WO2005109004A2 - Disease related protein aggregation - Google Patents

Abstract

Present invention demonstrates that the members of the FKBP family or FK506 Binding Proteins (FKBPs), more specifically FKBP12 or FKBP52 enhance the aggregation of α-SYN. Moreover, it was found that the effect of FKBP on α-SYN aggregation has its origin in the PPIase activity of the enzyme. Immunophilin ligands, specific inhibitors of the activity of FKBPs, inhibit stimulation of the aggregation of a α-SYN by FKBPs.

Description

DISEASE RELATED PROTEIN AGGREGATION

Background and Summary

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates generally to methods to determine molecules that prevent protein or peptide misfolding, aggregation or self-aggregation in mammalian cells and to determine molecules that block or slow down cytoplasmic protein or peptide aggregation or self-aggregation in mammalian cell, in particular in mammalian neuronal cells. More particularly the present invention relates to methods to determine molecules that prevent protein or peptide misfolding and aggregation of alpha-synuclein.

B. Description of the Related Art

The present invention provides methods to determine molecules that interact with proteins and peptides that are associated with protein folding diseases or diseases of amyloid aggregation. For instance human neurodegenerative diseases of amyloid type comprises aggregation of Ab peptides, prion proteins, α-synuclein, tau and proteins with polyglutamine extensions; these add to others that are associated with non-neurologic systemic disorders such as artherosclerosis (Ursini, F., et al Trends Mol. Med. 8, 370-374 (2002)).

In vitro, cell based assays and animal models are provided to screen for molecules or to screen for drugs with protein or peptide, in particular intracytoplasmic or intranuclear protein or peptide, anti-aggregation properties, in particular α-synuclein anti-aggregation properties, by assaying their capacity to interfere with the kinetics of PPIases more particular of FK506-Binding Protein (FKBP) induced alpha-synuclein aggregation. Furthermore the invention concerns methods and drugs to prevent or to diminish the formation of amyloid aggregation, more particular of alpha-synuclein self aggregates or the formation of alpha-synuclein deposition in Lewy bodies.

Present invention relates to compositions, methods for the treatment and a method for screening suitable molecules for a treatment of a range of conditions that are characterized by the formation of protein aggregation in cells, more particularly (cytoplasmic) protein aggregation in neural cells.

Protein aggregation disorders and more particular intracytoplasmic or intranuclear protein aggregation disorders are characterized by the intracytoplasmic or intranuclear accumulation of protein aggregates. Aggregates may accumulate, for example, in the cytoplasm of a cell.

Commonly, aggregates may form in neuronal cells, such as brain cells, for example in disorders such as Dementia with Lewy bodies (DLB), Huntington's disease and Parkinson's disease.

Dementia with Lewy bodies (DLB) is the second most common cause of neurodegenerative dementia in elderly people. It is part of the range of clinical presentations that share a neurological pathology based on abnormal aggregation of the synaptic protein α-SYN (a-syn) (I. McKeith et al., Lancet Neurol 3, 19-28 (2004)).

Parkinson's Disease (PD) is a neurodegenerative disease that affects about one percent of the people above the age of sixty-five. This directly illustrates the number one risk factor in PD, which is old age. The region of the brain that is most affected is the substantia nigra (SN) in the midbrain, more precise the dopaminergic neurons herein. When the first symptoms of PD begin to appear, already 80 percent of the dopaminergic neurons in the SN have disappeared. PD is caused by the degeneration of dopaminergic neurons in the substantia nigra. The pathogenic hallmark of PD is the accumulation and aggregation of a- synuclein in susceptible neurons. The cytoplasmic aggregates/inclusions characteristic of PD are called Lewy bodies and their major constituent is a-synuclein (Kahle, P.J. et al (2002) J. Neurochem. 82, 449-457). Lewy pathology is also found in dementia with Lewy Bodies (LB), the LB variant of Alzheimer's disease, in neurodegeneration with brain iron accumulation type I and in glial cytoplasmic inclusions of multiple system atrophy. These diseases are collectively known as synucleinopathies (Spillantini, M. G. et al (1997) Nature 388, 839840, Mezey, E et al. (1998) Mol. Psychiatry 3, 493-499).

α-SYN, an unfolded protein of 14 kDa that is ubiquitously present in the brain, plays a significant role in PD. Three facts have led to this conclusion: 1) A pathophysiological hallmark of PD is the presence of Lewy Bodies (LB) and Lewy neurites in the dopaminergic neurons of the Substantia Nigra (SN). LB's are eosinophilic cytoplasmic inclusions that contain predominantly aggregated α-SYN. 2) In three autosomal dominant familial forms of PD, a point mutation in α-SYN has been discovered: the A30P, A53T and E46K mutation (R. Kruger et al., Nat. Genet. 18, 106-108 (1998); M. H. Polymeropoulos et al., Science 276, 2045-2047 (1997) and J. J. Zarranz et al., Ann.Neurol. 55, 164-173 (2004)). These mutant forms A30P and A53T of α-SYN both oligomerize faster than the wild type form. Another important genetic finding is that α-SYN locus triplication can cause PD (A. B. Singleton et al., Science 302, 841 (2003)). 3) When laboratory animals (mice, rats or C.elegans) are overexpressing α-SYN, they exhibit pathological symptoms and neurological inclusions that are very similar to those observed in PD. Moreover, it has been shown that pathology appears when α- SYN begins to aggregate (P. J. Kahle, C. Haass, H. A. Kretzschmar, M. Neumann, J.Neurochem. 82, 449-457 (2002); P. J. Kahle et al, J.Neurosci. 20, 6365-6373 (2000).; M. Lakso et al., J.Neurochem. 86, 165-172 (2003); E. Lauwers et al., Brain Pathol. 13, 364-372 (2003) and M. K. Lee et al., Proc.Natl.Acad.Sci.U.S.A 99, 8968-8973 (2002)).

Huntington's disease (HD) is likely to remain static or decline in the near future. This will primarily reflect a reduction in the family size of both 'affected' and 'at-risk' populations due to a fear of developing HD and because of the increased incidence of genetic counseling. No drug is yet available to stop or reverse the progression of the disease.

Disorders of intracytoplasmic or intranuclear protein aggregation are associated with particular proteins or sets of proteins that misfold and aggregate in specific tissues. A possible cause is codon reiteration mutations, where protein misfolding is mediated by the abnormal expansion of a tract of repeated amino acids. (Paulson,H.L. (1999) Am. J. Hum. Genet. 64339-345).

Polyglutamine (polyQ) expansion diseases, exemplified by Huntington's disease (HD) is for instance disorder of cytoplasmic protein aggregation. HD is characterized by expansions of a polyQ stretch in exon 1 of the Huntington gene to more than 37 glutamines, and a short M-terminal fragment encoding the polyglutamine stretch is sufficient to cause aggregates in mice (Schilling, G. et al. (1999) Hum. Mol. Genet. 8 397-407) and in cell models (Wyttenbach, A. et al (2000) Proc. Natl. Acad. Sci. U S A 97 2898-2903). It is commonly believed that the mutant protein acquires its toxicity and its propensity to aggregate after cleavage, forming a short (so far, incompletely-defined) N-terminal fragment containing the polyglutamine stretch (Martindale,D. et al., (1998) Nat. Genet., 18, 150-154).

Recently, polyalanine (polyp) expansion mutations in the polyadenine binding protein 2 gene have been shown to cause OPMD, which is associated with aggregates in muscle cell nuclei (Brais, B et al. (1998). Nat. Genet.18, 164-167). This disease has been modelled in cell culture systems where aggregate formation is associated with cell death (Fan,X et al (2001) Hum. Mol. Genet. 10, 2341-2351). PolyA expansions of 19 or more repeats tagged with enhanced green fluorescent protein are sufficient to cause intracytoplasmic aggregate formation and cell death in cultured cells (Rankin,J. et al (2000) Biochem. J., 348, 15-19). Many of the codon reiteration diseases are dominantly inherited and genetic and transgenic studies suggest that they are generally due to galn-of-function mutations (for instance in polyp diseases) (Naraln,Y. et al (1999). J. Med. Genet., 36, 739-746).

Alzheimer's disease (AD) is the best known protein misfolding disease, affecting already 13 million people in US and Europe alone. As the disease progresses, plaque clusters made of a misfolded protein called "a-beta" accumulate in areas of the brain that control memory, mood and spatial awareness. A progressively dehabilitating disorder that results in steady degeneration of cognition and dementia and of which the research points to the formation of amyloid plaques in areas of the brain associated with memory function, primarily the hippocampus. Deposits of amyloid plaques are thought to be caused by the abnormal fragmentation of amyloid protein, a naturally occurring molecule associated with normal brain function. The consequence of plaque formation is the degeneration and death of neurons close to the plaque, and a resulting loss of cognitive function. There are few currently approved treatment options available for AD. AD is overwhelmingly a disease that afflicts the elderly, and it is the increasingly ageing population of major pharmaceutical markets in North America, Japan and Western Europe that makes AD one of the most promising development targets for pharmaceutical corporations.

Amyotrophic lateral sclerosis or Lou Gehrig's disease (ALS or A.L.S.) has also been demonstrated to be a protein misfolding disease, a class including Alzheimer's, Parkinson's, and Huntingon's diseases, in which protein aggregation is considered the underlying pathology. Similarly, proteinaceous intracellular aggregates, or inclusions, are a prominent feature in both the sporadic and familial forms of ALS. Blood can carry misfolded proteins. A plasma protein called transthyretin often misfolds into amyloid because of at least 80 mutations affecting different parts of the body. Some mutations lead to plaque buildup in the hands, feet, liver or heart. ALS is a fatal neurodegenerative disease characterized by the selective loss of motor neurons, leading to progressive and eventually complete paralysis without loss of cognitive function. ALS, the most common motor neuron disease in adults, is fatal within one to five years, and its onset usually occurs in mid-life. Two forms of ALS with slightly different pathologies exist: familial (fALS), which has earlier onset, and sporadic (sALS), which constitutes the vast majority of cases.

