terminus of

and/or P2 via an amide bond as in the generic structure of formula (IV):

Formula (IV)

wherein (Z) is a CPP and/or a detectable moiety (J). 28. The peptide or peptide analogue according to any one of the preceding claims, wherein (J) is conjugated to the monomeric or to the dimeric peptide or peptide analogue.

29. The peptide or peptide analogue according to any one of the preceding claims, wherein (J) is conjugated to the N-terminus of the peptide or peptide analogue.

30. The peptide or peptide analogue according to any one of the preceding claims, wherein (J) is a fluorophore.

3 1. The peptide or peptide analogue according to any one of the preceding claims, wherein (J) is a fluorophore selected from the group consisting of Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488 and ATTO 532 and 5/6 carboxy-tetramethyl rhodamine (TMR), 6-carboxyfluorescein (6- FAM), Alexa Fluor® 350, DY-415, ATTO 425, ATTO 465, Bodipy® FL, fluorescein isothiocyanate, Oregon Green® 488, Oregon Green® 514, Rhodamine Green™, 5'-Tetrachloro-Fluorescein, ATTO 520, 6-carboxy-4',5'- dichloro-2',7'-dimethoxyfluoresceine, Yakima Yellow™ dyes, Bodipy® 530/550, hexachloro-fluorescein, Alexa Fluor® 555, DY-549, Bodipy® TMR-X, cyanine phosphoramidites (cyanine 3 , cyanine 3.5, cyanine 5 , cyanine 5.5), ATTO 550, Rhodamine Red™, ATTO 565, Carboxy-X-Rhodamine, Texas Red (Sulforhodamine 101 acid chloride), LightCycler® Red 610, ATTO 594, DY-480-XL, DY-610, ATTO 610, LightCycler® Red 640, Bodipy 630/650, ATTO 633, Bodipy 650/665, ATTO 647N, DY-649, LightCycler® Red 670, ATTO 680, LightCycler® Red 705, DY-682, ATTO 700, ATTO 740, DY-782, IRD 700 and IRD 800, CAL Fluor® Gold 540 nm, CAL Fluor® Gold 522 nm, CAL Fluor® Gold 544 nm , CAL Fluor® Orange 560 nm, CAL Fluor® Orange 538 nm, CAL Fluor® Orange 559 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 569 nm, CAL Fluor® Red 591 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 635 nm, Quasar® 570 nm, Quasar® 548 nm, Quasar® 566 nm (Cy 3), Quasar® 670 nm, Quasar® 647 nm, Quasar® 670 nm (Cy 5), Quasar® 705 nm, Quasar® 690 nm, Quasar® 705 nm (Cy 5.5), Pulsar® 650 Dyes, SuperRox® Dyes. 32. The peptide or peptide analogue according to any one of the preceding claims, wherein (J) is a dye selected from a group consisting of Alexa Fluor* 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488 and ATTO 532 is conjugated to the C-terminal end of the monomeric or dimeric peptide or peptide analogue.

33. The peptide or peptide analogue according to any one of the preceding claims, wherein (J) is TMR.

34. The peptide or peptide analogue according to any one of the preceding claims, wherein TMR is conjugated to the N-terminal of the monomeric or dimeric peptide or peptide analogue.

35. The peptide or peptide analogue according to any one of the preceding claims, wherein the monomeric or dimeric peptide comprises or consists of a sequence corresponding to SEQ ID NO: 16 (FSIVGRYPRRRR).

36. The peptide or peptide analogue according to any one of the preceding claims, wherein the monomeric or dimeric peptide comprises or consists of a sequence corresponding to SEQ ID NO: 22 (FSIVGRYPRRRC).

37. The peptide or peptide analogue according to any one of the preceding claims, wherein the monomeric or dimeric peptide comprises or consists of a sequence corresponding to SEQ ID NO: 23 (FSIVGRYPRRRRC).

38. The peptide or peptide analogue according to any one of the preceding claims, wherein the monomeric or dimeric peptide comprises or consists of a sequence corresponding to SEQ ID NO: 19 (FSIVGRYPRRRRRRRRR).

39. The peptide or peptide analogue according to any one of the preceding claims, wherein the dimeric peptide comprises two identical peptides consists of a sequence corresponding to SEQ ID NO: 22 (FSIVGRYPRRRC).

