Modulators of alpha synuclein

Abstract

To provide diagnostic methods and methods for treating conditions arising from alpha-synucleopathies.MEANS FOR SOLVING THE PROBLEM: An aminopyrazole, that is, 4-[2-(4-bromophenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid or ester or amide thereof can be used to lower an amount of α-synuclein in cells of a subject for treating such conditions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 521,758, filed June 19, 2017, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to compositions and methods for modulating alpha-synuclein protein levels via the FAF1 gene and protein, and to inhibitors of FAF1 activity for modulating alpha-synuclein levels or aggregation or degeneration to treat or prevent alpha-synuclein-mediated synucleinopathies. The disclosure also includes related diagnostic methods and therapeutic methods using compounds that modulate the amount or activity of FAF1. [Background technology]

[0003] FAF1 is a protein that binds to the apoptosis receptor Fas and is called Fas-associated factor 1. FAF1 binds to Fas and promotes apoptosis when Fas interacts with its ligand, Fas ligand (FasL). Thus, FAF1 is a pro-apoptotic protein. FAF1 possesses an N-terminal 200-amino acid region that binds to Fas (Ryu SW et al. 2003, Yu C et al. 2016). FAF1 then activates the caspase cascade, thereby activating caspase-8 and the apoptotic pathway. FAF1 also activates JNK and binds to PARP1, thus stimulating the necrotic pathway (Yu C et al. 2016). However, in its inactive state, FAF1 is bound to HSP70 and maintained in an inactive form (Kim HJ et al. 2005, Gao X et al. 2015). FAF1 has been reported to be overexpressed in postmortem tissues from Parkinson's disease patients (Betarbet R et al. 2008).

[0004] Synucleinopathies are a group of neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein and its aggregates, such as Lewy body (LB) or Lewy neurite (LN) inclusions. Examples include Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systemic atrophy (MSA) (Spillantini MG and Goedert M 2000, Marti MJ et al. 2003).

[0005] Alpha-synuclein, genetically designated SNCA, is a small cytoplasmic protein with a size of 14 kDa. It is located primarily in synaptic vesicles in neuronal cells and functions in signal transduction and propagation (Snead D and Eliezer D 2014). The protein consists of three domains: an N-terminal domain with amphipathic helices, a middle domain with a flexible beta-sheet structure, and a C-terminal tail containing many acidic residues (Gallegos S et al. 2015). The protein is known to exist in soluble forms as a monomer or tetramer. However, alpha-synuclein can misfold and aggregate to form oligomers and even multimers, which, for various unknown reasons, aggregate into fibrillar structures called Lewy bodies and Lewy neurites. This process involves C-terminal proteolysis, phosphorylation of serine 129, monoubiquitination, and other modifications (Sato H et al. 2013, Oueslati A 2016). The reasons for alpha-synuclein misfolding and aggregation include protein accumulation due to overexpression of the protein, due to gene duplication or reduced degradation, or changes in the properties of the protein itself due to mutations. However, other non-genetic reasons are also known, such as environmental toxins, cellular stresses such as cytotoxicity, ER stress, and oxidative stress (Rockenstein E et al. 2014, Ingelsson M 2016). Furthermore, polymorphic variations in the SNCA locus affect alpha-synuclein levels and aggregation (Mata IF et al., 2010).

[0006] The toxicity of alpha-synuclein misfolded multimers and Lewy bodies has been studied and may be related to various pathogenic processes, particularly dysfunction in membrane trafficking, including exocytosis, ER-to-Golgi transport, ER stress, Golgi homeostasis, endocytosis, autophagy, and oxidative stress (Wang T and Hay JC 2016, Snead D et al. 2014). Furthermore, alpha-synuclein toxicity may also include mitochondrial toxicity and inflammation (Ingelsson M, 2016). Alpha-synuclein oligomers and Lewy bodies disrupt the proteasome pathway, leading to membrane disruption and pore formation, which in turn activates apoptosis in cells (Gallegos S et al. 2015).