Abnormal protein-protein interactions that result in the formation of intracellular and extracellular aggregates of proteinacious fibrils are a common neuropathological feature of several different sporadic and hereditary neurodegenerative diseases. Other neurogenerative diseases, for example, in Guam-Parkinsonism dementia complex, Dementia Pugilistica, adult Down Syndrome, subacute Sclerosing Panencephalitis, Pick's Disease, Corticobasal Degeneration, Progressive Supranuclear Palsy, Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex, Hallervorden- Spatz Disease, Neurovisceral Lipid Storage Disease, Mediterranean Fever, Muckle-Wells Syndrome, Idiopathetic Myeloma, Amyloid Polyneuropathy, Amyloid Cardiomyopathy, Systemic Senile Amyloidosis, Hereditary Cerebral Hemorrhage with Amyloidosis, Alzheimer's disease, Scrapie, Creutzfeldt- Jacob Disease, Fatal Familial Insomnia, Kuru, Gerstamnn-Straussler- Scheinker Syndrome, Medullary Carcinoma of the thyroid, Isolated Atrial Amyloid, Beta2-Microglobulin Amyloid in dialysis patients, Inclusion Body Myositis, Beta2-Amyloid deposits in muscle wasting disease, and Islets of Langerhans Diabetes Type2 Insulinoma have at least histologically been demonstrated to be associated with protein deposits or plaque build up.

Thus, there is a need in the art for treating these protein or peptide misfolding and aggregation disorders.

Some strategies of treating disorders of treating pathologies protein or peptide misfolding and aggregation in neuronal cells is stimulating or promoting neurite outgrowth by neurothrophic agents (Hamilton G.S. et al bioorganic & Medicinal Chemistry Letters. Vol. 7 No. 13 pp 1783 1790, 1997) or to stimulate autophagy to induce a clearance of intracellular protein aggregates (Rubinsztein D. et al. WO 2004/089369). Recent findings, however, indicate that amyloid aggregates of disease-unrelated proteins are cytotoxic in their pre-fibrillar organization, whereas mature fibrils are substantially harmless (Bucciantini, M. et al. Nature 416, 507-511 (2002)), as increasingly reported for other disease-related proteins and peptides (Nilsberth, C. et al. Nature Neurosci. 4, 887-893 (2001); Lashuel, H., et al. Nature 418, 291 (2002); Walsh, D. M. et al. Nature 416, 535- 539 (2002)).

The identification and characterization of several neurotoxic quaternary structure intermediates (referred to as protofibrils) that precede fibril formation and the finding that several pathogenic mutations promote protofibril formation indicate that protofibrils rather than the fibrils are the pathogenic species in these diseases. Removal of mature fibril formation could potentially be detrimental rather than beneficial.

The emergence of the toxic protofibril hypothesis indicates that simply inhibiting fibril formation is no longer a viable therapeutic strategy for these diseases, as it could result in the build-up of toxic intermediates, which would accelerate disease progression and pathogenesis.

As a consequence, therapeutic strategy should be aimed at preventing pre-fibrillar organization by protein or peptide misfolding and aggregation in cells and one should approach for screening anti-aggregation molecules and preventing early intermediates on the self-assembly pathway. Present invention provides such strategy to prevent, block or diminish the formation of the aggregation-prone conformational intermediate, further to prevent the formation of protofibrils and further to inhibit protofibril assembly to fibrils. By the present invention the onset of formation or the formation of such protofibrils or of such pre-fibrillar organization by protein or peptide misfolding and aggregation in cells can be prevented or diminished.

We for instance surprisingly found that an enzyme of the FK506 binding protein (FKBP) family, such as FKBP 12 or FKBP52 enhances the aggregation rate of cytoplasmic proteins or peptides in vitro and induces and speeds up the cytoplasmic protein or peptide aggregation in cells and have aggregation enhancing activities with regard to protein folding intermediates. For instance, α-SYN aggregation is greatly enhanced in vitro, in cell in particular in the cytoplasm and suggests this in vivo, when FKBP is present. This is in sharp contrast with the teachings of the state of the art that FKBP's induce protein refolding and have aggregation suppressing activities with regard to protein folding intermediates (Maruyama T. et al. Front Bioscience, 2004 May 1, 9, 1680-720); to exert protein folding and aggregation suppression activities in E. coli cells (Ideno A et al, Appl. Environ. Microbiology, 2002 Feb, 68 (2): 464 - 9); inhibited the thermal aggregation in vitro of two model substrates (Monaghan P, Bell A. Mol Biochem Parasitol. 2005 Feb; 139(2): 185-95).

By monitoring the expression of FKBP 12 and FKBP52 in the human neuroblastoma cells, both before and after the appearance of α-SYN aggregates, present invention demonstrated that FKBP 12 expression is situated in the whole cell with a higher expression in the nucleus and that the localization does not change when aggregates are formed (Fig 11). On the other hand, a high FKBP52 expression can be seen in the cytoplasm in cells without aggregates and after α-SYN aggregates are induced, the expression level of FKBP52 seems to decrease (Fig. 12). This change in expression pattern and/ or level demonstrates that interaction occurs between α-SYN and FKBP52.

There is also a need in the art for efficient and specific screening tools that identify druglike molecules that inhibit the aggregation of disease-associated proteins and the formation of protofibrils. Structure-based drug design has produced some small molecules that inhibit protein aggregation by stabilizing the native state against conformational changes that predispose the protein to self-assembly and aggregation. However, for the amyloidogenic proteins that lack a defined three-dimensional structure and/or small-molecule binding pockets, screening for aggregation inhibitors has been limited to biophysical-based (light scattering, electron microscopy, atomic-force microscopy and circular dichroism) and/or dye binding assays.

The present invention fulfils this need by providing in vitro, cell based and in vivo tools to screen for inhibitors of PPIase (EC 5.2.1.8), cyclophilin (CyP), FKBP, and parvulin and preferably for FKBP 12 and most preferably for FKBP52 inhibitors linked at the selection of suitable molecules based on their suppressing properties of specific intracellular proteins.

SUMMARY OF THE INVENTION

The invention is directed to the prevention protein or peptide misfolding and aggregation in cells and to blocking or slowing down intracytoplasmic or intranuclear protein or peptide aggregation in cell such as neuronal cells, in particular molecules that prevent aggregation of α-synuclein by means of molecules that act as selective inhibitors (or antagonists) of an FKBP, for instance FKBP12 and preferably FKBP52, such as antibodies and functional fragments derived thereof, RNAi (siRNA and shRNA) and DNA molecules (e.g. polynucleotide sequences), ribozymes that function to inhibit the translation of the FKBP.

Small molecules can also interfere by binding on the promoter region of the FKBP, preferably FKBP 12 or FKBP52, and inhibit binding of a transcription factor on said promoter region so that none of the FKBP mRNA is produced.

Also, the molecules in this invention comprise antagonists of the FKBP, in particular FKBP52 such as antibodies and functional fragments derived from these antibodies, anti- sense RNA, DNA molecules and ribozymes that function to inhibit the translation of the FKBP, particular FKBP52. By synthesis is meant the transcription of the FKBP, particular

FKBP52. Small molecules can bind on the promoter region of the FKBP and inhibit binding of a transcription factor or said molecules can bind said transcription factor and inhibit binding to the FKBP -promoter. By a FKBP it is meant also its variant forms, which occur as a result of RNA splicing.

The invention also provides the use of molecules that inhibit the expression and/or activity of the FKBP, in particular FKPB12 and FKBP52 and most preferably FKBP52 (herein after named FKBP) for the manufacture of a drug to prevent protein or peptide misfolding and aggregation in cells and to block or slow down intracytoplasmic or intranuclear protein or peptide aggregation in cell such as neuronal cells, in particular molecules that prevent alpha-synuclein aggregation.. Thus more specifically the invention relates to the use of molecules that neutralize the activity of the FKBP by interfering with its synthesis, translation and ligand-binding. By molecules it is meant peptides, tetrameric peptides, proteins, organic molecules, having the same neutralizing effect as stated above. Also, in this invention the molecules comprise antagonists of the FKBP such as anti-FKBP antibodies and fab's and single chains or other functional fragments derived thereof, anti- sense RNA and DNA molecules and ribozymes that function to inhibit the translation of the FKBP, all capable of interfering/or inhibiting the FKBP activity. By synthesis is meant the transcription of the FKBP. Small molecules can bind on the promoter region of the FKBP and inhibit binding of a transcription factor or these molecules can bind a transcription factor and inhibit binding to the FKBP -promoter so that there is no expression of the FKBP.

The term 'antibody' or 'antibodies' relates to an antibody characterized as being specifically directed against the FKBP or any functional derivative thereof, with above mentioned antibodies being preferably monoclonal antibodies; or an antigen-binding fragment thereof, of the F(ab')2, F(ab) or single chain Fv type, or any type of recombinant antibody derived thereof. These antibodies of the invention, including specific polyclonal antisera prepared against the FKBP or any functional derivative thereof, have no cross- reactivity to other proteins. The monoclonal antibodies of the invention can for instance be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat immunized against the FKBP or any functional derivative thereof, and of cells of a myeloma cell line, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the FKBP or any functional derivative thereof which have been initially used for the immunization of the animals. The monoclonal antibodies according to this embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Alternatively the monoclonal antibodies according to this embodiment of the invention may be human monoclonal antibodies. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice as described in PCT/EP 99/03605 or by using transgenic non-human animals capable of producing human antibodies as described in US patent 5,545,806. Also fragments derived from these monoclonal antibodies such as Fab, F(ab)'2 and ssFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses. An appropriate label of the enzymatic, fluorescent, or radioactive type can label the antibodies involved in the invention.

Small molecules, e.g. small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries. Random peptide libraries, such as the use of tetrameric peptide libraries such as described in WOO 185796, consisting of all possible combinations of amino acids attached to a solid phase support may be used in the present invention.

Also within the scope of the invention is the use of oligoribonucleotide sequences that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of FKBP mRNA. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site. Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridisation of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyse endonucleolytic cleavage of FKBP RNA sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences: GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. Both anti- sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors, which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize anti-sense RNA constitutively or inducible, depending on the promoter used, can be introduced stably into cell lines.

Another aspect of administration for treatment is the use of gene therapy to deliver the above mentioned anti-sense gene or functional parts of the FKBP gene or a ribozyme directed against the FKBP mRNA. Gene therapy means the treatment by the delivery of therapeutic nucleic acids to patient's cells. This is extensively reviewed by K. W. Culver, T. M. Vickers, J. L. Lamsam, H. W. Walling, T. Seregina, Br.Med.Bull. 51, 192-204 (1995); M. Evans, N. Affara, A. M. Lever, Br.Med.Bull. 51, 226-234 (1995); F. D. Ledley, Hum.Gene Ther. 6, 1129-1144 (1995); A. M. Lever, Br.Med.Bull. 51, 149-166 (1995).

