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WO2005109004A2 - Agregation de proteines associee a une maladie - Google Patents

Agregation de proteines associee a une maladie Download PDF

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Publication number
WO2005109004A2
WO2005109004A2 PCT/IB2005/001234 IB2005001234W WO2005109004A2 WO 2005109004 A2 WO2005109004 A2 WO 2005109004A2 IB 2005001234 W IB2005001234 W IB 2005001234W WO 2005109004 A2 WO2005109004 A2 WO 2005109004A2
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Prior art keywords
ppiase
protein
aggregation
cells
fkbp
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PCT/IB2005/001234
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WO2005109004A3 (fr
Inventor
Veerle Baekelandt
Zeger Debyser
Yves Engelborghs
Melanie Gerard
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Katholieke Universiteit Leuven
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Katholieke Universiteit Leuven
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Priority to EP05738697A priority Critical patent/EP1747468A2/fr
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Publication of WO2005109004A3 publication Critical patent/WO2005109004A3/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/99—Isomerases (5.)

Definitions

  • 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.
  • 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.
  • human neurodegenerative diseases of amyloid type comprises aggregation of Ab peptides, prion proteins, ⁇ -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)).
  • 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 ⁇ -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.
  • FKBP FK506-Binding Protein
  • 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.
  • 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.
  • DLB Dementia with Lewy bodies
  • Huntington's disease Huntington's disease
  • Parkinson's disease Parkinson's disease
  • DLB Dementia with Lewy bodies
  • Parkinson's Disease 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.
  • SN 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.
  • LB Lewy Bodies
  • 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).
  • ⁇ -SYN an unfolded protein of 14 kDa that is ubiquitously present in the brain, plays a significant role in PD.
  • 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 ⁇ -SYN.
  • SN Substantia Nigra
  • LB's are eosinophilic cytoplasmic inclusions that contain predominantly aggregated ⁇ -SYN.
  • a point mutation in ⁇ -SYN has been discovered: the A30P, A53T and E46K mutation (R. Kruger et al., Nat. Genet. 18, 106-108 (1998); M. H.
  • Huntington's disease 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.
  • 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).
  • 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).
  • polyp polyalanine 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).
  • 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).
  • AD Alzheimer's disease
  • a-beta a misfolded protein
  • 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.
  • AD is primarily 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 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.
  • 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.
  • fALS familial
  • sALS sporadic
  • 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, Gerstam
  • 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 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.
  • protofibrils neurotoxic quaternary structure intermediates
  • 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.
  • 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.
  • 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.
  • FKBP FK506 binding protein
  • 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.
  • FKBP FK506 binding protein
  • FKBP 12 and FKBP52 By monitoring the expression of FKBP 12 and FKBP52 in the human neuroblastoma cells, both before and after the appearance of ⁇ -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 ⁇ -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 ⁇ -SYN and FKBP52.
  • 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.
  • PPIase EC 5.2.1.8
  • CyP cyclophilin
  • FKBP cyclophilin
  • parvulin 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.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FKBP FKBP52
  • the invention relates to the use of molecules that neutralize the activity of the FKBP by interfering with its synthesis, translation and ligand-binding.
  • molecules it is meant peptides, tetrameric peptides, proteins, organic molecules, having the same neutralizing effect as stated above.
  • 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.
  • 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.
  • 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.
  • PBL peripheral blood lymphocytes
  • SCID severe combined immune deficiency
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • antisense cDNA constructs that synthesize anti-sense RNA constitutively or inducible, depending on the promoter used, can be introduced stably into cell lines.
  • 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).
  • Yet another embodiment of present invention involves the use of inhibitors of PPIase activity to manufacture a drug to prevent ⁇ -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 ⁇ -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 ⁇ -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 ⁇ -SYN.
  • Such neurotoxicity can for instance result in dementia with Lewy bodies.
  • FKBP12 and preferably FKBP52 inhibitors to manufacture a drug to prevent or inhibit the formation of ⁇ -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 ⁇ -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 ⁇ -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 ⁇ -SYN aggregates in a subject in need thereof.
