WO2012016963A1 - Peptides for the treatment of brain diseases - Google Patents

Abstract

The invention relates to peptides and combinations thereof which have the ability to interfere with the Ras-ERK signaling pathway in the brain. In particular the peptides can be fused to other amino acid sequences to allow brain penetration. The peptides are useful for the treatment of addiction to drugs of abuse, L-DOPA induced dyskinesia and related disorders, Ras-MAPK syndromes and Brain tumors.

Description

PEPTIDES FOR THE TREATMENT OF BRAIN DISEASES

Field of the invention

The present invention relates to peptides which have the ability to interfere with the Ras- ERK signaling pathway in the brain. In particular the peptides can be fused to other amino acid sequences to allow brain penetration. The peptides are useful for the treatment of addiction to drugs of abuse, L-DOPA induced dyskinesia and related disorders, Ras- MAPK syndromes and Brain tumors. Background art

The Ras-ERK cascade is a signaling pathway involved in a variety of cellular processes, from cell proliferation and survival, to differentiation and synaptic plasticity. In brain, activation of ionotropic and metabotropic receptors leads a switch of the small GTPases of the Ras family, which in turn activate the Raf/Mek/Erk protein kinase cascade. Sustained activation of these biochemical pathways leads to synaptic rearrangements requiring de novo gene expression and protein synthesis via, for instance, the CREB family of transcription factors. In addition, the Ras-ERK pathway is also involved in the cytoplasm in the control of protein translation, via for instance the activation of the rapamycin sensitive mTORCl complex (Orban et al, 1999) (Davis and Laroche, 2006).

There is growing evidence indicating that the Ras-ERK signaling pathway is not only implicated in normal functions of neural networks but that its activity is significantly upregulated in a number of brain diseases. More specifically, hyperactivity of Ras-ERK in neurons have been directly linked to four classes of brain diseases: drug addiction, L-DOPA induced dyskinesias, the Ras-MAPK syndromes and brain tumors (Brambilla, 2003; Fasano and Brambilla, 2002; Jenner, 2008; Kim and Choi, 2010; Samuels et al, 2009). In the first two diseases, the brain region directly implicated in the pathogenesis is the striatum, the input nucleus of the basal ganglia network. Crucially, one of the key factors in the striatum providing signal integration between glutamate and dopamine (DA) on ERK signaling is Ras-GRFl . Ras Guanine Exchange Factors (Ras-GEFs) like the neuronal specific, striatal enriched, Ras-GRFl, directly link NMD A receptors to Ras by catalysing the conversion from the inactive GDP -bound to the active GTP -bound form. The authors' recent evidence indicates that Ras-GRFl is a signaling integrator for DA and glutamate in the striatum and that its activity is crucial for behaviorally salient activity, such as behavioral responses to drugs of abuse, which all alter the function of dopamine signaling in the basal ganglia. The authors have already shown that in a mouse model in which Ras-GRFl, a specific activator of the Ras proteins in neurons of the central nervous system, is ablated (Ras-GRFl KO), both cellular and behavioral responses to psychostimulants (cocaine) and opiates (morphine) are considerably attenuated. In addition, in neurodegenerative models of Parkinson's Disease (PD), such the unilaterally 6-OHDA lesioned rodent and the MPTP -treated non-human primate ( HP), chronic treatment with L-DOPA, the gold standard pharmacological therapy for PD, leads to abnormal involuntary movements (AIM). Thus, L-DOPA-induced dyskinesia (LID) is a common debilitating complication of the dopamine replacement in PD which has been causally linked to abnormal long-term cellular adaptations in the basal ganglia associated to a supersensitivity of dopamine Dl receptors, and most notably to the hyperactivation of the Ras-ERK cascade. Related diseases to LID, also caused by chronic L-DOPA treatments, are pathological alterations of the reward mechanisms often found in parkinsonian patients, like dopamine dysregulation syndrome (DDS) and impulse control disorder (ICD). Thus, in a validated mouse model of PD in which the rodent LID behavioral correlate, abnormal involuntary movements (AIMs), can be induced by repeated L-DOPA treatment, Ras-GRFl deficient mice were significantly resistant to the development of dyskinesia. Furthermore, in a non-human primate (NHP) model of LID, Lentiviral Vectors (LV) expressing two dominant negative forms of Ras-GRFl (both sequences to be patented) cause a dramatic reversion of dyskinesia severity leaving intact the therapeutic effect of L-DOPA. These data not only highlight a central role of Ras- GRFl in governing critical aspects associated to dopamine-dependent adaptations in the striatum but also validate the first viable therapy for LID based on intracellular signaling modulation. Moreover, the authors have shown that one cell permeable peptide (CPP) which acts as inhibitory of the ERK pathway, upon systemic administration in the mouse model of LID, significantly prevent the appearance of AFMs.

Ras-MAPK syndromes are genetic diseases in which point mutations in various components of the Ras-ERK pathway lead to its hyperactivity in the brain. This class includes the following diseases: Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation- arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, NFl- like syndrome and Autism (del/ dup 16pl l .2).

Collectively, these diseases are characterized by a broad spectrum of mental retardation. It is thus conceivable that a partial inhibition of this signaling pathway using specific CPPs such as those outlined above can ameliorate the cognitive symptoms. Finally, most of the mutations found in patients affected by any Ras-MAPK syndrome are mild to severe oncogenic forms of components of this pathway, mutations which have also been found in tumors, including those of the nervous system.

US 7, 122,345 discloses NOV9 (that may have important structural and/or physiological functions characteristic of the Guanine Nucleotide Releasing protein family) as a portion of previously described Ras-GRFl able to bind the p21Ras protein and thereby to regulate the activity of ras gene products. The use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes is also disclosed.

US 2009215680 concerns peptides useful as MAP kinase/ERK pathway-specific inhibitors relative to a given substrate in a given subcellular compartment. In particular, a peptide comprising: at least one amino acid sequence which allows said peptide to penetrate into a cell; optionally, an intracellular targeting amino acid sequence chosen from NESs; optionally, an intracellular targeting sequence chosen from NLSs; an amino acid sequence corresponding to a docking domain sequence of a substrate of an ERK-type MAP kinase; optionally, at least one spacer sequence; optionally, an enzymatic cleavage sequence possibly surrounded by spacer sequences is claimed. Preferably, said docking domain sequence is chosen from the D or FXFP domains of the substrates of ERK-type MAP kinases. Preferably said sequence which allows said peptide to penetrate into a cell is chosen from the sequences of an HIV- TAT penetrating peptide, of penetratin, and the 7/11R or X7/11R sequences.

US 2005100972 concerns the use of whole or part of the GRFl protein, or of cells expressing whole or part of the GRFl protein, in methods for detecting compounds for preventing and/or treating pathologies or disorders of the central nervous system involving neuronal death, such as apoptosis, or related to leptin metabolism. The pathologies of the central nervous system are in particular cerebral ischemia, Parkinson's disease or Alzheimer's disease. Description of the invention

There is a large unmet medical need with substantial commercial potential for an effective product for the treatment of L-DOPA induced dyskinesia and brain related diseases.

Peptides that have the ability to interfere with the Ras-ERK signaling pathway in the brain are the object of the present invention. Brain delivery of the peptides is advantageously achieved through a specific tagging with cell penetrating peptide sequences, which allow both permeation through the brain blood barrier and through plasma membranes of neuronal cells.

Three different sets of peptide sequences are proposed:

1) (Group b) Peptides essentially consisting of aa sequence comprised in the following group: the catalytic domain of human Ras-GRFl (hRasGRFl CD, SEQ ID No. 1) and CDC25-like domains belonging to the list of Table 1, allelic variants, mutants and orthologous thereof. Specific and preferred examples are peptides VPYLGMYLTDLAFIEEGTPNYTEDGLVN (aa 973-1275 of hRasGRFl, aa. 217- 244 of SEQ ID No. 1) and its mouse orthologous sequence, wherein the aa A at pos. 12 of seq id No. 1 is substituted by aa V. Another embodiment of the invention is a mutant of the peptide of SEQ ID No. 1 wherein the aa W at pos 1056 of hRasGRFl is substituted by aa E (aa 97 of SEQ ID No. 1).