To achieve gene therapy there must be a method of delivering genes to the patient's cells and additional methods to ensure the effective production of any therapeutic genes. There are two general approaches to achieve gene delivery; these are non- viral delivery and virus-mediated gene delivery. Yet another embodiment of present invention involves the use of inhibitors of PPIase activity to manufacture a drug to prevent α-SYN self aggregation or deposition in Lewy bodies in a subject and more particularly to treat or prevent the neurotoxicity and/or cellular degeneration or dysfunction caused by abnormal aggregation of the synaptic protein α-SYN. Such neurotoxicity can for instance result in dementia with Lewy bodies.

Another embodiment of present invention involves the use of molecules which inhibit the activation of FKBP 12 and preferably the use of molecules which inhibit the activation of FKBP52 to manufacture a drug to prevent α-SYN self aggregation or deposition in Lewy bodies in a subject and more particularly to treat or prevent the neurotoxicity and/or cellular degeneration or dysfunction caused by abnormal aggregation of the synaptic protein α-SYN. Such neurotoxicity can for instance result in dementia with Lewy bodies.

The use of FKBP12 and preferably FKBP52 inhibitors to manufacture a drug to prevent or inhibit the formation of α-SYN aggregates is also the subject of present invention.

Another embodiment of present invention is the use of a molecule that inhibits the expression of FKBP 12 and preferably a molecule that inhibits the expression of FKBP 52 selected from the list consisting of an antisense molecule, a RNAi and a ribozyme, for the manufacture of a drug to prevent or treat a disorder of α-SYN aggregation in a subject in need thereof.

Yet another embodiment of present invention involves the use of a molecule that inhibits the activity of FKBP 12 or the use of a molecule that inhibits the activity of FKBP52 selected from the list consisting of an aptamer, an antibody, a fransdominant ligand, or a tetrameric peptide for the manufacture of a drug to prevent or treat a disorder of α-SYN aggregation in a subject in need thereof.

The invention also involves the use of a molecule that inhibits PPIase activity selected from the list consisting of an aptamer, an antibody, a fransdominant ligand, a tetrameric peptide for the manufacture of a drug to prevent or diminish the formation of α-SYN aggregates in a subject in need thereof. Yet another embodiment involves a method of preventing or diminishing α-SYN aggregation disorder in an animal, comprising administering to a mammal an effective amount of inhibitors of PPIase activity in an efficient dose to inhibit α-SYN aggregation and a method of preventing or treating an α-SYN aggregation disorder in an animal, comprising: administering to a mammal of an a FKBP 12 inhibitor in an efficient dose to inhibit α-SYN aggregation.

The present invention provides methods to determine protein aggregation formation through enzymatic process in an in vitro, cellular and in vivo system in order to identify novel inhibitors of said mechanism. Within the scope of this invention, methods to determine the alpha-synuclein anti-aggregation potential of molecules or to screen for drugs with alpha-synuclein anti-aggregation properties by assaying their capacity to interfere with the kinetics of PPIase or FK506-Binding Protein (FKBP) induced alpha- synuclein aggregation are forwarded, as well as methods and drugs to prevent or to diminish the formation of alpha-synuclein self aggregates or the formation of alpha- synuclein deposition.

Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Detailed Description

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following detailed description of the invention refers to the accompanying drawings. The same reference numbers in different drawings identify the same or similar elements. Also, the following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims and equivalents thereof. Some terms and definitions in the present invention will be defined first.

The term "pharmaceutically acceptable" is used adjectivally herein to mean that the modified noun is appropriate for use in a pharmaceutical product.

The term "treatment" refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly.

To inhibit the activity of the gene or the gene product of FKBP custom-made techniques are available directed at three distinct types of targets: DNA, RNA and protein. For example, the gene or gene product of FKBP can be altered by homologous recombination, the expression of the genetic code can be inhibited at the RNA levels by antisense oligonucleotides, interfering RNA (RNAi) or ribozymes, and the protein function can be altered by antibodies or drugs.

The term "a molecule that inhibits the expression" refers here to gene expression and thus to the inhibition of gene transcription and/or translation of a gene transcript (mRNA) such as for example an FKBP gene. Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher.

The term FK506-binding proteins may include, but are not limited to, the below listed FKBPs and FKBP homologues, which include a citation to the references which disclose them. This list is not intended to limit the scope of the invention. Mammalian FKBP- 12 Galat et aL, Eur. J. Biochem., 216:689707 (1993). FKBP- 12.6 Wiederrecht, G. and F. Etzkorn Perspectives in Drug Discovery and Design, 2:57-94 (1994). FKBP- 13 Galat et aL, supra; Wiederrecht and Etzkom, supra. FKBP-25 Galat et al., supra; Wiederrecht and Etzkorn, supra. FKBP-39 Wiederrecht and Etzkom, supra. FKBP-51 Baughman et al., Mol. Cell. Biol., 8, 4395-4402(1995). FKBP-52 Galat et al., supra. Bacteria Legionella pneumophilia Galat et al., supra. Legionella micadei Galat et al., supra. Chlamydia trachomatis Galat et al., supra. E. coli fkpa Home, S.M. and K.D. Young, Arch. Microbiol., 163:357-365 (1995). E. coli slyD Roof et al., J. Biol. Chem. 269:29022910 (1994). E. coli orf 149 Trandinh et al., FASEB J. 6:3410-3420 (1992). Neisseria meningitidis Hacker, J. and G. Fischer, Mol. Micro., 10:445-456 (1993). Streptomyces chrysomallus Hacker and Fischer, supra. Fungal yeast FKBP- 12 Cardenas et al., Perspectives in Drug Discovery and Design, 2:103-126 (1994). yeast FKBP- 13 Cardenas et al., supra, yeast NPR I (FPR3) Cardenas et al, supra. Neurospora Galat et al., supra.

Examples of exogenous forms of cyclophilin include but are not limited to human cyclophilin A, other human cyclophilins, the cyclophilins of other species (i.e., mouse, yeast) derivatized cyclophilin, recombinant cyclophilin, cyclophilin fusion proteins or peptide fragments expressing the domain of cyclophilin which binds to its host cell receptor.

The term "a molecule that inhibits the activity" refers to a molecule which inhibits the activity of FBKP, in this preferably FKBP12 or FKBP52. With "inhibition of expression" to gene expression is understood the inhibition of gene transcription and/or translation of a gene transcript (mRNA) such as for example the FKBP gene, in particular the FBKP 12 or the FKBP52 gene. Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60% , 80%, 90% or even higher. With "inhibiting activity" is referred to the protein that is produced such as FBKP, in this preferably FKBP52. The inhibition of activity leads to diminished interaction. Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher. The inhibition of activity leads to a diminished effect of FKBP 12 or FKBP52 on protein or peptide misfolding and aggregation in neuronal cells or intracytoplasmic or intranuclear protein or peptide aggregation in neuronal cell, for instance α-SYN aggregation. Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher.

The term 'drug to treat' relates to a composition comprising molecules described in this application and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat diseases as indicated above. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The 'drug' may be administered by any suitable method within the knowledge of the skilled man. The preferred route of administration is parental. In parental administration, the drug of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. However, the dosage and mode of administration will depend on the individual. Generally, the drug is administered so that the protein, polypeptide, peptide of the present invention is given at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10 μg kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably, it is given as a bolus dose. Continuous infusion may also be used and includes continuous subcutaneous delivery via an osmotic mini-pump. If so, the drug may be infused at a dose between 5 and 20 μgkg/minute, more preferably between 7 and 15 μg/kg/minute.

In the present invention, we show that protein or peptide misfolding and aggregation or intracytoplasmic or intranuclear protein or peptide aggregation in the neuronal cell, for instance of α-SYN, is greatly enhanced in vitro, in cell and suggest this in vivo when an enzyme of the FK506 binding protein (FKBP) family, such as FKBP 12 or FKBP52 is present.

This large mol.-wt. immunophilin, FK506 binding protein 4 or FKBP52, is a cochaperone protein, which exhibits peptidyl-prolyl cis-trans isomerase (PPIase) activity known to influence steroid hormone receptor functions, and to be down-regulated in stromal cells of HoxalO-/- mice. FKBP52 shows differential uterine cell-specific expression during the periimplantation period and it has been suggests that FKBP52 is important for the attainment of uterine receptivity and implantation. Further-more, FKBP52 shows differential cell-specific expression in the uterus in response to progesterone and/or estrogen consistent with its expression patterns during the periimplantation period. Collectively, these results and the female infertility phenotype of FKBP 52 suggest that a HoxalO-FKBP52 signalling axis is critical to uterine receptivity and implantation (Daikoku, Takiko et al. Molecular Endocrinology (2005), 19(3), 683-697).

Unlike FKBP 12, however, FKBP52 does not mediate the immunosuppressive actions of FK506 (Lebeau, M.C., et al. Biochemical and Biophysical Research Communications 203, pp. 750-755) and, due to its larger size, contains additional numerous functional domains. One such structure is a series of tetratricopeptide repeat (TPR) domains, which serve as binding sites for the ubiquitous and abundant mol. chaperone, Hsp90. It is this property as a TPR protein that best characterizes the known cellular roles of FKBP52. The active PPIase domain shares 55% sequence homology with its smaller, but better-studied cousin FKBP 12 (Callebaut, I., et al Proceedings of the National Academy of Sciences, USA 89, pp. 6270-6274).

The cDNA sequence of FKBP52 was first obtained from rabbit liver. Since then, sequence, hydrophobicity and crystal structure analyses have shown the immunophilin to be composed of four distinct domain.. The first two domains include a functional site for peptidyl-prolyl cis/trans isomerase (PPIase) activity and a PPIase-like region, both similar in structure to the PPIase domain of FKBP 12. Three tetratricopeptide repeat (TPR) domains occupy the third structural domain, while the fourth C-terminal domain contains a putative binding site for calmodulin (Lebeau et al. (1992) Journal of Biological Chemistry 267, pp. 4281-4284).