40. The peptide or peptide analogue according to any one of the preceding claims, wherein the dimeric peptide or peptide analogue comprises two identical peptides consists of a sequence corresponding to SEQ ID NO: 4 1 (YSIVGRYP). 4 1 The peptide or peptide analogue according to any one of the preceding claims, wherein the monomeric or dimeric peptide or peptide analogue is a

direct competitor of the gephyrin/GABA AR and gephyrin/GlyR interactions.

42. The peptide or peptide analogue according to any one of the preceding claims, wherein the first and/or the second peptide or peptide analogue has a K for gephyrin below 1 µΜ, such as below 900 nM, such as below 800 nM, such as below 700 nM, such as below 600 nM, such as below 500 nM, such as below 400 nM, such as below 300 nM, such as below 200 nM, such as below 100 nM.

43. The peptide or peptide analogue according to any one of the preceding claims, wherein the first and/or the second peptide or peptide analogue has a K for gephyrin below 1 µΜ.

44. A composition comprising the peptide or peptide analogue according to any one of claims 1 to 43.

45. The composition according to claim 44, wherein the composition is a pharmaceutical composition.

46. A peptide or peptide analogue according to any one of claims 1 to 43, or the composition according to any one of claims 44 to 45, for use as a medicament.

47. A peptide or peptide analogue to any one of claims 1 to 43 or the composition according to any one of claims 44 to 45, for use in a method of prophylaxis and/or treatment of a disorder selected from the group consisting of mental or behavioural disorders and diseases of the nervous system.

48. The peptide or peptide analogue for the use according to claim 46, wherein said mental or behavioural disorder is selected from the group consisting of anxiety disorders, autistic disorders, mood and affective disorders, mental and behavioural disorders due to psychoactive substance use, schizophrenia, schizotypal disorders, delusional disorders, organic mental disorders, neurotic disorders, stress-related disorders, somatoform disorders, anxiety disorders, behavioural syndromes associated with physiological disturbances and physical factors, disorders of adult personality and behaviour, mental retardation, disorders of psychological development, depression and behavioural and emotional disorders with onset usually occurring in childhood and adolescence.

49. The peptide or peptide analogue for the use of any one of claims 46 and 47, wherein said disease of the nervous system is selected from the group consisting of epilepsy, Alzheimer's disease, hyperekplexia, chorein deficiency, ischemic brain damage, inflammatory diseases of CNS, systemic atrophiesprimarily affecting CNS, extrapyramidal and movement disorders, Parkinson's disease, degenerative diseases of CNS, demyelinating diseases of CNS, episodic and paroxysmal disorders, insomnia, nerve-, nerve root and plexus disorders, polyneuropathies, disorders of the peripheral nervous system, diseases of myoneural junction and muscle, cerebral palsy and paralytic syndromes.

50. The peptide or peptide analogue for the use of any one of claims 46 to 49, wherein the use comprises administration of the composition in a therapeutically effective amount to a subject suffering from or suspected of suffering from a mental or behavioural disorder or from a disease of the nervous system.

5 1. A method of preventing and/or treating a mental or behavioural disorder or a disease of the nervous system, comprising administering a peptide or peptide analogue according to any one of claims 1 to 43, or a composition according to any of claims 44 to 45 to a subject in need thereof.

52. A method for modulating GABAAR- and GlyR-mediated fast synaptic inhibition comprising the steps of:

a . providing a sample comprising neurons wherein GABAAR , GlyR and gephyrin are present, b. providing a monomeric or dimeric peptide or peptide analogue as defined in any one of claims 1 to 43, c . contacting the peptide or peptide analogue comprised in said composition with said neurons, whereby said peptide enters in said neurons, and whereby said peptide is contacted with gephyrin,

thereby modulating the binding of gephyrin to GABAAR and/or GlyR and

modulating GABAAR- and GlyR-mediated fast synaptic inhibition.

The method according to claim 52, wherein the neurons are living interconnected neurons.

The method according to claim 52, wherein the neurons are in form of dissociated cultures, brain slices or a combination thereof.

55. The method according to any one of claims 52 and 54, wherein the method

reduces the synaptic GABAAR and/or GlyR concentration, thereby inhibiting

GABAAR- and GlyR-mediated fast synaptic inhibition.

56. The method according to any one of claims 52 to 55, wherein the method

enhances the synaptic GABAAR and/or GlyR concentration, thereby inducing

GABAAR- and GlyR-mediated fast synaptic inhibition.

57. The method according to any one of claims 52 to 56, wherein the monomeric or dimeric peptide or peptide analogue does not alter the properties of

GABAAR and GlyR.

58. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue alters the gephyrin receptor clustering activity.