Table 1

Coverage

SEQ Accession

name of the Name of the (on the

ID number / aa. Match

Gene Protein RasGRFl

N boundaries

sequence)

ras-specific

Identities = 303/303 guanine

Q13972 / 973- (100%), Positives =

1 RASGRF1 nucleotide- 100%

1275 303/303 (100%), Gaps = releasing

0/303 (0%)

factor 1

ras-specific

Identities = 222/302 guanine

NP_008840.1 / (74%), Positives =

2 RASGFR2 nucleotide- 99%

936-1237 268/302 (89%), Gaps = releasing

0/302 (0%)

factor 2

rap guanine Identities = 91/247 (37%), nucleotide NP_005303.2 / Positives = 136/247

3 RAPGEF1 80%

exchange 826-1064 (55%), Gaps = 10/247 factor 1 (4%) son of Identities = 72/245 (29%),

NP_005624.2 /

4 SOS1 sevenless 76% Positives = 137/245

782-1021

homolog 1 (56%), Gaps = 17/245 (7%)

ras-specific

RalGPS2 (Ral

guanine

GEF with PH Identities = 81/242 (33%), nucleotide- NP_689876.2 /

domain and Positives = 129/242

76%

releasing 52-290 (53%), Gaps = 12/242 SH3 binding

factor

motif 2) (5%)

RalGPS2

dentities = 72/240 (30%), son of

NP_008870.2 /

SOS2 Positives = 135/240

sevenless 76%

780-1017 (56%), Gaps = 11/240 homolog 2

(5%)

ras-specific

RalGPS l (Ral

guanine

GEF with PH Identities = 76/239 (32%), nucleotide- NP 001177659.1

domain and Positives = 127/239

75%

releasing / 53-288

SH3 binding (53%), Gaps = 13/239 factor (5%)

motif 1)

RalGPS l

ral guanine

Identities = 79/265 (30%), nucleotide

NP 055964.3 /

RGL1 dissociation Positives = 124/265

74%

271-534 (47%), Gaps = 39/265 stimulator-like

1 (15%)+F26

RASGRP3 ras guanyl- Identities = 67/238 (28%),

NP 056191.1 /

(CalGAG-GEF releasing 78% Positives = 123/238

148-383

III) protein 3 (52%), Gaps = 3/238 (1%) rap guanine Identities = 69/237 (29%), nucleotide NP 036426.3 /

RAPGEF5 Positives = 124/237

76%

exchange 498-730 (52%), Gaps = 10/237 factor 5 (4%)

ral guanine

Identities = 74/261 (28%), nucleotide

NP_001155088.1

RGL3 dissociation Positives = 127/261

77%

/ 245-504 (49%), Gaps = 28/261 stimulator-like

3 (1 1%)

ral guanine Identities = 74/259 (29%),

RALGDS nucleotide NP_001035827.1 Positives = 116/259

75%

(RALGEF) dissociation / 335-592 (45%), Gaps = 32/259 stimulator (12%)

rap guanine

RAPGEF4 Identities = 61/246 (25%), nucleotide NP_001093867.1

(cAMP-GEF 78% Positives = 119/246

exchange / 624-867

Π) (48%), Gaps = 9/246 (4%) factor 4

RASGRP1 RAS guanyl- Identities = 63/238 (26%),

NP_001122074.1

(CalDAG-GEF releasing 78% Positives = 114/238

/ 201-436

Π) protein 1 (48%), Gaps = 3/238 (1%) ras-GEF

Identities = 73/260 (28%), domain-

NP_778232.2 / Positives = 120/260 RASGEFIC containing 79%

196-444

family (46%), Gaps = 30/260

(12%)

member 1C

Identities = 76/255 (30%), ral-GDS- NP_705843.1 /

RGL4 Positives = 127/255

related protein 223-473 (50%). Gaps = 28/255

(11%) ral guanine

Identities = 65/267 (24%), nucleotide

NP_004752.1 / Positives = 117/267 RGL2 dissociation 75%

247-512 (44%), Gaps = 40/267 stimulator-like

(15%)

2

ras-GEF

domain- Identities = 61/194 (31%),

NP_689758.1 /

RASGEF1B containing 60% Positives = 92/194 (47%),

262-451

family Gaps = 15/194 (8%) member IB

rap guanine

Identities = 58/193 (30%), nucleotide NP_055062.1 /

RAPGEF2 63 Positives = 96/193 (50%), exchange 717-907

Gaps = 4/193 (2%) factor 2

rap guanine Identities = 66/233 (28%), nucleotide NP_001 157861.1 Positives = 112/233 RAPGEF6 74%

exchange / 860-1073 (48%), Gaps = 25/233 factor 6 (11%)

NP_722541.1

RASGRP2 RAS guanyl- Identities = 65/242 (27%),

NP_001092140.1

(CalDAG GEF releasing 78% Positives = 117/242

/ 150-387

I) protein 2 (48%), Gaps = 9/242 (4%)

NP 001092141.1

ras-GEF

domain- Identities = 58/194 (30%),

NP_660356.2 /

PvASGEFIA containing 60% Positives = 91/194 (47%),

270-459

family Gaps = 15/194 (8%) member 1A

rap guanine

Identities = 54/233 (23%), nucleotide NP_057423.1 /

RAPGEFL1 75 Positives = 115/233

exchange 221-452

(49%), Gaps = 6/233 (3%) factor-like 1

rap guanine

NP_006096.2 Identities = 51/189 (27%),

RAPGEF3 nucleotide

NP_001092002.1 Positives = 90/189 (48%), (cAMP-GEF I) exchange

/ 613-799 Gaps = 4/189 (2%) factor 3

RAS guanyl-

Identities = 54/241 (22%), releasing NP_001139674.1

RASGRP4 78' Positives = 105/241

protein 4 / 183-420

(44%), Gaps = 5/241 (2%) isoform b

RAS guanyl-

Identities = 54/241 (22%), releasing NP_733749.1 /

RASGRP4 78% Positives = 105/241

protein 4 197-434

(44%), Gaps = 5/241 (2%) isoform a

RAS guanyl-

Identities = 43/169 (25%), releasing NP_001139677.1

RASGRP4 55 Positives = 77/169 (46%), protein 4 / 233-400

Gaps = 3/169 (2%) isoform e

protein very Identities = 36/172 (21%),

NP_689856.6 /

KNDC 1 KIND isoform 56% Positives = 82/172 (48%),

1533-1702

a Gaps = 3/172 (2%)

RAS guanyl-

Identities = 33/153 (22%), releasing NP_001139676.1

RASGRP4 50% Positives = 66/153 (43%), protein 4 / 197-330

Gaps = 19/153 (12%) isoform d RAS guanyl-

Identities = 27/125 (22%), releasing NP_001139675.1

30 RASGRP4 Positives = 56/125 (45%), protein 4 / 197-319

Gaps = 2/125 (2%) isoform c

RAS guanyl-

Identities = 26/107 (24%), releasing NP_001139679.1

31 RASGRP4 Positives = 43/107 (40%), protein 4 7 232-337

Gaps = 3/107 (3%) isoform g

2) (Group a) Peptides essentially consisting of the interacting domains of ERK kinases with their intracellular partners, of aa sequences selected from the group of SEQ ID No. 32 to SEQ ID No 64, as in Table 2, and functional derivatives thereof.