  • Yet another embodiment involves a method of preventing or diminishing ⁇ -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 ⁇ -SYN aggregation and a method of preventing or treating an ⁇ -SYN aggregation disorder in an animal, comprising: administering to a mammal of an a FKBP 12 inhibitor in an efficient dose to inhibit ⁇ -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.
  • 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.
  • FKBP FK506-Binding Protein
  • pharmaceutically acceptable is used adjectivally herein to mean that the modified noun is appropriate for use in a pharmaceutical product.
  • 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.
  • RNA DNA
  • RNA protein
  • 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.
  • RNAi interfering RNA
  • 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.
  • mRNA gene transcript
  • Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher.
  • 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.
  • 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.
  • a molecule that inhibits the activity refers to a molecule which inhibits the activity of FBKP, in this preferably FKBP12 or FKBP52.
  • inhibittion 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.
  • mRNA gene transcript
  • Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60% , 80%, 90% or even higher.
  • inhibitting activity is referred to the protein that is produced such as FBKP, in this preferably FKBP52. The inhibition of activity leads to diminished interaction.
  • 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 ⁇ -SYN aggregation.
  • said inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher.
  • '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.
  • 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.
  • the dosage and mode of administration will depend on the individual.
  • the drug is administered so that the protein, polypeptide, peptide of the present invention is given at a dose between 1 ⁇ g/kg and 10 mg/kg, more preferably between 10 ⁇ g kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg.
  • 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 ⁇ gkg/minute, more preferably between 7 and 15 ⁇ g/kg/minute.
  • FKBP FK506 binding protein
  • 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.
  • PPIase peptidyl-prolyl cis-trans isomerase
  • 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.
  • 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.
  • TPR tetratricopeptide repeat
  • 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.
  • PPIase peptidyl-prolyl cis/trans isomerase
  • 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).
  • FKBP 12 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).
  • NEF nerve growth factor
  • FKBP 12 clearly enhances the aggregation of ⁇ -SYN in neuronal cells.
  • FKBPs are members of the immunophilines, enzymes that bind to immunosuppresantia and have a peptidyl-prolyl isomerase (PPIase) activity.
  • FKBPs bind selectively to FK506 (Tacrolimus), a powerful immunosuppressant. FK506 strongly and specifically inhibits the PPIase activity of most FKBPs.
  • 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.
  • 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.
  • this role is exerted through enhancing the aggregation rate of ⁇ -SYN because folding of ⁇ -SYN implies in many cases the initiation of aggregation of the protein.
  • disorders of peptide misfolding and aggregation or protein or peptide aggregation in a cell more particularly in a neuronal cell 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.
  • oculapharyngeal muscular dystrophy OPMD
  • polyp polyadenine expansion mutation in the polyadenine binding protein 2 gene.
  • 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 ⁇ -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.
  • pCHMWS lentiviral vector transfer plasmid
  • pRSET B a prokaryotic expression vector with N-terminal His-tag. Sequencing confirmed the correct sequence and reading frame of the gene.
  • DH5 ⁇ -Tl resistant heat sensitive E. coli cells were transformed with the plasmid. In the presence of lOO ⁇ g/mL ampicillin, transformed cells were grown to an 8L culture at 30°C.
  • the culture 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 22 ⁇ M 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 ⁇ -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.
  • FCS Fluorescence Spectroscopy
  • the point mutation S42C was introduced in the ⁇ -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 ⁇ -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.
  • 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).
  • Example 5 PPIase enhances the aggregation rate of ⁇ -SYN
  • 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).
  • FKBP 12 a protein that often co-localizes with alpha-synuclein in Lewy bodies and Lewy neurites in PD brains (M.
  • a cell culture model for PD was used (Hasegawa T. accelerated a-snc aggregation brain research 1013 (2004) 51- 59).
  • 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.
  • FeCl 2 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- ⁇ - 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 ⁇ -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 FeCl 2 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 ⁇ M FeCl 2 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 ⁇ -SYN aggregate positive cells when SHSY5Y cells are put under moderate oxidative stress.
  • FKBP 12 and FKBP52 in the cells were monitored, both before and after the appearance of ⁇ -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 ⁇ -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 ⁇ -SYN and FKBP52.
  • FK506 is dissolved in suitable vehicle (1% Cremophor EL, 4% ethanol) and administered in volumes 10 ⁇ L per g body weight by oral gavages once daily. Gavage is the preferred method of oral administration for oil soluble drugs.
  • suitable vehicle 1% Cremophor EL, 4% ethanol
  • 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.
  • 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.
  • ⁇ 0.05