Some studies suggest a role for the immunophilin, FKBP 12, in neurite outgrowth. For instance after crush injury of facial and sciatic nerve in rats leads to markedly increased FKBP12 levels in the respective nerve nuclei (Avramut M & Achin C L Abstract 778.3 & Biosis Abstract 200400145712). In the light of this mechanism these authors suggest that FK506 acts as a neuroprotective molecule, preventing aggregation. Certain FKBP 12 ligands act as neurothrophic agents (for instance they promote neurite outgrowth in chicken sensory neurons) with a comparable potency as neurothrophic growth factors such as nerve growth factor (NGF) (Hamilton et al. Bioorganic & Medicinal Chemistry Letters Vol.7 No. 13, pp. 1783 - 1790, 1997).

Present invention demonstrates that the members of the FKBP family, and more particularly FKPB52 enhance the formation of the aggregation-prone conformational intermediate, the formation of protofibrils and the protofibril assembly to fibrils in this way resulting into a cell degenerative effect, in neuronal tissues. For instance FKBP 12 clearly enhances the aggregation of α-SYN in neuronal cells. FKBPs are members of the immunophilines, enzymes that bind to immunosuppresantia and have a peptidyl-prolyl isomerase (PPIase) activity. In an unfolded protein, generally around 80% of the prolines in a protein are in the cis conformation, while in a folded protein around 80% are in the trans conformation. The PPIase activity is believed to help this cis-to-trans conformation change of proline, a very energy demanding and thus often rate-limiting step in protein folding. Other members of the immunophilines in humans are the cyclophilins and Pinl, an enzyme that is believed to play a role in Alzheimers disease (P. J. Lu, G. Wulf, X. Z. Zhou, P. Davies, K. P. Lu, Nature 399, 784-788 (1999).

All FKBPs bind selectively to FK506 (Tacrolimus), a powerful immunosuppressant. FK506 strongly and specifically inhibits the PPIase activity of most FKBPs. A tissue distribution of different human FKBPs revealed that two forms are expressed in high amounts in the brain; FKBP12 and FKBP52. Further research showed that the regions in the brain with the highest amounts of protein are the SN and the deep grey matter for both FKBPs.

Data of the present invention have demonstrated a clear role for the FKBP and more particularly FKBP 52 in disorders of peptide misfolding and aggregation of intracytoplasmic or intranuclear protein or peptide aggregation in cells, more particulary in neuronal cells. This cell aggregates can lead to cell degeneration disorders such as PD, LB dementia, CAD, OPMD, HD, A.L.S. and artherosclerosis. Here we demonstrate that this role is exerted through enhancing the aggregation rate of α-SYN because folding of α-SYN implies in many cases the initiation of aggregation of the protein. In vitro aggregation studies with recombinant purified proteins showed that α-SYN aggregation is enhanced when a protein of the FKBP family is present. Both an E.coli PPIase and the human FKBP 12, for instance, clearly enhance α-SYN aggregation. The same will be true for other members of this family. Moreover we demonstrated that FK506 clearly counteracts the effect of the bacterial PPIase and human FKBP 12 on α-SYN aggregation. Also human FKBP52 has been demonstrated to affect the aggregation of α- SYN. Disorders of peptide misfolding and aggregation or protein or peptide aggregation in a cell more particularly in a neuronal cell, which can be treated in accordance with the invention, include codon reiteration mutation disorders, in particular polyQ expansion disorders such as Huntington's disease, spinocerebellar ataxias types 1, 2, 3, 6, 7, and 17, Kennedy's disease and dentatorubral- pallidoluysian atrophy. These disorders are characterized by the aggregation of mutant proteins which contain an expanded tract of repeated glutamine residues. For example, HD is characterized by an expanded polyQ stretch in exon 1 of the Huntington gene.

The disorders of peptide misfolding and aggregation or protein or peptide aggregation in a cell, more particularly in a neuronal cell, which can be treated in accordance with the invention, also include polyA expansion disorders. These disorders are characterized by the aggregation of mutant proteins which contain an expanded tract of repeated alanine residues. For example, oculapharyngeal muscular dystrophy (OPMD) is characterized by a polyadenine (polyp) expansion mutation in the polyadenine binding protein 2 gene.

Other Disorders of peptide misfolding and aggregation or protein or peptide aggregation in a cell, more particularly in a neuronal cell, which may be treated in accordance with the invention may include α-synucleinopathies such as Parkinson's disease, LB variant Alheimer's disease and LB dementia. These are disorders characterized by the accumulation of cytoplasmic aggregates called Lewy bodies, which comprise a-synuclein.

Disorders of peptide misfolding and aggregation or protein or peptide aggregation in a cell, more particularly in a neuronal cell, that may be treated in accordance with the invention also include prion disorders such as CAD.

Examples

Example 1. Cloning and purification of α-SYN

A lentiviral vector transfer plasmid, pCHMWS, containing the gene of α-SYN was already present in the lab Lauwers et al. 2002; Brain Pathol; 13; 364-372). With the restriction sites Xhol and Hindlll the α-SYN gene was cloned in pRSET B, a prokaryotic expression vector with N-terminal His-tag. Sequencing confirmed the correct sequence and reading frame of the gene. DH5α-Tl resistant heat sensitive E. coli cells were transformed with the plasmid. In the presence of lOOμg/mL ampicillin, transformed cells were grown to an 8L culture at 30°C. When reaching an OD of 0.8, the culture was induced with 1 mM IPTG and left shaking for another 3 hours. Cells were harvested by centrifugation at 10000 g during 15 minutes (Sorvall® RC-24 Refrigerated Superspeed Centrifuge; Du Pont Instruments, Wilmington, VS). Pellets were solubilized in 35 mL sonication buffer (20 mM Hepes (Sigma, MO, VS), 100 mM NaCl, ImM PMSF (Aldrich), 0.05 mM EDTA (Chimica, Geel), pH 7.4) and frozen overnight.

The cell suspension was sonicated with a sonifier (Model 450 Sonifier, Branson Ultrasonics Corp. Danburym CT, VS) until the cell suspension lost its high viscosity (40 seconds, 35 times). This cell lysate was heated to 65°C during 45 minutes and then centrifuged (30 min., 4°C, 39100g, Sorvall® RC-24 Refrigerated Superspeed Centrifuge). The supematans was filtered through a 22μM filter (millipore) and applied on a FPLC (Bio-RAD) NiNTA column. Washing buffer for the column is 20 mM Hepes, 20 mM imidazole, 100 mM NaCl, pH 7.4. Elution buffer contained 20 mM Hepes, 250 mM imidazole, 100 mM NaCl, pH 7.4. The fractions containing protein were analysed with SDS-PAGE (NuPage gels, BIS-TRIS 4-12%, Invitrogen). Fractions containing α-SYN (migrates at 19 kDa) were pooled and applied on a FPLC anionic exchange column (washing buffer 20 mM Hepes, 100 mM NaCl, pH 8; elution buffer 20 mM Hepes, 1M NaCl, pH 8) to remove an E. coli impurity. Elution fractions containing protein are again analysed on SDS-PAGE and the fractions containing pure protein are pooled and applied on a desalting column (HiPrep TM 26/10 Desalting) prewashed with 20 mM ammoniumbicarbonate. The peak fraction from this column is pooled and the concentration of α-SYN is determined at an OD of 276 nM (ε = 5800 M^cm^"1). Aliquots containing 1 mg α-SYN are frozen overnight at -70° and then lyophilised (Heto DRY WINNER model DW3 Heto Holetn A S).

Example 2. Fluorescence Correlation Spectroscopy measurements

To visualise the aggregation of α-SYN, a technique called Fluorescence Correlation

Spectroscopy (FCS) was used. In this technique the diffusion of a fluorescent molecule, travelling through a fixed volume of about 0.2 femtoliter, is followed with an inverted confocal microscope. A very low concentration of the fluorescent molecules combined with the small detection volume gives a very good signal-to-noise ratio, which permits sensitive detection at the single molecule level. The measured diffusion time of the fluorescent molecule through the detection volume is linked via the diffusion coefficient to the size of the molecule.

To be able to do FCS with α-SYN, the point mutation S42C was introduced in the α-SYN gene. Purification of this mutant protein proceeded in exactly the same way as the wild type form with the exception that, before running the anionic exchange column, the protein is chemically labelled with the fluorescent dye Bodipy Maleimide 493/503 (Molecular Probes, Eugene, US). This thiol-labelling is very specific for the inserted cystein because wild type α-SYN does not contain any cysteins. A 10 time molar excess of dye is added to the protein. The sample is stirred in a dark tube at room temperature for two hours. Afterwards, the sample is applied on the anionic exchange column as described above. For some measurements, it was necessary to fluorescently label human FKBP 12. To be able to discriminate between the fluorescent light coming from the Bodipy dye attached to α-SYN and the light coming from labeled FKBP 12, a red dye was chosen. FKBP 12 was stirred in a dark tube at room temperature for two hours with Cy5 NHS-ester (Amersham biosciences, Uppsala, Sweden). To separate unreacted free dye from labeled protein, the sample was applied on a PD10 desalting column (Amersham Pharmacia, Uppsala, Sweden).

In every FCS measurement the ratio of non-labelled wild type protein to labelled S42C α- SYN is about 1000:1. Before each experiment, all aggregates are removed from the sample. This is done by a pH jump from 7 to 11 and back for 10 minutes. This dissolves small oligomers formed during purification. A subsequent centrifugation step removes the larger aggregates. When incubating α-SYN at 37°C under continuous stirring for several hours, the diffusion time of the protein increases as larger complexes of α-SYN molecules are formed which move slower through the detection volume. This increase in the diffusion time is afterwards fitted to a sigmoidal model with 4 parameters (SigmaPlot version 8.0). Example 3 Results on Purification of α-SYN

After making first attempts to purify α-SYN, a consistent impurity was observed. When the same sample was applied both on a NUPAGE gel and on western blot, one band was not present in the westem blot. Mass-spectrometry analysis allowed unambiguous identification of the impurity as E. coli FKBP-type peptidyl-prolyl cis-trans isomerase slyD (PPIase). This is a heat-stable histidine-rich enzyme, which explains why it was co- purified. When running an anionic exchange column after the Ni-NTA column, the PPIase is successfully removed (Fig. 2).