Table 2: aa sequences of peptides of the invention

Peptide Sequences Domain Gene

Group 2 Domain Gene

PGIMLRRLQKGNLPV SEQ ID No. 32 D (RB I) MKP-3

LEQYYDPTDEPVAE SEQ ID No. 33 common ERK-1

Docking

LEQYYDPSDEPIAE SEQ ID No. 34 common ERK-2

Docking

LAKYHDPDDEPDCA SEQ ID No. 35 common ERK-5

Docking

MPKKKPTPIQLNPAPDGSAVNGTSSAETNL SEQ ID No. 36 D MEK- 1

MLARRKPVLPALTINPTIAEGPSPTSEGAS SEQ ID No. 37 D MEK-

2

ETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFA SEQ ID No. Ras QQCMLEILDTAGTEQFTAMRDLYMKNGQGFALVYS SEQ ID Rap 1 No. 39

SPAKLSFQFPSGSAQVHI SEQ ID No. 40 FXFP Elk-1

SPARLQGANTLFQFPSVLN SEQ ID No. 41 FXFP Sap-1

SPARLQGPSTLFQFPTLLN SEQ ID No. 42 FXFP Sap-2

MAVLDRGTSTTTVFNFPV SEQ ID No. 43 FXFP MKP-1

PNPSPGQRDSRFSFPD SEQ ID No. 44 FXFP KSR

SLTPTAAHSGSHLFGFPP SEQ ID No. 45 FXFP Gata-2

KGRKPRDLELPLSPSLL SEQ ID No. 46 D Elk-1

RSKKPKGLGLAPTLAPTLVI SEQ ID No. 47 D Sap-1

KAKKPKGLEISAPPLLVL SEQ ID No. 48 D Sap-2

SSILAQRRVRKLPSTTL SEQ ID No. 49 D Rsk-1

RRSTLAQRRGIKKITSTAL SEQ ID No. 50 D Rsk-2

SSNLAQRRGMKRLTSTRL SEQ ID No. 51 D Rsk-3

KSRLARRRALAQAGRSRD SEQ ID No. 52 D Mnk-1

QSKLAQRRQRASLSSATPV SEQ ID No. 53 D Mnk-2

KAPLAKRRKMKKTSTSTE SEQ ID No. 54 D Msk-1

RFSTIVRRRAKGAKGAG SEQ ID No. 55 D MKP-1

PGIMLRRLQKGNLPVRAL SEQ ID No. 56 D MKP-3 LPALLLRRLRRGSLSVR SEQ ID No. 57 D MKP-4

GLQERRGSNVSLTLDM SEQ ID No. 58 D Step

LVTTTPTSTQFLYPKVAA SEQ ID No. 59 FXFP JunD

CTTYTSSFVFTYPEEADSFPS SEQ ID No. 60 FXFP c-Fos

SNGVITTTPTPPGQYFYPRG SEQ ID No. 61 FXFP JunB

MLKKD ALTL SLAEQGAA SEQ ID No. 62 D JunD

SGAALCALGKECFLELAPDF SEQ ID No. 63 D Ets-1

NGQMLCMLGKERFLELAPDF SEQ ID No. 64 D Ets-2

3) Dominant negative sequences of Ras-GRFl as, i.e., the peptide essentially consisting of the binding domain of human Ras-GRFl to NR2B (hRasGRF 1 -NR2B BD, SEQ ID No. 65) and allelic variants, mutants and orthologous thereof, as in Table 3.

Table 3

The proposed sets of peptide sequences may be advantageously fused to carriers allowing the brain delivery, in particular to peptides consisting of aa sequences selected from SEQ ID No. 66 to SEQ ID No. 81, as in Table 4. Table 4

Peptide Sequences Domain Gene

Cell penetrating peptides Gene

GRKKRRQRRR SEQ ID No. 66 HIV-TAT

RQIKIWFQNRRMKWKK SEQ ID No. 67 Penetratin

RRRRRRR SEQ ID No. 68 X7R

XRRRRRRRX SEQ ID No. 69 X7R (oe)

XRRRXRRRR SEQ ID No. 70 X7R (oe)

RRRXRRRRX SEQ ID No. 71 X7R (oe)

RRRRRRRXX SEQ ID No. 72 X7R (oe)

XXRRRRRRR SEQ ID No. 73 X7R (oe)

RRRRRRRRRRR SEQ ID No. 74 X11R

XRRRRRXRRRRRR SEQ ID No. 75 Xl lR (oe)

RRRRRXRRRRRRRX SEQ ID No. 76 Xl lR (oe)

GAYDLRRRERQSRLRRRERQSR SEQ ID No. 77 DPV15b

SRRARRSPRHLGSG SEQ ID No. 78 DPV10

LRRERQSRLRRERQSR SEQ ID No. 79 DPV15

VKRGLKLRHVRPRVTRMDV SEQ ID No. 80 DPV1047 RKKRRRESRKKRRRES SEQ ID No. 81

Peptides of the invention are valuable therapeutic means, either alone or in combinations to treat brain disorders as:

i) Addiction to drugs of abuse (psychostimulants, opiates, ethanol, nicotine, cannabinoids, hallucinogens, inhalants, phencyclidine, new drugs);

ii) L-DOPA induced dyskinesia and related disorders (dyskinesia, tardive dyskinesia, dopamine dysregulation syndrome, impulsive control disorder, dystonia);

iii) Ras-MAPK syndromes [Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, NFl-like syndrome and Autism (del/ dup 16pl 1.2)];

iv) Brain tumors. In particular, it is proposed the combined use of two or more peptides (combined therapy) for the treatment of above diseases, preferably for the treatment of dyskinesia, more specifically, the combination of at least one peptide of Group a) and of at least a peptide of Group b).

It is therefore an object of the invention a peptide belonging to any of the following groups:

a) a peptide essentially consisting of the interacting domain of an ERK kinase with its intracellular partner, consisting of an aa sequence selected from the group of SEQ ID No. 32 to SEQ ID No 64 or functional derivatives thereof;

b) a peptide essentially consisting of an aa sequence comprised in the catalytic domain of human Ras-GRF 1 (hRasGRF 1 CD, SEQ ID No. 1), or in one of CDC25-like domains belonging to the list of Table 1, allelic variants, mutants and orthologous thereof;

c) or a combination of at least two peptides wherein the first peptide belongs to group a) and the second peptide belongs to group b).

In a preferred embodiment the peptide of the invention essentially consists of aa of SEQ ID No. 32 and/or of the aa. 217-244 of SEQ ID No. 1 or of its mouse orthologous sequence, wherein the aa. A at pos. 12 is substituted by aa. V. In an alternative embodiment the peptide essentially consists of SEQ ID No. 1 wherein the aa W at pos 97 is substituted by aa E.

For the purposes of the present invention, the term derivative denotes any molecule obtained by modification, of a genetic and/or chemical nature, of these sequences and which retains the desired activity. Modification of a genetic and/or chemical nature should be understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for different purposes, such as, in particular, that of increasing the affinity of the peptide for its interaction site, that of improving its levels of production, that of increasing its resistance to proteases, that of increasing its therapeutic efficacy or of reducing its side effects, that of endowing it with novel pharmacokinetic and/or biological properties, that increasing circulatory half-life in the body of the patient, that of enhancing bioavailability and/or enhancing efficacy and/or specificity. In addition, non-peptide peptidomimetics for improving stability, for example less susceptible to biological degradation must also be included as well as the synthesis of the said peptide sequences using D-amino acids instead of the natural L-amino acids, which may increase stability and resistance to degradation.

In particular allelic variants, refer to variants of peptides in the same species, orthologous of peptides of the invention refer to variants in different species. Examples of orthologous are mouse Ras-GRFl having the sequence of SEQ ID No 82 TDDTLKYRVICFLEEVMHDPDLLTQERKAAANIIRTLTLEETTEQHSMLEEVILMTE GVKTEPFENHPALEIAEQLTLLDHLVFKSIPYEEFFGQGEMKAEKYERTPYIMKTT KHFNHVSNFIASEIIRNEDISARASAIEKWVAVADICRCLHNYNAVLEITSSINRSAIF RLKKTWLKVSKQTKSLLDKLQKLVSSDGRFKNLRESLRNCDPPCVPYLGMYLTD L IEEGTPNYTEDGLVNFSKMRMISHIIREIROFOOTTYKIDPOPKVIOYLLDESFM LDEE SL YE S SLLIEPKLPT .

In a preferred embodiment of the invention the peptide is fused to a cell penetrating peptide sequence, preferably selected from the group of SEQ ID No. 66 to SEQ ID No. 81. A specific object of the invention refers to a peptide having essentially the sequence MGRKKRRQRRRPPQAPGIMLRRLQKGNLPVSRYPYDVPD - SEQ ID No. 83 and/or the sequence GRKKRRQRRRPPCVPYLGMYLTDLVFIEEGTPNYTEDGLVN - SEQ ID No. 85, or of a combination thereof wherein the first peptide has the sequence MGRKKRRQRRRPPQAPGIMLRRLQKGNLPVSRYPYDVPD - SEQ ID No. 83 and the second peptide has the sequence

GRKKRRQRRRPPCVPYLGMYLTDLVFIEEGTPNYTEDGLVN - SEQ ID No. 85. It is a further object of the invention at least one of the peptides as above disclosed for medical use, particularly for the treatment of brain related disorders as: Addiction to drugs of abuse (psychostimulants, opiates, ethanol, nicotine, cannabinoids, hallucinogens, inhalants, phencyclidine, new drugs); L-DOPA induced dyskinesia and related disorders (dyskinesia, tardive dyskinesia, dopamine dysregulation syndrome, impulsive control disorder, dystonia); Ras-MAPK syndromes [Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, NF1- like syndrome and Autism (del/ dup 16pl 1.2)]; Brain tumors.