59. The method according to any one of claims 52 to 57, wherein the monomeric

or dimeric peptide or peptide analogue alters GABAAR and/or GlyR clustering at post-synaptic sites.

60. The method according to any one of claims 52 to 57, wherein the monomeric

or dimeric peptide or peptide analogue alters GABAAR and/or GlyR clustering at extra-synaptic sites. 6 1. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue alters gephyrin clustering.

62. The method according to any one of claims 52 to 57, wherein the monomeric

or dimeric peptide or peptide analogue alters GABAAR and/or GlyR diffusion properties within the post-synaptic neuronal membrane.

63. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue alters fast synaptic transmission by

altering the exchange rate of desensitized GABAARs and/or GlyRs.

64. The method according to any one of claims 52 to 57, wherein the monomeric

or dimeric peptide or peptide analogue alters the turnover rate of GABAARs and/or GlyRs.

65. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue alters the turnover of gephyrin.

66. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue alters the neuronal balance of fast synaptic inhibition and excitation.

67. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue specifically alters the diffusion properties and/or exchange rates and and/or turnover rates of a specific

subset of GABAARs and/or GlyRs subtypes.

68. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue specifically alters the diffusion properties and/or exchange rates and and/or turnover rates of a specific

subset of GABAARs and/or GlyRs subtypes.

69. The method according to any one of claims 52 to 57, wherein the monomeric or dimeric peptide or peptide analogue specifically alters the diffusion

properties and/or exchange rates and and/or turnover rates of GABAARs and/or GlyRs subtypes and/or gephyrin within a specific subset of neuronal cells and/or specific brain regions.

70. The method according to any one of claims 52 to 58, whereby said composition is provided at a concentration below 10 µΜ, such as below 9 µΜ, such as below 8 µΜ, such as below 7 µΜ, such as below 6 µΜ, such as below 5 µΜ, such as below 4 µΜ, such as below 3 µΜ , such as below 2 µΜ, such as below 1 µΜ, such as below 100 nM.

7 1. A method for isolation of the inhibitory postsynaptic proteome from brain lysate comprising the steps of: a . providing brain lysate, b. providing a peptide or peptide analogue as defined in any one of claims 1 to 43, c . immobilizing the peptide or peptide analogue on a resin, thereby obtaining an immobilised composition, d . contacting said brain lysate with said immobilized composition, e . eluting the bound gephyrin and its binding partner, thereby isolating proteins of the inhibitory post-synaptic density.

72. The method according to claim 7 1, wherein the brain lysate is of non-human animal origin.

73. The method according to any one of claims 7 1 and 72, further comprising the step of analysing the eluate by electrophoresis, mass spectrometry, electron microscopy, small angle X-ray scattering or a combination thereof.

74. A method for the exclusive labeling of inhibitory synapses comprising the steps of: a . providing primary neurons, b. providing a peptide or peptide analogue comprising a dye conjugate as defined in any one of claims 1 to 43, and c . visualization of the gephyrin bound peptide by microscopy. 75. The method according to claim 74, wherein the neurons are in form of primary hippocampal neurons

76. The method according to any one of claims 74 and 75, wherein the peptide or peptide analogue is provided at a concentration between 0.05 nM and 50 nM.

77. The method according to any one of claims 74 and 76, wherein super- resolution microscopy is used for the visualization of the gephyrin bound peptide.

78. The method according to any one of claims 74 to 77, wherein the super- resolution microscopy technique is selected from a group composed of structured illumination microscopy, stimulated-emission-depletion fluorescence microscopy, photoactivation localization microscopy and stochastic optical reconstruction microscopy.

79. The method according to any one of claims 74 and 76, wherein conventional fluorescence microscopy is used for the visualization of the gephyrin bound peptide.

80. A method for improving affinity of a monomeric peptide or peptide analogue for gephyrin, said method comprising the step of linking said monomeric peptide to a second peptide through a linker, thereby obtaining a dimeric peptide or peptide analogue with improved affinity.

8 1. The method of claim 80, wherein the first peptide or peptide analogue and/or the second peptide or peptide analogue are as defined in any one of claims 1 to 42.

82. The method of any one of claims 80 and 8 1, wherein the affinity of the dimeric peptide or peptide analogue for gephyrin is increased at least 10-fold compared to the affinity of the monomeric peptide or peptide analogue for gephyrin, such as at least 25-fold, such as at least 50-fold, such as at least 100-fold, such as at least 250-fold, such as at least 500-fold, such as at least 750-fold, such as at least 1000-fold compared to the affinity of the monomeric peptide or peptide analogue. 83. The method of any one of claims 80 to 82, wherein the dimeric peptide or peptide analogue has a K for gephyrin below 1 nM, such as below 900 pM, such as below 800 pM, such as below 700 pM, such as below 600 pM, such as below 500 pM, such as below 400 pM, such as below 300 pM, such as below 200 pM, such as below 100 pM.