Example 4 Results on the quality control of the recombinant α-SYN

To determine the quality of the purified α-SYN, an aggregation test was performed in the presence of spermine, a polycation that enhances the aggregation rate of α-SYN through the shielding of the negative charges in the C-terminus. Normally, the electric repulsion between strongly negatively charged α-SYN molecules inhibits the formation of fibrils in vitro. The results were in accordance with the experiments performed by Anthony et al. (T. Antony et al., J Biol.Chem. 278, 3235-3240 (2003). The half-time of the aggregation of α- SYN in the presence of 250 μM spermine was 2.2 hours while Anthony et al. found a half- time of aggregation of 5 hours in the presence of 100 μM spermine (Fig. 3).

Example 5 PPIase enhances the aggregation rate of α-SYN

FCS experiments on the α-SYN with and without the contaminating PPIase showed that the sample with the PPIase aggregated within 8 hours while the pure α-SYN did not (data not shown). Therefore the PPIase was purified separately following the same protocol as for the purification of α-SYN using non-transfected DH5α-Tl cells. Aggregation experiments on α-SYN (70 μM) with 6 nM PPIase or 6 nM ovalbumine as a control showed that the PPIase did have a positive effect on the aggregation rate of α-SYN (Fig. 4).

To determine the specificity of the effect on the aggregation, a specific inhibitor for the enzymatic activity of PPIase was added to the samples. FK506 is a molecule that inhibits the PPIase activity of the FKBPs. In the samples where FK506 was added, the formation of large aggregates was inhibited. If a 7 time molar excess of FK506 to PPIase was added, aggregation was inhibited completely (Fig. 5).

Example 6 Human FKBP 12 enhances the aggregation rate of a-snc

The previous experiments have shown that the E. coli FKBP, PPIase, has a strong and specific effect on the aggregation of human α-SYN. To determine a possible physiological effect, the same experiments were repeated with a human FKBP form. Two members of the FKBP family are expressed in high levels in the brain, FKBP 12 and FKBP52 (respectively 12 and 52 kDa). Both forms are found preferentially in the substantia nigra and the deep grey matter, which are also the regions most affected in PD. We investigated the possible effect of FKBP 12 (a protein that often co-localizes with alpha-synuclein in Lewy bodies and Lewy neurites in PD brains (M. Avramut and C. L. Achim, Physiol Behav. 77, 463-468 (2002)).on α-SYN aggregation by using a human recombinant GST- FKBP12 fusion protein. When adding increasing concentrations of FKBP 12 to α-SYN, the aggregation rate of α-SYN increased (Fig. 6 and Fig. 7). In the presence of the inhibitor FK506, the effect of FKBP 12 on the aggregation of α-SYN decreased (Fig. 8). When labelling both proteins; α-SYN with a green dye (Bodipy Maleimide 493/503) and human FKBP 12 with a red dye (Cy5 NHS ester), the diffusion time of both proteins can be followed in an FCS measurement during the aggregation of α-SYN. When adding 1 μM FKBP 12 to 70 μM α-SYN, it can be seen that the diffusion time of α-SYN increases during the incubation at 37°C, while the diffusion time of FKBP12 stays constant (Fig 9). This demonstrates that the effect of FKBP 12 on α-SYN is due to an enzymatic effect and not due to direct binding of FKBP 12 to α-SYN because if the latter was true, the diffusion time of FKBP 12 would also increase when the protein binds to aggregated α-SYN. Moreover since inhibition of FKBP 12 for instance by FK506 results in vitro in diminishing α-SYN aggregation one can expect that the prevention of α-SYN aggregation is a direct action unrelated to α-SYN clearance or degradation by dysfunction of the cell mechanism of autophagy or proteasomal (Webb et al. The Journal of Biology Chemistry Vol. 278, No 27, July 4, pp 25009 - 25013, 2003). Example 7 Cell culture experiments

To investigate the biological relevance of above mentioned results, a cell culture model for PD was used (Hasegawa T. accelerated a-snc aggregation brain research 1013 (2004) 51- 59). Human neuroblastoma SHSY5Y cells are plated on round glass plates (= day 0). On day 1, cells are differentiated for 6 days with 10 μM retinoic acid. From day 7 - 12, cells are exposed to 0 - 5 mM FeCl₂. Retinoic Acid induces differentiation of the cells, thereby making them non-growing and non-dividing and thus a better model for human brain cells. RA works by inhibition of the mitochondria and is therefore also a source of oxidative stress. FeCl₂ is used to induce extra oxidative stress to the cells. From day 1, cells are also treated with various concentrations FK506 (0-160 nM). Every two days, media are replaced. At day 12, cells are fixed and incubated overnight with primary antibody (anti-α- SYN and anti-FKBP12 or anti-FKBP52). After extensive washing the plates are incubated for three hours with secondary antibody (Alexa Fluor 488 or 633 conjugated antibodies). Thereafter, the glass plates are mounted on a cover glass. Fluorescence is detected with the 488 Argon Ion and the 633 Helium Neon laser with the LSM software of Confocor II from Zeiss.

The percentage of cells containing α-SYN aggregates in the cytoplasm is determined. To obtain this percentage, ~800 cells were counted per condition. The result of this experiment is given in Figure 10. It can clearly be seen that there is a difference between cells receiving FK506 treatment and cells not receiving FK506 when not adding FeCl₂ to the medium; i.e. when the only source of oxidative stress is retinoic acid. Adding higher concentrations of FK506 does not result in a further large decrease in the percentage of aggregate containing cells. The same effect, though less pronounced, can also be seen when 0,2 μM FeCl₂ is added to the medium. When adding more FeCl , the effect of FK506 disappears. The result of this experiment shows that FK506 treatment results in a lower amount of α-SYN aggregate positive cells when SHSY5Y cells are put under moderate oxidative stress.

The expression of FKBP 12 and FKBP52 in the cells was monitored, both before and after the appearance of α-SYN aggregates. It can be seen that FKBP 12 expression is situated in the whole cell with a higher expression in the nucleus. The localization does not change when aggregates are formed (Fig 11). On the other hand, a high FKBP52 expression can be seen in the cytoplasm in cells without aggregates. After α-SYN aggregates are induced, the expression level of FKBP52 seems to decrease (Fig. 12). This change in expression pattern and/ or level demonstrates that interaction occurs between α-SYN and FKBP52.

Example 8. Mouse model

To asses the possible effect of immunophilin ligands on the aggregation of α-SYN in vivo, the α-SYN mouse model of Lauwers et al. was used (E. Lauwers et al., Brain Pathol. 13, 364-372 (2003)). A Lentiviral vector containing the α-SYN cDNA was constructed and injected in the striatum of black six mice. The mice were divided into 5 groups, described in Fig. 13

2 μL of vector was stereotactically injected in the striatum of 11 weeks old black six female mice. Treatment with FK506 begins 1 week following surgery. Dose selection was based on the results of previous studies to include a high functionally active dose, a lower functionally active dose and a borderline/possibly inactive dose. This selection allows for high probability of dose-response characterization.

FK506 is dissolved in suitable vehicle (1% Cremophor EL, 4% ethanol) and administered in volumes 10 μL per g body weight by oral gavages once daily. Gavage is the preferred method of oral administration for oil soluble drugs. The gavage needles for mice +/- 25 g are 22-gage, with a tip of 1.25 mm and are 25 mm long. They are used with a 1 mL syringe.

Long plasma half-life (>20 hours) and linear pharmacokinetic profile (CYP3A4 metabolised) allow for once daily administration. Bioavailability via the oral route is acceptable at 50%. Control animals in groups 1 and 2 receive the drug vehicle similarly. FK506 (Prograf®) was purchased from Fujisawa Healthcare, Inc. (Germany). Capsules were opened and the content was dissolved in the vehicle solution in concentrations of 0.05 mg/mL, 0.2 mg/mL and 0.8 mg/mL. Treatment was performed daily for 5 months. After 5 months, animals were perfused transcardially and the brain was fixed in 4% paraformaldehyde overnight. For further storage, brains were put in PBS with 0.1% sodium azide. All brains were sliced in 50 μm coronal slices with a Leica VT 1000S vibratome and stained immunohistochemically. In the striatum, every fifth section was treated with 3% hydrogen peroxide and incubated overnight with a polyclonal rabbit anti α-SYN (1:5000, Chemicon, Temecula Calif USA) in 10% normal swine serum. Then the sections were incubated with biotinylated secondary antibody (DAKO, Glostrup, Denmark), followed by incubation with Strept-ABC-HRP complex (DAKO). Detection was with diaminobenzidine (DAB) using H2O2 as a substrate.

These stained sections were analysed in two ways. First the volume of the transduced brain area was quantified by a stereological procedure based on the Cavalieri principle. For each animal, serial sections (minimum five) with an interval of 500 μm centered around the injection site were analyzed by means of a Bioquant image Analysing System (R&M Biometrics, Nashville, TN, USA) connected with a CCD video camera to the microscope. A point-counting grid was placed over the screen upon which the entire transduced brain region was displayed from a low-power objective. An example of such a region in shown in Fig. 14. In this figure, it is also possible to follow the needle tract made during injection. Points overlying α-SYN positive cells and fibers were counted. The transduced volume was calculated by multiplying the sum of the counted points with the distance between the counted sections and the area associated with each point on the grid. The results of this experiment are displayed in Fig. 15. This shows that the transduced volume does not differ when treating the animals with different concentrations of FK506, but that there is a smaller volume present when mice were not treated with FK506. This is also confirmed via statistical testing. When performing a non-parametric Kruskal Wallis test (α = 0.05), there is no difference between groups 2, 3 and 4 (groups receiving FK506 treatment): p- value = 0.456. The interpretation of this result is as follows. When injecting all mice with an equal volume of lentiviral vector, an equal amount of cells are transduced with α-SYN. If FK506 protects against the formation of α-SYN aggregates, which ultimately leads to the death of the cell, less neurons will die when mice are treated with FK506. This hypothesis is confirmed by the data. A second means of analysis was to investigate the amount of α-SYN positive cells exhibiting Lewy-body like aggregates in the cytoplasm. Therefore, slices were investigated with a lOOx magnifying lens. 50 neurons expressing α-SYN were counted and for each neuron, it was determined if aggregates were present. An example of α-SYN positive cells with and without aggregates are displayed in Fig. 16. The results of this experiment are shown in Fig. 17. There again, it can be seen that FK506 has an influence on the percentage of aggregate positive cells but that increasing the dose has no further effect. Statistical analysis (non parametric Kruskal Wallis test) showed that there was indeed no difference between groups 2, 3 and 4 (p-value = 0.884), but that there was a significant difference between groups receiving and not receiving FK506 treatment (p-value = 0.018). This shows effectively that FK506 treatment causes a reduction in the amount of α-SYN aggregates formed. From our in vitro and in cell data we believe that this reduction is caused through inhibition of an FKBP protein, in particular FKBP52, that normally accelerates formation of α-SYN aggregates. A summary of the results from the mouse experiment are given in Fig. 18