It is a further object of the invention a pharmaceutical composition comprising at least one of the peptides as above disclosed and suitable diluents and excipients and carriers.

Preferably the pharmaceutical composition further comprises a peptide essentially consisting a binding domain of human Ras-GRF 1 to NR2B (hRasGRF 1 -NR2B BD, SEQ

ID No. 65) or allelic variants, mutants and orthologous thereof

The pharmaceutical composition is preferably for direct administration into the brain.

Further object of the invention is a nucleic acid recombinant vector for gene therapy comprising under the control of suitable regulative sequences a nucleotide sequence encoding the peptide or a combination as above discloses.

A further object is a method of treatment of addiction to drugs of abuse (psychostimulants, opiates, ethanol, nicotine, cannabinoids, hallucinogens, inhalants, phencyclidine, new drugs), of L-DOPA induced dyskinesia and related disorders (dyskinesia, tardive dyskinesia, dopamine dysregulation syndrome, impulsive control disorder, dystonia), as treatment for Ras-MAPK sindromes [Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation- arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous, Legius syndrome, NFl-like syndrome and Autism (del/ dup 16pl l .2), of brain tumors comprising administering to a subject in need thereof at least one of peptides according to claim 1 to 10 in appropriate amount and dosages. A further object is a method of gene therapy treatment of addiction to drugs of abuse (psychostimulants, opiates, ethanol, nicotine, cannabinoids, hallucinogens, inhalants, phencyclidine, new drugs), of L-DOPA induced dyskinesia and related disorders (dyskinesia, tardive dyskinesia, dopamine dysregulation syndrome, impulsive control disorder, dystonia), as treatment for Ras-MAPK sindromes [Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous, Legius syndrome, NFl-like syndrome and Autism (del/ dup 16pl 1.2), of brain tumors comprising administering the nucleic acid recombinant vector as above disclosed.

The invention will be now illustrated by means of non-limiting examples referring to the following figures.

Fig. 1 Attenuated temporal development of L-dopa-induced Abnormal Involuntary Movements during the chronic drug treatment in RasGRFl KO mice. Animals received 2 injections a day and tested for AEVIs once a day. Two-Way ANOVA for repeated measure indicated a significant difference between groups in the responses to L- dopa (Time x Group interaction, P< 0001). More specifically, L-dopa treated RasGRFl KO showed a severe attenuation of AEVIs score in comparison to WT littermates.

Fig. 2 Severe DA depletion induced by 6-OHDA lesion in WT and Ras-GRFl KO mice. (A, B) The TH optical density was measured throughout the striatum and values are expressed as a percentage of the optical density on the intact side in sham and lesioned animals. (C) More than 90% reduction of TH-positive fiber density was seen in lesioned animals without difference in genotype (p>0.5). (D-G) In the Substantia Nigra pars compacta counting of TH-positive neurons was carried out on 3 sections per animal and only sections in which the lateral part of the SN was clearly separated by the medial terminal nucleus (MTN) were selected. (H) No significant difference was seen in the mean±sem of number of TH-positive cells in the intact (I) and lesion side (L) of both groups.

Fig. 3 Strong reduction of p-ERK and AFosB accumulation in lesioned striata of RasGRFl KO treated with L-dopa. (a) An aberrant phospho-ERK activation was observed in dyskinetic WT animals while a severe reduction was seen in Ras-GRFl KO animals, (b) FosB/AFosB accumulation is severely attenuated in the lesioned striata of Ras-GRFl KO animals in comparison to littermate controls (*p< 0001 genotype difference).

Fig. 4 Low dose of the MEK inhibitor SL327 did not alter motor responses and cellular events in 6-OHDA-lesion mice acutely treated with L-dopa. Lesioned animals were injected with a low, medium or high dose of SL327 (10, 30, 50 mg/kg, respectively) or vehicle 30 min before a challenge of L-dopa (6 mg/kg). (A) contralateral rotations were counted over 60 minute/session. 30 and 50 mg/kg of the MEK inhibitor strongly decreased L-dopa induced turning behaviour whereas the lowest dose was ineffective. One way anova and post hoc Tukey's HSD test 4 p< 001 Vehicle + L-dopa vs SL327 30 mg/kg + L- dopa and Vehicle + L-dopa vs SL327 50 mg/kg + L-dopa. (B) Coronal sections from mice sacrificed 20 min after L-dopa injection were immunostained for p-ERKl/2 and optical density was analysed. The medium and the highest dose of SL327 clearly inhibited ERK activation in lesioned striata. One way anova and post hoc Tukey's HSD test, 4 p< 01 Vehicle + L-dopa vs SL327 30 mg/kg + L-dopa and Vehicle + L-dopa vs SL327 50 mg/kg + L-dopa. (C) Representative autoradiographs showing distribution pattern of FosB mRNA in striatal sections of animals sacrificed 3 hrs later L-dopa injection. The DA- depleted striatum is shown to the right in all panels. As expected, FosB mRNA levels were significantly induced ipsilaterally to the lesion after L-dopa administration. This treatment effect was maintained in combination with 10 mg of SL327, but was markedly reduced with the other two doses. One way anova and post hoc Tukey's HSD test, 4 p< 001 Vehicle + L-dopa vs SL327 30 mg/kg + L-dopa and Vehicle + L-dopa vs SL327 50 mg/kg + L-dopa.

Fig. 5 Suboptimal treatment of Ras-GRFl KO mice with SL327 caused an enhanced reduction of the AIMs. (A) Cumulative AIMs after 9 days of escalating doses of L-dopa expressed as mean of three days/dose showed a stronger attenuation of dyskinesia in Ras- GRFl KO animals when pretreated with a low dose of SL327 (10 mg/kg, i.p.) in comparison with Ras-GRFl KO mice L-dopa treated only. Repeated measures and post hoc Tukey's HSD test, # p< 001 WT L-dopa (n=10) vs Ras-GRFl KO L-dopa (n=9) and t p< 01 Ras-GRFl KO L-dopa vs Ras-GRFl KO SL327 L-dopa (n=10). (B) Representative photomicrographs of phospho-ERKl/2 immunoreactive cells (upper panels) and FosB/AFosB expression (lower panels) in the dorsolateral part of the striatum after 9 days of L-dopa treatment in combination with SL327 (10 mg/kg). (C) Quantification of p- ERKl/2 positive cells (mean±sem) in the intact (I) and lesioned (L) dorsolateral striatum of Ras-GRFl mutants and control animals. Two-way ANOVA revealed a significant reduction in the number of pERK positive cells in lesioned striatum of Ras-GRFl mutants in comparison to littermate controls (Tukey's HSD test, # p< 001 genotype effect) and a greater effect in lesioned striatum of Ras-GRFl KO SL327 pre-treated mice (Tukey's HSD test t p< 001 treatment effect). (D) Quantification of FosB/AFosB positive cells (mean±sem) in the intact (I) and lesioned (L) striata of Ras-GRFl KO mice. Two-way ANOVA showed significant differences between WT and Ras-GRFl KO mice L-dopa treated (Tukey's HSD test, # p< 001 genotype effect). A significant reduction in the number of FosB//AFosB immunoreactive cells was also observed in Ras-GRFl KO treated with SL327 in comparison to Ras-GRFl KO only L-dopa injected. (Tukey's HSD test t p< 001 treatment effect).

Fig. 6 Expression of Ras-GRFl in MSN subpopulations and its protein levels in dyskinetic animals. Immunofluorescence of Ras-GRFl (red), EGFP (green) and nuclear labelling with DAPI (blu) of striatonigral neurons (direct pathway) of M4-EGFP mice (A) and striatopallidal neurons (indirect pathway) of A2A-EGFP mice (C). The graph provided quantitative data of the percentage number of GFP positive cells over the total Ras-GRFl - positive neurons indicating that Ras-GRFl is equally expressed in each subpopulation (E). As expected, Ras-GRFl was not expressed either in the direct pathway of M4-EGFP Ras- GRFl KO (B) or in the indirect pathway of A2A EGFP Ras-GRFl KO mice (D). Scale bar 20um. Protein levels of Ras-GRFl, Ras-GRF2 and phospho-ERKl/2 in intact (I) and lesioned striata (L) of wildtype mice after 9 days of L-dopa treatment were determined by Western blot analysis (F). pl40 ^Ras"GRF1 (G) as well as pl35 ^Ras"GRF2 (H) levels were not altered in dopamine denervated striata after saline or L-dopa treatment whereas phosphorylation of ERKl/2 is enhanched only in L-dopa treated striata (one-way ANOVA, # p< 01 (I).