  • p- value 0.456.
  • Rl is a C 1 -C9, straight or branched chain alkyl or alkenyl group optionally substituted with C 3 -C 8 cycloalkyl, C 3 or C 5 cycloalkyl, C 5 -C , cycloalkenyl, or Ari, where said alkyl, alkenyl, cycloalkyl or cycloalkenyl groups may be optionally substituted with C 1 -C 4 alkyl, C 1 -C 4 alkenyl, or hydroxy.
  • 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, C ⁇ ,-C 6 , straight or branched alkyl or alkenyl, C 1 -C 4 , alkoxy or C 1 -C 4 , alkenyloxy, phenoxy, benzyloxy, and amino;
  • X is oxygen, sulfur, methylene (CH 2 ), or H 2 ;
  • Y is oxygen or NR 2 , where R is hydrogen or C ⁇ -C 6 , alkyl; and Z is a Ci-C ⁇ straight or branched chain alkyl or alkenyl, wherein the alkyl chain is substituted in one or more positions with Ar, as defined above, C ⁇ -C 8 , cycloalkyl, cycloalkyl connected by a C ⁇ -C 6 straight or unbranched alkyl or alkenyl chain, or Ar 2 where Ar 2 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-C ⁇ , straight or branched alkyl or alkenyl, C1-C 4 al
  • R 3 is selected from the group consisiting of straight or branched alkyl Ci-Gg optionally substituted with C 3 -C 8 cycloalkyl, or Ar, as defined above;
  • X 2 is O or NR 5 , where R 5 is selected f rom the group consisting of hydrogen, -C 5 straight or branched alkyl and alkenyl;
  • R 4 is selected from the group consisting of phenyl, benzyl, -C 5 straight or branched alkyl or alkenyl, and C1-C 5 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
  • 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) ⁇ ro ⁇ yl 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)
  • immunosuppressant drugs 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.
  • rapamycin binds potently to FKBP12 but does not inhibit calcineurin, it can serve as an antagonist to FK506.
  • rapamycin binds potently to FKBP12 but does not inhibit calcineurin, it can serve as an antagonist to FK506.
  • 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)).
  • 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.
  • 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
  • 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)
  • 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).
  • FKBP-52 a mouse FKBP-52
  • Sf9 cells Spodoptera frugiperda insect cells
  • ES Alnemri et al. Proceedings of the National Academy of Sciences (1993), Vol 90, 6839-6843
  • (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.
  • 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, ⁇ -synuclein, ⁇ - synuclein proteins or of synaptophysin, presenilin, huntingtin, ubiquitin, glial fibrillary acidic protein and tau eventually after transduction with the respective lentiviral vector.
  • neuroblastoma cells e.g. SKNSH cells
  • SKNSH cells neuroblastoma cells
  • SKNSH cells that overexpress wild type, mutant or antisense protein of target, for instance from the group of prion-, tau-, amyloid,
  • 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
  • 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.
  • 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 ⁇ -SYN (17 kDa), at a2 and a3 double or triple aggregates of ⁇ -SYN and at bl a co-eluted E. coli impurity. The standard MW reference is presented in Lane 3.
  • FIG. 1 NUPAGE BIS-TRIS gel of recombinant ⁇ -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 ( ⁇ sec) and thus the size of ⁇ -SYN oligomers to multimers (70 ⁇ M) in the presence of 250 ⁇ M spermine using Fluorescence Correlation Spectroscopy (FCS). Half-time of the aggregation of ⁇ -SYN in the presence of 250 ⁇ M spermine was 2.2 hours.
  • FIG. 4 Aggregation of ⁇ -SYN (70 ⁇ M) followed with FCS in the presence of 250 ⁇ M 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.
  • FIG. 7 Aggregation of ⁇ -SYN (70 ⁇ M) followed with turbidity with no additives (pink circles), in the presence of 0.3 ⁇ M FKBP 12 (blue triangles), 15 ⁇ M FKBP 12 (red triangles) and 200 ⁇ M spermine (black circles).
  • FK506 inhibits the aggregatory effect of human FKBP 12 on ⁇ -SYN Turbidity measurement of the aggregation of ⁇ -SYN (70 ⁇ M) without additives and, in the presence of 0.5 ⁇ M FKBP12 and 12 ⁇ M or 120 ⁇ M FK506. To accelerate the aggregation process, 60 ⁇ M of spermine was added to each sample.
  • Figure 9 The diffusion time of ⁇ -SYN increases during aggregation while the diffusion time of FKBP12 does not. 70 ⁇ M WT ⁇ -SYN and ⁇ 7 nM S42C ⁇ -SYN (labelled with a green dye) was incubated at 37°C with 1 ⁇ M FKBP12 (labelled with a red dye).
  • Figure 10 Cell counts on SHSY5Y cells containing cytoplasmic ⁇ -SYN aggregates. Cells have been treated for 6 days with 10 ⁇ M Retinoic Acid (RA) and afterwards 6 days with FeCl 2 . A total of ⁇ 800 cells was counted per condition. All counts were performed blind and standard deviations account for counts performed by different persons.
  • RA Retinoic Acid
  • FIG. 11 Fixed and stained SHSY5Y cells. ⁇ -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 ⁇ -SYN aggregation.
  • FIG. 12 Fixed and stained SHSY5Y cells. ⁇ -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 ⁇ -SYN aggregates.
  • Figure 13 Description of groups in in vivo mouse experiment to asses the influence of FK506 on the formation of ⁇ -SYN aggregates
  • Figure 14 Picture of a section in the striatum. ⁇ -SYN positive areas can clearly be distinguished in dark brown.
  • Figure 15 Result of Cavalieri test.
  • the 4 groups that received ⁇ -SYN lentiviral injection in the striatum are the 4 groups that received ⁇ -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.
  • the transduced volume is listed. Error bars show mean +/- 2 standard deviations.
  • Figure 16 Picture of two ⁇ -SYN positive neurons. The cell on the left shows no ⁇ -SYN aggregates whereas the cell on the right does.
  • Figure 17 Result of cell counts.
  • 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.
  • the percentage of aggregate positive cells is listed. Error bars show mean +/- 2 standard deviations.
  • Figure 18 Summary of results from the mouse experiment