Example 9 PPIase inhibitors

Small molecule PPIase inhibitors have been described in WO9640633 as derivatives of the basic structures formule 1

where Rl is a C₁-C9, straight or branched chain alkyl or alkenyl group optionally substituted with C₃-C₈ cycloalkyl, C₃ or C₅ cycloalkyl, C₅-C , cycloalkenyl, or Ari, where said alkyl, alkenyl, cycloalkyl or cycloalkenyl groups may be optionally substituted with C₁-C₄ alkyl, C₁-C₄ alkenyl, or hydroxy. and where Ar, is selected from the group consisting of 1- napthyl, 2-napthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2thienyl, 3-thienyl, 2-, 3-, or 4- pyridyl, or phenyl, having one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, triflucromethyl, Cι,-C₆, straight or branched alkyl or alkenyl, C₁-C₄, alkoxy or C₁-C₄, alkenyloxy, phenoxy, benzyloxy, and amino;

X is oxygen, sulfur, methylene (CH₂), or H₂;

Y is oxygen or NR₂, where R is hydrogen or Cι-C₆, alkyl; and Z is a Ci-Cβ straight or branched chain alkyl or alkenyl, wherein the alkyl chain is substituted in one or more positions with Ar, as defined above, Cι-C₈, cycloalkyl, cycloalkyl connected by a Cι-C₆ straight or unbranched alkyl or alkenyl chain, or Ar₂ where Ar₂ is selected from the group consisting of 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2- thiazolyl, 2-thienyl, 3-thienyl, 2-, 3-, or 4-pyridyl, or phenyl, having one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nifro, trifluoromethyl, Ci-Cβ, straight or branched alkyl or alkenyl, C1-C₄ alkoxy or C1-C4 alkenyloxy, phenoxy, benzyloxy, and amino; Z may also be the fragment:

where

R₃ is selected from the group consisiting of straight or branched alkyl Ci-Gg optionally substituted with C₃-C₈ cycloalkyl, or Ar, as defined above; X₂ is O or NR₅, where R₅ is selected f rom the group consisting of hydrogen, -C₅ straight or branched alkyl and alkenyl;

R₄ is selected from the group consisting of phenyl, benzyl, -C₅ straight or branched alkyl or alkenyl, and C1-C₅ straight or branched alkyl or alkenyl substituted with phenyl; or pharmaceutically acceptable salts or hydrates thereof

PPIase inhibitors can also be molecules of the group consisting of

3-(2,5-dimethoxyphenyl)-l-propyl (2S)-l-(3,3dimethyl-l,2-dioxopentyl)-2- pyrrolidinecarboxylate, 3-(2,5-dimethoxyphenyl)-l-prop (E)-enyl (2S) (3,3-dimethyl-l,2-dioxopentyl) pyrrolidinecarboxylate,

2-(3 ,4,5-trimethoxyphenyl)- 1 -ethyl (2S)-l-(3 ,3 -dimethyl- 1 ,2-dioxoρentyl) pyrrolidinecarboxylate,

3-(3-Pyridyl)-l-propyl (2S) (3,3-dimethyl-l,2dioxopentyl) pyrrolidinecarboxylate, 3-(2-Pyridyl)-l-propyl (2S)-I-(3, 3 -dimethyl- l,2dioxopentyl) pyrrolidinecarboxylate, 3-(4-Pyridyl)-l-propyl (2S)-I-(3,3-dimethyl-i,2dioxopentyl) pyrrolidinecarboxylate, 3-ρhenyl-l-propyl (2S) (2-tert-butyl-l,2dioxoethyl) pyrrolidinecarboxylate, 3 -phenyl- 1 -propyl (2 S)-l-(2-cyclohexylethyl- 1 ,2dioxoethyl) pyrrolidinecarboxylate, 3-(3-pyridyl)-l-propyl (2S)-l-(2-cyclohexylethyll ,2-dioxoethyl) pyrrolidinecarboxylate, 3 -(3 -pyridyl)- 1 -propyl (2S)-l-(2-tert-butyl-l,2dioxoethyl)-2- pyrrolidinecarboxylatel 3,3-diphenyl-i-propyl (2S) (3,3-dimethyl-l,2dioxopentyl) pyrrolidinecarboxylate, 3 -(3 -pyridyl)- 1 -propyl (2S) (2-cyclohexyl-l,2dioxoethyl) pyrrolidinecarboxylate, 3 -(3 -Pyridyl)-i-proρyl (2 S)-N-([2-thienyl] glyoxyl)pyrrolidinecarboxylate, 3,3-Diphenyl-i-propyl (2S)-I-(3,3-dimethyl-l ,2dioxobutyl) pyrrolidinecarboxylate, 3,3-Diphenyl-l-propyl (2S)-l-cyclohexylglyox yl2-pyrrolidinecarboxylate, and 3,3-Diphenyl-l-propyl (2S) (2-thienyl)glyoxyl2-pyrrolidinecarboxylate.

Inhibitors of PPIase can be also selected of the group of (lr)-l-Cyclohexyl-3 -Phenyl- 1- Propyl (2s)-l-(3,3-Dimethyl- l,2-Dioxopentyl)-2-Piperidinecarboxylate, (lr)-l,3- Diphenyl-1 -Propyl (2s)-l-(3,3-Dimethyl-l,2- Dioxopentyl)-2-Piperidinecarboxylate, (21s)- l-Aza-4,4-Dimethyl-6,19-Dioxa-2,3,7,20- Tetraoxobicyclo[19.4.0]pentacosane, and (21s)- 1 -Aza-4,4-Dimethyl-6, 19-Dioxa-2,3 ,7,20- Tetraoxobicyclo[ 19.4.0]pentacosane.

Inhibitors of FKBP 12 can be selected of the group of L-Proline, 1 -(3 ,3 -dimethyl- 1,2- dioxopentyl)-, 3-(3-pyridinyl)ρroρyl ester (9CI), L-Leucine, N-[[(3R)-4-[(4- methylphenyl)sulfonyl]-3-thiomorpholinyl]carbonyl]-, ethyl ester (9CI) and L-Leucine, N- [[(3R)-4-[(4-methylphenyl)sulfonyl]-3-thiomorpholinyl]carbonyl]-, ethyl ester (9CI)

There are three principal structural classes of immunosuppressant drugs related in structure to cyclosporin A, FK506, and rapamycin. Though FK506 and cyclosporin A bind to distinct FKBP's, they both act as immunosuppressants by inhibiting calcineurin. Rapamycin binds with very high affinity to FKBP 12, but the drug-immunophilin complex does not in turn bind to calcineurin. Instead, immunosuppressing actions result from the rapamycin-FKBP12 complex binding to a recently identified and cloned protein designated RAFT-1 (rapamycin and FK506 target) and also designated FRAP (E. J. Brown et al., Nature 369, 756-758 (1994); Y. Chen et al., Biochem.Biophys.Res Commun. 203, 1-7 (1994) and D. M. Sabatini, H. Erdjument-Bromage, M. Lui, P. Tempst, S. H. Snyder, Cell 78, 35-43 (1994)). Because rapamycin binds potently to FKBP12 but does not inhibit calcineurin, it can serve as an antagonist to FK506. There exist non-immunosuppressive derivatives of rapamycin. One of these, WAY- 124,466, a triene derivative of rapamycin, binds with high affinity to FKBP 12 and inhibits PPIase activity, but is devoid of immunosuppressant actions. Cyclosporin A is a large cyclic undecapeptide. The mere addition of a methyl group to an alanine at the 6 position results in an agent that does not inhibit calcineurin and lacks immunosuppressive effects, though it inhibits the PPIase activity of cyclophilin to a similar extent as cyclosporin A (J. P. Steiner et al., Proc.Natl.Acad Sci U.S.A 94, 2019-2024 (1997)).

Other inhibitors of peptidyl-prolyl isomerase or PPIase enzyme activity are for instance selected of molecules such as pipecolic acid derivative of Way- 124,666, rapamycin, FK506, Rap-Pa, SLB-506, GPI1046, L685818, molecule 3-84, and molecules 86-88 as described in US20020052372.

Other inhibitors of peptidyl-prolyl isomerase or PPIase enzyme activity are for instance pipecolic acid derivative of Way- 124,666, rapamycin, FK506, Rap-Pa, SLB-506, GPI1046, L685818, molecule 3-84, and molecule 86-88 as described in US20020052372

Others are the molecules provided in the publications: the neurotrophic N-glyoxylprolyl ester molecules having an affinity for FKBP-type immunophilins (US20040049046A1, US6140357A1); neurotrophic low molecular weight, small molecule sulfonamide molecules having an affinity for FKBP- type immunophilins (US20030114492A1, US6245783B1); neurotrophic pipecolic acid derivative molecules having an affinity for FKBP-type immunophilins (US20030114365A1); neurotrophic low molecular weight, small molecule heterocyclic ester and amides having an affinity for FKBP-type immunophilins (US20030032635A1); neurotrophic N-glyoxyl-prolyl ester molecules having an affinity for FKBP-type immunophilins (US6500959B1, US5859031A1); neurotrophic pipecolic acid derivative molecules having an affinity for FKBP-type (US6500843B2); neurotrophic low molecular weight, small molecule N-oxides of heterocyclic esters amides, thioesters, and ketones having an affinity for FKBP-type immunophilins (US6486151B2, US20010036942A1); low molecular weight, small molecule cyclic esters and amides having an affinity for FKBP-type immunophilins (US6462072B1); neurotrophic low molecular weight, small molecule N-linked ureas and carbamates of heterocyclic thioesters having an affinity for FKBP-type immunophilins (US20020049199A1, US6184243B1, US6274607B1); neurotrophic low molecular weight, small molecule N-linked sulfonamides of heterocyclic thioesters having an affinity for FKBP-type immunophilins (US20020049193A1, US6121273A1, US6294551B1); inliibitory activity of novel α,α-difluoroacetamido molecules that are neurotrophic agents (i.e. molecules capable of stimulating growth or proliferation of nervous tissue) and that bind to immunophilins such as FKBP12 and inhibit their PPIase activity (US6239146B1, US6096762A1); inhibitory activity of novel pyrrolidinemethyl diamide and carbamate molecules that are neurotrophic agents (i.e. molecules capable of stimulating growth or proliferation of nervous tissue) and that bind to immunophilins such as FKBP 12 (US6228872B1, EP1129070A1); neurotrophic low molecular weight, small molecule heterocyclic esters and amides having an affinity for FKBP-type immunophilins (US6218544B1); nematode "cyclophilin- like proteins (CLP)", in a method for identifying molecules capable of binding to and or inhibiting the peptidyl-prolylcistransisomerase activity of these proteins (US6127148A1); neurotrophic low molecular weight, small molecule piperidine and pyrrolidine sulfonamide molecules having an affinity for FKBP- type immunophilins (US5968957A1, WO0112622A1); neurotrophic molecules having an affinity for FKBP-type immunophilins (US5614547A1).