Fig. 7 Dominant negative LV constructs were able to reduce ERK activation in mouse striatum, (a) stereotaxic injections of LV-GFP, LV-Ras-GRF 1 -NR2B-BD, LV-Ras- GRF1-CD ^W1056E, LV-ERK2^K52R (Low and High titer) or LV-Mix-GFP were performed into dorsal striatum of WT mice. 4 weeks later, mice were challenged once with saline (upper panel) or 20 mg/kg of cocaine (lower panel) and 20 minutes later were perfused. Brain sections were immunostained with GFP (green) and pERK (red), (b) The graph showed quantitative data of the number of double pERK/GFP positive neurons indicating that all Ras-GRFl dominant negative constructs and LV-ERK2 (High titer) as well as the LV-Mix-GFP, effectively reduced ERK activation.

Fig. 8 Dominant negative LV constructs were able to reduce dyskinesia when injected in mouse striatum. Stereotaxic injections of LV-GFP, LV-Ras-GRFl-NR2B-BD, LV- Ras-GRF 1 -CD ^W1056E, L V-ERK2^K52R (Low and High titer) or LV-Mix-GFP were done into dorsal striatum of 6-OHDA lesioned mice (n= 9 each group). 4 weeks later, mice were treated with L-dopa as in Figure 1. Mean ± sem of the last dose (6 mg/Kg) are shown. The graph showed the cumulative AFMs score indicating that all Ras-GRFl dominant negative constructs and LV-ERK2^K52R (High titer) as well as the LV-Mix-GFP, effectively reduced dyskinetic symptoms.

Fig. 9 Effect on colony formation of TAT-fused peptides with MKP3-DD (RBI) in the presence of H-Ras^G12V.

Administration of a vector expressing the CPP TAT-MKP3-DD (RBI) significantly attenuates cell transformation in NIH3T3 cells by an oncogenic form of H-Ras.

Fig.10. S6 ribosomal protein phosphorylation is prevented by RBI peptide incubation in a model of mature striatal slices. Double immunolabeling of phospho S6 ribosomal protein (Ser235/236) (red) and NeuN (green) in adult striatal slices stimulated or not with glutamate 100 μΜ for 10 min in the presence of the vehicle or 50μΜ RBI peptide. The data from the quantification are represented in the graph as mean±SEM. Statistical analysis were performed using two-way ANOVA and post-hoc comparisons between groups using Bonferroni test (**** pO.0001).

Fig.ll. S6 ribosomal protein phosphorylation is prevented by RB3 peptide incubation in a model of mature striatal slices. Double immunolabeling of phospho S6 ribosomal protein (Ser235/236) (red) and NeuN (green) in adult striatal slices stimulated or not with glutamate 100 μΜ for 10 min in the presence of the vehicle or 50μΜ RB3 peptide. The data from the quantification are represented in the graph as mean±SEM. Statistical analysis were performed using two-way ANOVA and post-hoc comparisons between groups using Bonferroni test (**** pO.0001).

Fig. 12 Score of the Abnormal Involuntary Movements (AIMs) elicited in mice treated with L-dopa or saline at increasing doses within 9 days in the presence of RBI and RB3 peptides, alone or in combination. Dose for days 1-3 : 3mg/kg, days 4-6: 6mg/kg, days 7-9: 12mg/kg. Mice were treated (lOmg/kg) with RBI, RB3, RB 1+RB3 or SCRAMBLE peptide as a control. ALO = sum of Axial, Limb and Orololingual AIM scores. RB 1+RB3 injected mice showed the maximal reduction of AIM score in comparison to controls.

Fig. 13 RBI peptide significantly reduces cocaine-mediated responses in conditioned place preference. Cocaine dose: 15 mg/Kg. Dose of either RBI or scramble peptide (SCR): 20 mg/Kg. Administration of RB I to the cocaine-paired group significantly inhibits place preference to the drug (SCR cocaine, n=9 vs RB I cocaine, n=10 one-way ANOVA, drug effect *p< 05) but does not affect basal condition in the saline treated animals (SCR,n=9 and RBI, n=9).

Methods

Production of Lentiviral vectors. All LVs used have already been reported (Vantaggiato et al, 2006) (Fasano et al, 2010, submitted). Ectopic expression of the DN constructs of RasGRFl, both epitope-tagged, is achieved through the use of an LV under the control of the hPGK promoter which also co-expresses GFP (via an IRES). In the first LV, LV-Ras- GRF1- R2B-BD, a 230aa portion encompassing the PH2 domain located at the N-term portion of RasGRFl has been shown to bind NR2B containing receptor and act as inhibitor of glutamate mediate ERK activation (Krapivinsky et al, 2003). Targeting the R2B-Ras- GRFl interaction is particularly appealing since it is known that this receptor subunit is expressed at high levels in the striatum and involved in LID (Gardoni et al., 2006). In the second LV, LV-Ras-GRFl-CD ^⁰⁵⁶^ two point mutants in the C-term catalytic domain (300aa) of RasGRFl are able to displace endogenous Ras-GRFl molecule from Ras proteins, thus blocking Ras-mediated activation of ERKs (Vanoni et al, 1999).

Mouse studies. Generation of Ras-GRFl KO mice has been originally described (16, 18). Ras-GRFl KO and littermate controls were kept in mix background (C57BL/6 x 129SVJ) and housed under a 12 hour-light-dark cycle with ad libitum access to the food.

Lesion Surgery. Ras-GRFl WT (n=20) and KO (n=20) mice were anesthetized with Isofluorane (Baxter Medical AB, Sweden) and secured in a stereotaxic frame equipped with a mouse adaptor. Ι μΐ of 6-OHDA-HCL (3 μg/μl) was injected into the right ascending MFB at the following coordinates according to the mouse brain atlas: AP -0.7; L -1.2; DV -4.7 from dural surface using a glass capillary attached to a ΙΟμΙ Hamilton syringe. Severity of DA denervation was assessed at the end of the experiments analyzing striatal levels of tyrosine hydroxylase (TH) and nigral cell loss using immunohistochemical labelling. Motor impairment and AIM induction. Two weeks after lesion, mice were evaluated in the open field in order to estimate the success rate of lesion. Starting from day 18, they were treated twice a day, for 9 consecutive days with an escalating L-dopa dosing regimen (1.5, 3, 6 mg/kg) plus benserazide (12 mg/kg) (Ras-GRF 1 WT, n=9 and Ras-GRF 1 KO, n=9) or with saline (Ras-GRF 1 WT, n=6 and Ras-GRF 1 KO, n=6). In a second experiment, the same protocol of increasing L-dopa treatment was applied once a day in combination with a low dose (10 mg/kg) of MEK inhibitor, SL327. Abnormal involuntary movements were scored using a 0-4 rating scale according to validated mouse model of LID. Every morning, mice were individually placed in large transparent boxes and observed for 1 min every 20th min during 180 min after L-dopa injection.

Immunohistochemical methods. At the end of the behavioral testing sessions, after animal killing and perfusion, loss of DA innervation is measured with TH staining and fiber density in the striatum will be quantified using the Image J software as previously described (Westin et al, 2007). Immunohistochemistry is used to determine whether behavioral performances in response to L-DOPA are accompanied by increased in p-ERK and FosB/AFosB expression within the medial and lateral portions of dorsal striatum, as described (Westin et al, 2007).