84. A method of manufacturing a peptide or peptide analogue according to any one of claims 1 to 43 comprising: a . providing a resin; b. providing a solution comprising amino acid residues; c . coupling a first amino acid residue to the resin; d . coupling a second amino acid residue to the first one, and coupling each following amino acid residues to the previous one to form a peptide comprising at least 12 proteinogenic or non-proteinogenic amino acid residues according to any one of claims 1 to 43; e . providing cleavage of the so formed peptide from the resin.

A . CLASSIFICATION O F SUBJECT MATTER INV. A61K38/00 C07K14/705 ADD.

According to International Patent Classification (IPC) o r to both national classification and IPC

B . FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K C07K

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) EPO-Internal

C . DOCUMENTS CONSIDERED TO B E RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

HANS MICHAEL MAR ET AL: "Desi gn and 1-84 Synthesi s of Hi gh-Affi ni t y Dimeri c Inhi bi tors Targeti ng the Interacti ons between Gephyri n and Inhi bi tory Neurotransmi tter Receptors" , ANGEWANDTE CHEMI E INTERNATIONAL EDITION , 20 November 2014 (2014-11-20) , pages n/a-n/a, XP055339233 , DE ISSN : 1433-7851 , DOI : 10. 1002/ani e . 201409043 ci ted i n the appl i cati on the whol e document -/-

X| Further documents are listed in the continuation of Box C . □ See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date o r priority date and not in conflict with the application but cited to understand "A" document defining the general state of the art which is not considered the principle o r theory underlying the invention to be of particular relevance "E" earlier application o r patent but published o n o r after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel o r cannot b e considered to involve a n inventive "L" documentwhich may throw doubts o n priority claim(s) orwhich is step when the document is taken alone cited to establish the publication date of another citation o r other "Y" document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve a n inventive step when the document is "O" document referring to a n oral disclosure, use, exhibition o r other combined with one o r more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than the priority date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

26 January 2017 06/02/2017

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 N L - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Lopez Garci a , F INTERNATIONAL SEARCH REPORT PCT/DK2016/050369

Box No. I Nucleotide and/or amino acid sequence(s) (Continuation of item 1.c of the first sheet)

With regard to any nucleotide and/or amino acid sequence disclosed in the international application, the international search carried out on the basis of a sequence listing:

forming part of the international application as

j j in the form of an Annex C/ST.25 text file. □ on paper or in the form of an image file. □ furnished together with the international application under PCT Rule 13fer1 (a) for the purposes of international search only in the form of an Annex C/ST.25 text file.

c . j j furnished subsequent to the international filing date for the purposes of international search only:

j j in the form of an Annex C/ST.25 text file (Rule 13fer1 (a)).

j j on paper or in the form of an image file (Rule 13fer1 (b) and Administrative Instructions, Section 7 13).

2 . In addition, in the case that more than one version or copy of a sequence listing has been filed or furnished, the required statements that the information in the subsequent or additional copies is identical to that forming part of the application as filed or does not go beyond the application as filed, as appropriate, were furnished.

3 . Additional comments:

Form PCT/ISA/21 0 (continuation of first sheet (1)) (January 201 5) C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

HANS MICHAEL MAR ET AL: 1-84 "Gephyri n-bi ndi ng pepti des v i sual i ze postsynapti c si tes and modul ate neurotransmi ssi on" , NATURE CHEMICAL BIOLOGY, vol . 13 , no. 2 , 28 November 2016 (2016-11-28) , pages 153-160, XP055339240, GB ISSN : 1552-4450, DOI : 10. 1038/nchembi o.2246 the whol e document - & HANS MICHAEL MAR ET AL: 1-84 "SUPPLEMENTARY INFORMATION . -Gephyri n b i ndi ng pepti des v i sual i ze postsynapti c si tes and modul ate neurotransmi ssi on" , NATURE CHEMICAL BIOLOGY, vol . 13 , no. 2 , 28 November 2016 (2016-11-28) , pages 153-160, XP055339241 , GB ISSN : 1552-4450, DOI : 10. 1038/nchembi o.2246 the whol e document

page 2 of 2