Enabling methods for expressing or overexpression of FKBP is cell lines have been described. Below we list references to methods of FKBPs (over)expression, which include a citation to the references which disclose them. This list is not intended to limit the scope of the invention. For instance, immunophilin FKBP-52 (a mouse FKBP-52) has been overexpressed in Spodoptera frugiperda insect cells (Sf9 cells) with the baculovirus expression system (ES Alnemri, et al. Proceedings of the National Academy of Sciences (1993), Vol 90, 6839-6843); Christopher H. G. et al. Biochem. J. (2003) 370 (579-589) describe the cloning of cDNA encoding human FKBP 12 and FKBP 12.6 and the expression of FKBP12 and FKBP12.6 in Chinese Hamster Ovary ( CHOhRyR2 cell lines) cells; Insect cells (Sf9) have been used for co-expressing of a FKBP (FKBP 12) with other recombinant factors (Ondrias K et al. Annals of the New York Academy of Sciences 853:149-156 (1998)) Also technologies of (over)expression of other PPIases are available. Bugli F, et al. Protein Expr Purif. 1998 Apr;12(3):340-6 describe the cloning and biochemical characterizations and expression in Sf9 insect cells of recombinant cyclophilin proteins (CYPs) from Schistosoma mansoni; Recombinant cyclophilin 40 has been expressed in E. coli (L. Kieffer et al. J. Biol. Chem. 1992 267 5503 and L. Kieffer et al. J. Biol. Chem. 1993 268 12303); Hak-Sun YU, et al The Korean Journal of Parasitology Vol. 40, No. 3, 131-138, September 2002 disclose the cloning and expression of Giardia intestinalis cyclophilin; Santosa Anne Navarrete et al., Biology of Reproduction 62, 1-7 (2000) have described the expression of Cyclophilin 18 in Rabbit Blastocysts.

The most preferred cells for screening molecules which inhibits the aggregation or the aggregation rate of cellular proteins (or peptides) or of protein (or peptide) folding intermediates of cellular proteins (or peptides) are neuronal cell types, in particular neuroblastoma cells (e.g. SKNSH cells) that overexpress wild type, mutant or antisense protein of target, for instance from the group of prion-, tau-, amyloid, β-synuclein, α - synuclein proteins or of synaptophysin, presenilin, huntingtin, ubiquitin, glial fibrillary acidic protein and tau eventually after transduction with the respective lentiviral vector. These cell lines can be used for evaluating the effect on cellular protein aggregation of molecules, in particular small molecules and including nucleic acids that inhibit PPIases, such as FKBPs.

The screening tool can also be based on one or more target cells from the group consisting of blast cells, cloned cells, fertilized ova, placental cells, keratinocytes, basal epidermal cells, hair shaft cells, hair-root sheath cells, surface epithelial cells, basal epithelial cells, urinary epithelial cells, salivary gland cells, mucous cells, serous cells, von Ebner's gland cells, mammary gland cells, lacrimal gland cells, ceruminous gland cells, eccrine sweat gland cells, apocrine sweat gland cells, Moll gland cells, sebaceous gland cells, Bowman's gland cells, Brunner's gland cells, seminal vesicle cells, prostate gland cells, bulbourethral gland cells, Bartholin's gland cells, Littre gland cells, uterine endometrial cells, goblet cells of the respiratory or digestive tracts, mucous cells of the stomach, zymogenic cells of the gastric gland, oxyntic cells of the gastric gland, insulin- producing β cells, glucagon- producing α cells, somatostatin- producing δ cells, pancreatic polypeptide- producing cells, pancreatic ductal cells, Paneth cells of the small intestine, type II pneumocytes of the lung, Clara cells of the lung, anterior pituitary cells, intermediate pituitary cells, posterior pituitary cells, hormone secreting cells of the gut or respiratory tract, thyroid gland cells, parathyroid gland cells, adrenal gland cells, gonad cells, juxtaglomerular cells of the kidney, macula densa cells of the kidney, peripolar cells of the kidney, mesangial cells of the kidney, brush border cells of the intestine, striated duct cells of exocrine glands, gall bladder epithelial cells, brush border cells of the proximal tubule of the kidney, distal tubule cells of the kidney, nonciliated cells of ductulus efferens, epididymal principal cells, epididymal basal cells, hepatacytes, fat cells, type I pneumocytes, pancreatic duct cells, nonstriated duct cells of the sweat gland, nonstriated duct cells of the salivary gland, nonstriated duct cells of the mammary gland, parietal cells of the kidney glomerulus, podocytes of the kidney glomerulus, cells of the thin segment of the loop of Henle, collecting duct cells, duct cells of the seminal vesicle, duct cells of the prostate gland, vascular endothelial cells, synovial cells, serosal cells, squamous cells lining the perilymphatic space of the ear, cells lining the endolymphatic space of the ear, choroids plexus cells, squamous cells of the pia-arachnoid, ciliary epithelial cells of the eye, comeal endothelial cells, ciliated cells having propulsive function, ameloblasts, planum semilunatum cells of the vestibular apparatus of the ear, interdental cells of the organ of Corti, fibroblasts, pericytes of blood capillaries, nucleus pulposus cells of the intervertebral disc, cementoblasts, cementocytes, odontoblasts, odontocytes, chondrocytes, osteoblasts, osteocytes, osteoprogenitor cells, hyalocytes of the vitreous body of the eye, stellate cells of the perilymphatic space of the ear, skeletal muscle cells, heart muscle cells, smooth muscle cells, myoepithelial cells, red blood cells, megakaryocytes, monocytes, connective tissue macrophages, Langerhan's cells, osteoclasts, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cells, plasma cells, helper T cells, suppressor T cells, killer T cells, immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, killer cells, rod cells, cone cells, inner hair cells of the organ of Corti, outer hair cells of the organ of Corti, type I hair cells of the vestibular apparatus of the ear, type II cells of the vestibular apparatus of the ear, type II taste bud cells, olfactory neurons, basal cells of olfactory epithelium, type I carotid body cells, type II carotid body cells, Merkel cells, primary sensory neurons specialized for touch, primary sensory neurons specialized for temperature, primary neurons specialized for pain, proprioceptive primary sensory neurons, cholinergic neurons of the autonomic nervous system, adrenergic neurons of the autonomic nervous system, peptidergic neurons of the autonomic nervous system, inner pillar cells of the organ of Corti, outer pillar cells of the organ of Corti, inner phalangeal cells of the organ of Corti, outer phalangeal cells of the organ of Corti, border cells, Hensen cells, supporting cells of the vestibular apparatus, supporting cells of the taste bud, supporting cells of olfactory epithelium, Schwann cells, satellite cells, enteric glial cells, neurons of the central nervous system, astrocytes of the central nervous system, oligodendrocytes of the central nervous system, anterior lens epithelial cells, lens fiber cells, melanocytes, retinal pigmented epithelial cells, iris pigment epithelial cells, oogonium, oocytes, spermatocytes, spermatogonium, ovarian follicle cells, Sertoli cells, and thymus epithelial cells, or combinations thereof.

Suitable animal models and methods of locoregional transgenese to study, validate or screen for molecules which inhibits the aggregation or the aggregation rate of cellular proteins (or peptides) or of protein (or peptide) folding intermediates of cellular proteins (or peptides) have been described by Debyser et al US20040093623 Non-human animal disease models. It is particularly possible to overexpress or silence the protein of target in selected tissues of said animal models.

Drawing Description

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:

Figure 1. A. SDS-Page (NUPAGE gel) analysis of the NiNTA-column eluted protein fractions on Lane 1 & 2, presenting at al the approximate MW of α-SYN (17 kDa), at a2 and a3 double or triple aggregates of α-SYN and at bl a co-eluted E. coli impurity. The standard MW reference is presented in Lane 3. B. Western blot analysis of the recombinant α-SYN (Lane 1 & 2).

Figure 2. NUPAGE BIS-TRIS gel of recombinant α-SYN after anionic exchange column with the almost total disappearance of the E. coli impurity. Mass spectrometry analysis allowed unambiguous identification of the impurity as E. coli FKBP-type peptidyl-prolyl cis-trans isomerase slyD (PPIase). Figure 3. Aggregation is shown as increase in the diffusion time (μsec) and thus the size of α-SYN oligomers to multimers (70 μM) in the presence of 250μM spermine using Fluorescence Correlation Spectroscopy (FCS). Half-time of the aggregation of α-SYN in the presence of 250 μM spermine was 2.2 hours.

Figure 4. Aggregation of α-SYN (70 μM) followed with FCS in the presence of 250 μM spermine (pink triangles), 6 nM PPIase (blue circles) or 6 nM ovalbumine (red triangles). Spermine is used as a positive control and ovalbumine (28 kDa) is used as a negative control to eliminate a possible effect of protein concentration. Half-time of the aggregation in the presence of PPIase is 2.5 hours.