LV testing in mice. WT (n=60) mice were anesthetized with Isofluorane and secured in a stereotaxic frame. Ι μΐ of 6-OHDA-HCL (3μ§/μ1) was injected into the right ascending MFB. Two weeks after 6-OHDA lesion, stereotaxic injections (Ι μΐ x 2) of LV-GFP (n=9), LV-Ras-GRF 1 - R2B-BD (n=9), LV-Ras-GRFl-CD ^W1056E (n=9), LV-ERK2^K52R (n=18), (Low and High titer) and a mix of the three LV (LV-Mix-GFP, (n=9)) containing high titer of the two LV-Ras-GRF 1 and a low titer of LV-ERK2K52R, were done into dorsal striatum. 4 weeks later, mice were treated with L-dopa (1.5, 3, 6 mg/kg) plus benserazide (12 mg/kg) and AIMs scoring were evaluated every day.

Immunohistochemical methods. At the end of the behavioral testing sessions, after animal killing and perfusion, loss of DA innervation is measured with TH staining and fiber density in the striatum will be quantified using the Image J software as previously described (Westin et al, 2007). Immunohistochemistry is used to determine whether behavioral performances in response to L-DOPA are accompanied by increased in p-ERK and FosB/AFosB expression within the medial and lateral portions of dorsal striatum, as described (Westin et al, 2007). Preparation of Peptides. Cell permeable peptides against the ERK pathway as well as the scrambled (SCR) ones (ineffective) are custom synthesized by GE ECUST EUROPE (Luxembourg). The sequences of the two tested peptides (RBI and RB3) and their scrambled versions (SCR-RB1 and SCR-RB3) are:

fusedRBl : MGRKKRRQRRRPPQAPGIMLRRLQKG LPVSRYPYDVPD - SEQ ID No. 83;

SCR-RB1 : MGRKKRRQRRRPPQALSLKRLRSRGMNRTSATQSRYPYD - SEQ ID No.

84;

fusedRB3 : GRKKRRQRRRPPCVPYLGMYLTDLVFIEEGTPNYTEDGLVN - SEQ ID No. 85;

SCR-RB3 : GRKKRRQRRRPPCFEVYPDSGDYTYEGELNGTLMVVPTN - SEQ ID No. 86.

For all in vitro and in vivo experiments we required batches of 100 mg, highly purified (>99%). For the production of the two long Ras-GRFl polypeptides, we expressed in bacteria a fusion protein containing the TAT sequence fused to the relevant Ras-GRF l domain (NR2B or CDC25) and a hystidine tag. Bacterial lysates were loaded on standard high capacity (100 mg) Hys-tag affinity columns and the relevant peptide were purified. Pending on the initial quality of the peptide eluted, we also considered a second purification step by FPLC.

Peptide testing in mice. Unilaterally lesioned mice are generated using 6-OHDA injected in the medium forebrain bundle, as described (Fasano et al., 2010, submitted; Lundblad et al, 2004; Lundblad et al, 2005). Two weeks after lesion, we tested a dose of 10 mg/kg (i.p.) of each peptide and its scrambled form. Groups of 12-15 animals are used. Peptides are given daily, 4h before L-DOPA treatment, and AIMs will be monitored (effect on LID formation).

For the conditioned place preference test, an unbiased procedure was performed as previously described (Fasano et al, 2009). The place preference apparatus consisted of two different Plexiglas compartments separated by a central neutral area. It was located in a sound proof testing room with low luminosity (25 lux). No group showed initial preference for any chamber. Treatments were counterbalanced between compartments. The schedule consists of three different phases: pre-conditioning phase : mice were placed in the middle of the neutral area and their location recorded for 18 min; conditioning phase : mice were treated for 6 consecutive days with alternate drug (cocaine, 15 mg/Kg)days 1, 3 and 5) or saline (days 2, 4, and 6) injection, each paired with one of the two compartments. Saline treated mice received saline on all 6 days; both saline and cocaine groups either received 20 mg/Kg of RBI peptide or SCR (inactive scramble peptide) 2 hours before saline/cocaine injections; post-conditioning phase: the test was conducted 24 hours after the final conditioning session. Place preference was quantified in terms of time spent in drug-paired side. A score was calculated for each mouse as the difference between post- conditioning and pre-conditioning time spent in drug-paired compartment.

In vitro work. Initial screening assay of the peptides was performed with a Ras-dependent proliferation assay in NIH 3T3 fibroblasts as described (Vantaggiato et al, 2006). Scrambled peptides demonstrated inability to do so.

Ex-vivo work on acute brain slices. Mouse brains are kept in ice-cold sucrose-based dissecting solution oxygenated with 95% 0₂ and 5% C0₂ and subsequently mounted on the vibratome stage. 200 μπι-thick slices are prepared and transferred into the brain slice chamber and let them recover for 1 hour at 32°C, with a constant perfusion of carboxigenated ACSF in the presence of the relevant CPP (50μΜ) or the scramble peptide. Brain slice stimulation is performed with 100 μΜ glutamate in the chamber for 10 minutes. After a rapid fixation in 4%PFA for 15 min at room temperature slices are incubated in the cryoprotectant overnight at 4°C. Following criostate sectioning at 18μπι, slices are incubate with anti-phospho-S6 ribosomal protein (Thr235/236) antibodies and anti NeuN antibodies followed by secondary antibodies. Single and double-labeled images are obtained using a laser scanning confocal microscopy, equipped with the corresponding lasers and the appropriate filters sets to avoid the cross-talk between the fluorochromes. Images are obtained with a 40X and 63 X objectives. Neuronal quantification is performed with ImageJ software by counting phospho-S6 immunoreactive neurons among NeuN positive neurons in each slice. Statistical analysis were performed using two-way ANOVA and post-hoc comparisons between groups were made using Bonferroni test (**** pO.0001).

Results

Ras-ERK pathway and Ras-GRFl in the treatment of L-dopa induced Dyskinesia and related disorders

The authors took advantage of the availability of Ras-GRFl deficient mice (Ras-GRF l KO) (Brambilla et al, 1997) to generate a unilateral 6-hydroxydopamine (6-OHDA)-lesion model of PD and LID (Fasano et al, 2010). The authors injected the neurotoxin 6-OHDA into the right medial forebrain bundle of both Ras-GRFl KO mice and littermate controls. Two weeks after surgery, the authors measured spontaneous ipsilateral rotations as an efficacy index of the lesion. The rotational behavior was similar in the two genotypes (WT, 24.7±1.4; Ras-GRFl KO, 28.1±2.5 turns/10 min), suggesting an equivalent sensitivity to the neurotoxic damage (Figure 1A). Post-mortem immunohistochemical analysis confirmed that the high ipsilateral rotational scores corresponded to more than 90% depletion of both DA fiber terminals in the striatum and DA cell bodies in the substantia nigra without any genotypic difference (Figure 2, A-H).

To elicit axial, limb, and orolingual ATMs, a validated model of LID in mice (Lundblad et al, 2004) (Lundblad et al, 2005), the authors administered an ascending dose regimen of L-dopa (1.5, 3, 6 mg/kg, twice daily) to both 6-OFIDA-lesioned WT and Ras-GRFl mutant animals for 9 consecutive days. Daily scoring of ATMs revealed a gradual development of dyskinetic symptoms in both genotypes, which affected the trunk, limb and orolingual regions on the side of the body contralateral to the lesion. However, for all doses of L- dopa, the ATMs score were significantly reduced in Ras-GRFl KO mice in comparison to their littermate controls (Figure IB).

These results demonstrate that the absence of Ras-GRFl strongly attenuates LID induction in mice. Consistently with previous experimental evidence (Lundblad et al, 2004; Pavon et al, 2006; Santini et al, 2007; Westin et al, 2007), the authors observed abnormally high levels of ERKl/2 phosphorylation and FosB/AFosB induction in the dorsolateral lesioned striatum of WT mice. In marked contrast, low levels of ERKl/2 phosphorylation and FosB/AFosB expression were observed in the Ras-GRFl mutants despite their almost complete striatal denervation (Figure 3).

Since AFMs expression in Ras-GRFl mutant mice was only attenuated but not completely abolished, the authors next explored the possibility of a pharmacogenetic interaction between Ras-GRFl loss and a direct chemical inhibition of the core component of ERK signaling pathway. Systemic administration of high doses (>50 mg/kg) of the SL327, a specific chemical inhibitor of MEK1/2, the upstream kinases of ERKl/2, has been already shown to efficiently inhibit ERK signaling in the striatum (Santini et al, 2007; Valjent et al, 2000). In a pilot experiment with 6-OHDA-lesioned drug-naive mice the authors used 3 three different doses of SL327 (10, 30 and 50 mg/kg, i.p.) to study cellular and behavioral effects upon an acute L-dopa challenge. The authors found that only the lowest dose of SL327 did not alter L-dopa induced contralateral turning behavior nor abolished ERKl/2 phosphorylation and FosB/AFosB expression in the striatum (Figure 4). The authors next asked whether this ineffective low dose of SL327 in WT mice may cause a further reduction of the AIMs in a Ras-GRFl deficient genetic background.