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Abstract

La présente invention démontre que les membres de la famille des protéines de liaison FKBP ou FK506, plus précisément la FKBP12 ou la FKBP52, augmentent l'agrégation de l'α-SYN. De plus, il s'avère que l'effet de la FKBP sur l'agrégation de l'α-SYN découle de l'activité PPIase de l'enzyme. Les ligands de l'immunophiline, des inhibiteurs spécifiques de l'activité des FKBP, inhibent la stimulation de l'agrégation de l'α-SYN par les FKBP.
PCT/IB2005/001234 2004-05-06 2005-05-06 Agregation de proteines associee a une maladie Ceased WO2005109004A2 (fr)

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US20140163021A1 (en) * 2009-09-24 2014-06-12 Institut National De La Sante Et De La Recherche Medicale (Inserm) FKBP52-Tau Interaction as a Novel Therapeutical Target for Treating the Neurological Disorders Involving Tau Dysfunction
CN102549438B (zh) * 2009-09-24 2014-10-29 国家健康与医学研究院 Fkbp52-tau相互作用作为新颖治疗靶点用于治疗涉及tau机能失调的神经障碍
JP2015092186A (ja) * 2009-09-24 2015-05-14 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル タウ機能不全を伴う神経障害を処置するための新規な治療ターゲットとしてのｆｋｂｐ５２−タウ相互作用
US9128104B2 (en) * 2009-09-24 2015-09-08 Institut National De La Sante Et De La Recherche Medicale (Inserm) FKBP52-Tau interaction as a novel therapeutical target for treating the neurological disorders involving tau dysfunction
US9518995B2 (en) 2009-09-24 2016-12-13 Institut National De La Sante Et De La Recherche Medicale (Inserm) FKBP52-Tau interaction as a novel therapeutical target for treating the neurological disorders involving Tau dysfunction
US20170056500A1 (en) * 2009-09-24 2017-03-02 Institut National De La Sante Et De La Recherche Medicale (Inserm) FKBP52-Tau Interaction as a Novel Therapeutical Target for Treating the Neurological Disorders Involving Tau Dysfunction
US20180161432A1 (en) * 2009-09-24 2018-06-14 Inserm (Institut National De La Sante Et De La Recherche Medicale) Fkbp52-tau interaction as a novel therapeutical target for treating the neurological disorders involving tau dysfunction
WO2014188197A1 (fr) * 2013-05-24 2014-11-27 Chronos Therapeutics Limited Tacrolimus pour emploi dans le traitement de maladies caractérisées par le dépôt d'agrégats de protéines dans les cellules neuronales
JP2016528171A (ja) * 2013-05-24 2016-09-15 クロノス セラピューティクス リミテッドＣｈｒｏｎｏｓ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｌｉｍｉｔｅｄ 神経細胞におけるタンパク質凝集体の沈着を特徴とする疾患の治療に使用するためのタクロリムス
JP2019104746A (ja) * 2013-05-24 2019-06-27 クロノス セラピューティクス リミテッドＣｈｒｏｎｏｓ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｌｉｍｉｔｅｄ 神経細胞におけるタンパク質凝集体の沈着を特徴とする疾患の治療に使用するためのタクロリムス
WO2017190016A1 (fr) * 2016-04-29 2017-11-02 University Of South Florida Cyclophiline 40 utilisée dans la réduction des fibrilles neurotoxiques et dans le traitement des maladies neurodégénératives

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