Figure 5. Aggregation of α-SYN (70 μM) followed with FCS in the presence of 6 μM PPIase and no FK506 (black circles), 6 μM FK506 (red triangles) and 42 μM FK506 (blue triangles).

Figure 6. Aggregation of α-SYN (70 μM) followed with FCS in the presence of 30 nM FKBP12 (yellow), 120 nM FKBP12 (pink) and 1.2 μM FKBP12 (green). As a negative control to correct for the increased total protein concentration, 140 μM α-SYN was incubated at 37°C (red)

Figure 7. Aggregation of α-SYN (70 μM) followed with turbidity with no additives (pink circles), in the presence of 0.3 μM FKBP 12 (blue triangles), 15 μM FKBP 12 (red triangles) and 200 μM spermine (black circles).

Figure 8. FK506 inhibits the aggregatory effect of human FKBP 12 on α-SYN Turbidity measurement of the aggregation of α-SYN (70 μM) without additives and, in the presence of 0.5 μM FKBP12 and 12 μM or 120 μM FK506. To accelerate the aggregation process, 60 μM of spermine was added to each sample.

Figure 9. The diffusion time of α-SYN increases during aggregation while the diffusion time of FKBP12 does not. 70 μM WT α-SYN and ~7 nM S42C α-SYN (labelled with a green dye) was incubated at 37°C with 1 μM FKBP12 (labelled with a red dye). Figure 10. Cell counts on SHSY5Y cells containing cytoplasmic α-SYN aggregates. Cells have been treated for 6 days with 10 μM Retinoic Acid (RA) and afterwards 6 days with FeCl₂. A total of ~800 cells was counted per condition. All counts were performed blind and standard deviations account for counts performed by different persons.

Figure 11. Fixed and stained SHSY5Y cells. α-SYN is seen in green and FKBP 12 is seen in red. These SHSY5Y cells have been put under oxidative stress and it can be seen that the distribution of FKBP12 does not change after induction of α-SYN aggregation.

Figure 12. Fixed and stained SHSY5Y cells. α-SYN is seen in green and FKBP59 is seen in red. In these SHSY5Y cells under oxidative stress it can be seen that the distribution of FKBP59 is different in cells with and without α-SYN aggregates.

Figure 13: Description of groups in in vivo mouse experiment to asses the influence of FK506 on the formation of α-SYN aggregates

Figure 14: Picture of a section in the striatum. α-SYN positive areas can clearly be distinguished in dark brown.

Figure 15: Result of Cavalieri test. In the x-axis, the 4 groups that received α-SYN lentiviral injection in the striatum. Group 1 is the group that was treated with vehicle, groups 2, 3 and 4 were treated with FK506 0.5, 2 and 8 mg/kg/day respectively. In the y- axis, the transduced volume is listed. Error bars show mean +/- 2 standard deviations.

Figure 16: Picture of two α-SYN positive neurons. The cell on the left shows no α-SYN aggregates whereas the cell on the right does.

Figure 17: Result of cell counts. In the x-axis, the 4 groups that received α-SYN lentiviral injection in the striatum. Group 1 is the group that was treated with vehicle, groups 2, 3 and 4 were treated with 0.5, 2 and 8 mg/kg/day respectively. In the y-axis, the percentage of aggregate positive cells is listed. Error bars show mean +/- 2 standard deviations. Figure 18: Summary of results from the mouse experiment

Claims

DISEASE RELATED PROTEIN AGGREGATION

1) A screening system for selecting molecules, characterised in that said system is designed to select a molecule that 1) is a ligand of a PPIase or inhibits the activity or expression of a PPIase and 2) inhibits the aggregation or the aggregation rate of cellular proteins (or peptides) or of protein (or peptide) folding intermediates of cellular proteins (or peptides).

2) The screening system of claim 1, characterised in that said system is designed to select a molecule that 1) is a ligand of a PPIase or inhibits the activity or expression of a PPIase and 2) inhibits the aggregation or the aggregation rate of intracellular proteins (or peptides) or of protein (or peptide) folding intermediates of intracellular proteins (or peptides).

3) The screening system of claim 1, characterised in that said system is designed to select a molecule that 1) is a ligand of a PPIase or inhibits the activity or expression of a PPIase and 2) inhibits the aggregation or the aggregation rate of intracytoplasmic proteins (or peptides) or of protein (or peptide) folding intermediates of intracytoplasmic proteins (or peptides).

4) The screening system of claim 1, characterised in that said system is designed to select a molecule that 1) is a ligand of a PPIase or inhibits the activity or expression of a PPIase and 2) inhibits the aggregation or the aggregation rate of cytoplasmic proteins (or peptides) or of protein (or peptide) folding intermediates of cytoplasmic proteins (or peptides).

5) The screening system of claim 1, characterised in that said system is designed to select a molecule that 1) is a ligand of a PPIase or inhibits the activity or expression of a PPIase and 2) inhibits the aggregation or the aggregation rate of a intranuclear protein (or peptides) or of protein (or peptide) folding intermediates of intranuclear proteins (or peptides). 6) The screening system of claim 1, characterised in that said system is designed to select a molecule that 1) inhibits the activity or expression of a PPIase and 2) inhibits the deposition of proteins, protein fragments and peptides in beta-pleated sheets and/or fibrils and/or aggregates.

7) The screening system of the claims 1 to 6, wherein the PPIase are selected of the group consisting of cyclophilin, FKBP and Parvulin.

8) The screening system of the claims 1 to 6, wherein the PPIase is a FKBP.

9) The screening system of the claims 1 to 6, wherein the PPIase is FKBP 12.

10) The screening system of the claims 1 to 6, wherein the PPIase is FKBP52.

11) The screening system of the claims 1 to 10, wherein the target protein or peptide is of the group consisting of synaptophysin, prion PrP protein, presenilin, huntingtin, beta- amyloid, α-synuclein, β-synuclein, ubiquitin, glial fϊbrillary acidic protein and tau.

12) The screening system of the claims 1 to 11, wherein said system comprises an in vitro means for in vitro enhancing of protein aggregation by a PPIase and for selecting PPIase ligands or PPIase inhibiting molecules that inhibit the formation of aggregates or the formation rate of the aggregates.

13) The screening system of the claims 1 to 11, wherein said system comprises an in vitro means for in vitro enhancing of protein aggregation by a PPIase and for selecting PPIase ligands or PPIase inhibiting molecules that interact with early folding intermediates.

14) The screening system of the claims 1 to 13, wherein said system comprises a cell based means comprising at least one cell wherein the protein aggregation is enhanced by a PPIase and further a means for contacting said cell with a PPIase ligand or PPIase inhibiting molecule to select said molecules based on its effect on the formation of aggregates in said cell. 15) The screening system of the claims 1 to 14, wherein said system comprises an animal model means comprising at least one non human animal model, wherein a protein aggregation is enhanced in cells or tissues of the non human animal model by a PPIase and further comprising a means for treating said animal with a PPIase ligand or PPIase inhibiting molecule to select said molecule based on its effect on the formation of aggregates in said cell or tissue of the animal.

16) The screening system of the claims 14 to 16, comprising or the in vitro means, or the cell based means or the animal model means of any of the previous claims or a combination thereof.

17) The screening system of the claims 14 to 16, wherein the in vitro aggregation of said protein of target is demonstrated by an increase in the diffusion time for instance by means of Fluorescence Correlation Spectroscopy.

18) The screening system of the claims 14 to 17, wherein the in vitro aggregation of said protein of target is demonstrated by a turbidity measurement.

19) The screening system of the claims 14 to 18, wherein a polyamine molecule, in particular N,N'-bis(3-aminopropyl)butane-l,4-diamine or an analogue thereof, is used as a positive control for in vitro aggregation.

20) The screening system of the claims 14 to 19, wherein a non aggregating inducing or inhibiting protein such as ovalbumine is used as a negative control to eliminate a possible effect of protein concentration.

21) The screening system of the claims 14 to 20, wherein a cell count is carried of cells with and without intracellular aggregates of the cellular protein of target.

22) The screening system of the claims 14 to 21, wherein a histological examination has been carried to visualize in distinct tissue zones the formation of aggregates of the protein of target. 23) The screening system of the claims 14 to 22, wherein cells in distinct zones of tissues of animal models are by means of lentiviral vector transfer transduced by a polynucleotide encoding the protein of target.

24) The screening system of the claim 23, wherein the percentage of aggregation positive cells is counted.

25) The use of PPIase ligands or molecules that inhibit or neutralise the activity or expression of a PPIase of the group of antibodies and functional fragments derived thereof, RNAi (siRNA and shRNA) and DNA molecules (e.g. polynucleotide sequences), aptamers and ribozymes to manufacture a drug to treat or a medicament to prevent or inhibit the formation of aggregation-prone conformational intermediates in cells or to prevent or inhibit the formation of protofibrils in cells or to prevent or inhibit the protofibril assembly to fibrils in cells.

26) The use of claim 25, wherein the PPIase ligand is a FKBP ligand.

27) The use of claim 25, wherein the PPIase ligand is of FKBP 12.

28) The use of claim 25, wherein the PPIase ligand is of FKBP52.

29) The use of claim 25, wherein the molecule inhibits or neutralises the activity of a FKBP or functions to inhibit the translation or synthesis of a FKBP.

30) The use of claim 25, wherein the molecule inhibits or neutralises the activity of FKBP 12 or functions to inhibit the translation or synthesis of FKBP 12.

31) The use of claim 25, wherein the molecule inhibits or neutralises the activity of FKBP52 or functions to inhibit the translation or synthesis of FKBP52.

32) The use of the claims 25 to 31, wherein the protein or peptide of target is a protein or peptide of the group consisting of synaptophysin, prion PrP protein, presenilin, huntingtin, beta-amyloid, alpha- synuclein, beta-synuclein, ubiquitin, glial fibrillary acidic protein and tau.

33) The use of a small molecule that interferes by binding on the promoter region of the FKBP, preferably FKBP12 or FKBP52, and inhibit binding of a transcription factor on said promoter region so that none of the FKBP mRNA or that antagonises the activity of an FKBP, preferably FKBP 12 or FKBP52 to manufacture a drug to prevent or inhibit the formation of aggregation-prone conformational intermediates in cells or to prevent or inhibit the formation of protofibrils in cells or to prevent or inhibit the cytoplasmic or nuclear protofibril assembly to fibrils.