Hemiparkinsonian Ras-GRFl KO and control mice pre-treated once daily with 10 mg/kg of SL327 were injected 30 min later with escalating doses of L-dopa (1.5, 3, 6 mg/kg, same regimen as above). As expected, SL327 did not change the dyskinesia profile in control animals but greatly attenuated the AIMs scores in Ras-GRFl mutants (Figure 5). This was paralleled by a significant inhibition of phospho-ERKl/2 and FosB/AFosB expression by SL327 in the same group (Figure 5). These results provide the basis for a potential LID therapy based on a combined Ras-GRFl and ERK signaling inhibition.

Postsynaptic changes in striatal medium spiny neurons (MSNs) following dopamine depletion and chronic L-dopa are thought to be responsible for dysregulation of neurotransmission within the basal ganglia. In particular, recent evidence indicates that LID results from a supersensitivity of Dl receptors, which are preferentially expressed in the striatonigral MSN population, leading to a selective hyperactivation of ERK signaling (8) (22). By using two types of bacterial artificial chromosome (BAC) transgenic mice expressing enhanced green fluorescent protein (EGFP) specifically in striatonigral or striatopallidal MSN namely, M4-EGFP mice (striatonigral neurons, direct pathway) and adenosine receptor A2A-EGFP mice (striatopallidal neurons, indirect pathway), the authors addressed the question of the cellular localization of Ras-GRFl in the striatum. A Ras-GRFl positive signal was found in both GFP-labelled neurons in both mouse lines (Figure 6, A and C). Quantitative dual-fluorescence confocal analysis indicated that Ras- GRFl was found equally expressed in both pathways, being the Ras-GRFl/EGFP double positive cells roughly 50% of the total Ras-GRFl positive cells (53.6% direct pathway, 46.4%) indirect pathway, Figure 6 E). Ras-GRFl expression, as expected, was found absent in both M4-EGFP/Ras-GRF 1 KO and A2 A-EGFP/Ras-GRF 1 KO double mutants (Figure 6 B and D). These data indicate that the specific involvement of Ras-GRFl in LID may not be due to its selective expression in striatonigral MSN but rather to its specific engagement by Dl receptors.

In addition to Dl receptor supersensitivity, several downstream changes in gene expression and protein levels of key signalling molecules have been reported in striata of dyskinetic animals (23) (24). Interestingly, a recent report indicated that the expression of two striatal enriched regulators of Ras-ERK signalling, CalDAG-GEFl and 2 is inversely affected in dyskinetic rats (25). To determine whether Ras-GRFl and its close homolog Ras-GRF2 may similarly be altered, the authors assessed the levels of these proteins pl40^Ras"GRF1 and pl35^Ras"GRF2 in the dyskinetic striata by western blot analysis (Figure 6 F). Differently from what observed for CalDAG-GEFs, the striatal levels of Ras-GRFl/2 did not appear to change in L-DOPA-treated dyskinetic animals relative to saline-treated controls, although, as expected, ERK1/2 phosphorylation was massively upregulated in the former group (Figure 6 G-I). These data are important since they indicate that Ras-GRFl/2 levels are not regulated by the dyskinetic physio-pathological process, which could have posed significant problems to a potential therapy targeting this molecular pathway.

The data shown so far indicate that, in a mouse model of PD, the absence of Ras-GRFl in the brain, also combined with a low dose of a MEK inhbitor (SL327) significantly attenuates LID and the associated molecular and synaptic abnormalities.

In an effort to translate these findings into a clinically relevant therapy, the authors set out to determine whether a partial inhibition of Ras-GRFl in the striatum should be able to revert already established dyskinetic symptoms. Thus, the authors generated lentiviral vectors (LV), which express two dominant negative constructs for Ras-GRFl under the control of the strong and ubiquitous human phosphoglycerol kinase (PGK) promoter in addition to the GFP marker (Amendola et al, 2005; Indrigo et al., 2010, in press). These two constructs have been designed using the human sequence of Ras-GRFl and tested in the mouse striatum (Figure 7). The first construct expresses the binding domain of Ras- GRFl on the R2B subunit of the NMD A receptor (Ras-GRFl -NR2B-BD), thus blocking glutamate-mediated activation of Ras-GRFl (Krapivinsky et al, 2003). The second dominant negative construct is instead a point mutation in the catalytic domain (CDC25 domain) of Ras-GRFl (Ras-GRFl -CDC25^W1056E) which sequesters Ras proteins and thus blocks activity of the endogenous Ras-GRFl (Vanoni et al, 1999). Expression of both constructs at high level (-5x1010 TU/ml, Ι μΐ injection/striatum) showed a strong inhibitory effect on the ability of 20 mg/kg of cocaine to activate ERK1/2, confirming that in the mouse these LV are efficacious in preventing the activation of this signaling pathway. In order to confirm that the potential therapeutic effect mediated by the inhibition of Ras-GRFl may be further enhanced by a concomitant suboptimal manipulation of the core MEK-ERK component of the pathway, the authors also tested the ability of a strong dominant negative mutant of ERK2, ERK2K52R, to block ERK activation (Vantaggiato et al, 2006). A high titer of LV-ERK2K52R (-5x1010 TU/ml) is fully capable of blocking phosphorylation of ERK1/2 in response to cocaine while a lower titer (<lxl010 TU/ml) was ineffective by itself. Interestingly, when a mix of the three LV (LV-Mix-GFP, 1 : 1 : 1), containing high titer of the two LV-Ras-GRFl and a low titer of LV-ERK2K52R, was injected in the mouse striatum, the authors still found a significant reduction of pERKl/2 levels.

The same LVs used in Figure 7 were injected in the same conditions in the dorsal portion of the striatum of 6-OHDA lesioned mice. Escalating doses of L-dopa caused a significant reduction of ATMs expression in the two dominant Ras-GRFl mutants as well as in the high titer LV-ERK2K52R but not in the low titer of LV-ERK2K52R. Importantly, the LV- Mix-GFP consisting of high titer of the two LV-Ras-GRFl and a low titer of LV- ERK2K52R (1 : 1 : 1), showed even a stronger effect (Figure 8).

Targeting the Ras-ERK Pathway with cell permeable peptides

The authors designed small polypeptides over interacting sequences between ERK proteins and their effectors (either upstream or downstream) which should be able to disrupt their interaction thus effectively interfering with ERK signaling. These sequences are essentially the docking (D) domains found in the effectors or the CD domains found on ERK1/2 (Enslen and Davis, 2001; Sharrocks et al, 2000). In addition the authors have designed peptide sequences belonging to Ras and Ras related proteins which correspond to interacting domains with Ras effectors, including guanine nucleotide exchange factors such as Ras-GRFl and related molecules (Cullen and Lockyer, 2002; Grewal et al., 1999). To assay the effect of the polypeptides on Ras activity the authors produced expression vectors that allowed expression of these molecules fused with TAT peptide and HA tag. The authors transfected NIH3T3 cells to perform a colony formation assay (Guha et al, 1997): together with these constructs, the cells were transfected with an oncogenic form of Ras (G12V) to verify theirs ability to inhibit the transforming phenotype. Some peptides exhibited intriguing effects, for example the peptide mimicking the DD of MKP3 (hereafter named RBI) was able to significantly (p>0.05) revert the hyper-proliferation normally induced by RasG12V oncogene, without affecting the proliferation in normal conditions, as shown in Figure 9.

In order to study biochemically the function of these cell permeable peptides, including RB I, the authors have also set up an ex-vivo system in which brain slices can be freshly prepared from adult mice, incubated in a perfusing chamber with the peptides and stimulated with appropriate agonist and antagonists. As shown in Figure 10, striatal slices previously incubated with 50μΜ RB I have been challenged with glutamate and induction of ERK signaling was monitored at the single cell level using phospho-specific antibodies either against ribosomal protein S6 (pS6). Quantification of double pS6 (in red) and anti- NeuN staining (in green) allow us to monitor changes in ERK activity. Importantly, pre- treating the slices with RB 1, completely blocks S6 phosphorylation, indicating that this cell permeable peptide is very effective at this concentration in blocking ERK activity.

In addition, as shown in Figure 11, a new cell permeable peptide, named RB3, which was identified as a portion of the larger Ras-GRFl catalytic domain, has also been shown to block ERK activation, using this ex-vivo adult brain slice system.

After having verified the effective capacity of RBI peptide to block the Ras-ERK hyperactivation in vitro and in the ex-vivo acute slice system the authors tested his efficacy in vivo, in a mouse model of dyskinesia. 6-OHDA lesions were performed as previously described. Two weeks after surgery, the authors measured spontaneous ipsilateral rotation as an efficacy index of the lesion. The authors then divided the animals in four balanced groups and performed the first treatment with RBI, RB3, or RB 1+RB3 and compare them to the s SCRAMBLE inactive peptide (lOmg/kg, i.p.). Four hours after treatment the authors verified whether the injection of the peptides affected spontaneous rotation behavior, observing no differences between the two groups (data not shown). Dyskinesia was induced by a single daily L-DOPA/benserazide i.p. injection (3mg/kg for day 1-3, 6mg/kg for days 4-6 and 12mg/kg for days 7-12; 12mg/kg benserazide). Four hours before L-DOPA administration lOmg/kg of either the active or SCRAMBLE peptides were injected i.p. and AFMS scoring was performed as described above. As shown in Figure 12, both RB I and RB3 were effective in reducing AFM scoring, and they significantly synergize by causing a combine AIM reduction of about 80% in coparison to the SCARMBLE treated animals.

Ras-ERK pathway and Ras-GRFl in the treatment of drug addiction

The key data using the Ras-GRFl KO mouse strain have already been published (Fasano et al, 2009). Those data have validated Ras-GRFl as target for treating addiction to drugs of abuse. In addition, we also performed, as shown in figure 13 a test with RBI peptide which showed, in conditioned place preference (CPP) a significant inhibition of the response to this drug. Moreover, always in the present invention, the two Ras-GRFl specific peptide sequences have been expressed via lentiviral vector technology (see section on Dyskinesia, Figure 7) and proved to effectively reduce ERK activation in the striatum of mice challenged with cocaine. These data demonstrate the therapeutic use of the peptides of the invention in the treatment of addiction to drug of abuse.

Ras-ERK pathway in the treatment of Ras-MAPK syndromes and brain tumors

The authors have recently validated in the laboratory a mouse model for the Ras-MAPK syndrome, the K-Ras G12V "floxed" knock in mutant (Guerra et al., 2003). This model can also be used to cause brain specific tumors. Importantly, Figure 9 indicated that the CPP of the present invention are able to block Ras dependent cell transformation in vitro. At present, no CPP against the Ras-ERK cascade have either been tested in animal models of these genetic diseases or of brain tumors.

It is believed that the use of the cell permeable peptides of the invention would "rebalance" ERK activity in the brain and thus will rescue the behavioral deficits associated to the brain diseases. It is very important to underlie that a valid therapy based on Ras-GRFl and ERK inhibition would require to not completely block this signaling pathway but only reduce the abnormally high component of it. In order to achieve this, as a major invention presented here, we propose to simultaneously target Ras-GRFl or Ras-GRFl -like proteins and the downstream components of the pathway, to maximize the therapeutic effect, as combined therapy approach. Altogether, the results make the combined therapy approach using Ras-GRFl and ERK specific therapeutic cell permeable peptides of the invention the first "viable" option for the treatment of brain diseases characterized by an abnormal activation of the Ras-ERK pathway.

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Claims

Claims

1. A peptide belonging to any of the following groups:

a) a peptide essentially consisting of the interacting domain of an ERK kinase with its intracellular partner, consisting of an aa sequence selected from the group of SEQ ID

No. 32 to SEQ ID No 64 or functional derivatives thereof;

b) a peptide essentially consisting of an aa sequence comprised in the catalytic domain of human Ras-GRF 1 (hRasGRF 1 CD, SEQ ID No. 1), or in one of CDC25-like domains belonging to the list of Table 1, allelic variants, mutants and orthologous thereof; or c) a combination of at least two peptides wherein the first peptide belongs to group a) and the second peptide belongs to group b).

2. The peptide according to claim 1 essentially consisting of aa of SEQ ID No. 32 and/or of the aa. 217-244 of SEQ ID No. 1 or of its mouse orthologous sequence, wherein the aa. A at pos. 12 is substituted by aa. V.

3. The peptide according to claim 1 consisting of SEQ ID No. 1 wherein the aa W at pos 97 is substituted by aa E.

4. The peptide according to any of previous claims that is fused to a cell penetrating peptide sequence.

5. The peptide according to claim 4 wherein the cell penetrating peptide sequence is selected from the group of SEQ ID No. 66 to SEQ ID No. 81.

6. The peptide according to claim 5 having essentially the sequence MGRKKRRQRRRPPQAPGIMLRRLQKGNLPVSRYPYDVPD - SEQ ID No. 83 and/or the sequence GRKKRRQRRRPPCVPYLGMYLTDLVFIEEGTPNYTEDGLVN - SEQ ID No. 85.

7. The combination according to any of previous claims wherein the first peptide has the sequence MGRKKRRQRRRPPQAPGIMLRRLQKGNLPVSRYPYDVPD - SEQ ID No. 83 and the second peptide has the sequence

GRKKRRQRRRPPCVPYLGMYLTDLVFIEEGTPNYTEDGLVN - SEQ ID No. 85.

8. The peptide or a combination thereof as any of previous claims for medical use.

9. The peptide or a combination thereof as any of previous claims for the treatment of brain related disorders.

10. The peptide or a combination thereof as in claim 9 wherein the brain related disorder belongs to the group of : addiction to drugs of abuse (psychostimulants, opiates, ethanol, nicotine, cannabinoids, hallucinogens, inhalants, phencyclidine, new drugs); L- DOPA induced dyskinesia and related disorders (dyskinesia, tardive dyskinesia, dopamine dysregulation syndrome, impulsive control disorder, dystonia); Ras-MAPK syndromes [Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio- cutaneous syndrome, Legius syndrome, NFl-like syndrome and Autism (del/ dup 16pl 1.2)]; Brain tumors.

11. A pharmaceutical composition comprising at least one of the peptides according to any of claims 1 to 7 and suitable diluents and excipients and carriers.

12. The pharmaceutical composition according to claim 11 further comprising a peptide essentially consisting a binding domain of human Ras-GRFl to NR2B (hRasGRFl-NR2B BD, SEQ ID No. 65) or allelic variants, mutants and orthologous thereof

13. The pharmaceutical composition of claim 11 or 12 for direct administration into the brain.

14. A nucleic acid recombinant vector for gene therapy comprising under the control of suitable regulative sequences a nucleotide sequence encoding the peptide or a combination thereof according to any of claims 1 to 10.

15. A method of treatment of addiction to drugs of abuse (psychostimulants, opiates, ethanol, nicotine, cannabinoids, hallucinogens, inhalants, phencyclidine, new drugs), of L-

DOPA induced dyskinesia and related disorders (dyskinesia, tardive dyskinesia, dopamine dysregulation syndrome, impulsive control disorder, dystonia), as treatment for Ras- MAPK sindromes [Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation- arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous, Legius syndrome, NFl-like syndrome and Autism (del/ dup 16pl l .2), of brain tumors comprising administering to a subject in need thereof at least one of peptides according to claim 1 to 10.

16. A method of gene therapy treatment of addiction to drugs of abuse (psychostimulants, opiates, ethanol, nicotine, cannabinoids, hallucinogens, inhalants, phencyclidine, new drugs), of L-DOPA induced dyskinesia and related disorders (dyskinesia, tardive dyskinesia, dopamine dysregulation syndrome, impulsive control disorder, dystonia), as treatment for Ras-MAPK sindromes [Noonan syndrome, LEOPARD syndrome, hereditary gingival fibromatosis 1 syndrome, neurofibromatosis 1 syndrome, capillary malformation- arteriovenous malformation syndrome, Costello syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous, Legius syndrome, Fl-like syndrome and Autism (del/ dup 16pl l .2), of brain tumors comprising administering the nucleic acid recombinant vector of claim 14.