[0007] "Synucleinopathies" are a group of neurodegenerative disorders with a range of clinical manifestations, including Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), atypical Parkinson's disease, and pure autonomic failure (PAF) (Spillantini MG and Goedert M 2000, Marti MJ 2003). These diseases are caused by Lewy bodies, Lewy neurites, or aggregates of alpha-synuclein in neurons and glia. Clinically, they are characterized by a chronic and progressive decline in motor, cognitive, and autonomic functions, depending on the distribution of the lesions.

[0008] Parkinson's disease (PD) is the second most common neurodegenerative disease after AD (Feng LR et al. 2010) and is generally considered a movement disorder. PD's pathological hallmark is the selective progressive degeneration and loss of dopaminergic neurons in the substantia nigra (SNc), particularly the midbrain's substantia nigra pars compacta (SNPC), which is involved in movement disorders (Luk KC et al. 2012). However, symptoms are often accompanied by the emergence of autonomic, cognitive, and psychiatric problems (Jankovic J 2008).

[0009] Another hallmark of PD is the presence of cytoplasmic inclusions in dopaminergic neurons, called Lewy bodies (LBs) and Lewy neurites (LNs), which are found in postmortem tissue from over 90% of Parkinson's disease patients. The main protein component of LBs is misfolded and aggregated alpha-synuclein (Angot E et al. 2012).

[0010] Several genetic alterations, including point mutations and gene duplications, in the SNCA gene result in familial Parkinson's disease (PD), i.e., are thought to be the cause of PD. The dominant inheritance of these mutations suggests a gain-of-function mechanism, such as altered protein properties or increased protein levels leading to aggregation (Devine MJ et al. 2011, Appel-Cresswell S et al. 2013, Kasten M and Klein C 2013, Petrucci S et al. 2016). Specific polymorphisms or mutations in the distal enhancer region within the SNCA gene, which may increase its expression and thus its aggregation, have been reported to be associated with PD (Mata IF et al. 2010, Soldner F et al. 2016).

[0011] In addition to mutations in the SNCA gene, many other mutations have been associated with Parkinson's disease and related synucleinopathies (Wang CD and Chan P 2014, Kalinderi K et al. 2016, Federoff M 2015, Nussbaum R 2017). Some of these genes have been implicated in alpha-synuclein processing and its accumulation. For example, mutations in the glucocerebrosidase gene (GBA) are present in 23% of cases of dementia with Lewy bodies and 21% of cases of PD (Sidransky E et al. 2009, Jesus S et al. 2016). GBA variants have been reported to increase SNCA accumulation by altering alpha-synuclein processing (Cullen V et al. 2011, Fernandes HJ 2016). Other known genes associated with these diseases include parkin, LRRK2, DJ-1, and PINK1. Although the role of these genes in the pathogenesis of Parkinson's disease and other synucleinopathies is largely unknown, these genes have functions in post-translational modification and processing of proteins, including ubiquitination, membrane trafficking, autophagy, and protein folding, and overexpression of alpha-synuclein results from mutations in some of these genes (Walden H and Muqit MMK 2017, Galegos S 2015).

[0012] Dementia with Lewy bodies (DLB) is the second most common form of dementia and is characterized by clinical symptoms of progressive cognitive decline accompanied by dementia and fluctuating cognition, particularly visual hallucinations. Postmortem tissue reveals numerous Lewy bodies (LBs) in the cerebral cortex as well as the substantia nigra, and these LBs contain alpha-synuclein (Gaser F et al. 2005, Goeder M et al. 2017).

[0013] Multiple system atrophy (MSA) is a fatal neurodegenerative disorder characterized by a variable combination of autonomic failure, parkinsonism, and cerebellar ataxia (Daniela Kuzdas-Wood et al. 2014). It is a rare disease with a prevalence of 1.9–4.9 per 100,000, a survival time of 6–9 years (Stefanova et al. 2009), and a mean age of adult onset of 57 years. Pathologically, widespread glial cytoplasmic inclusions (GCIs) are observed throughout the CNS (Trojanowski JQ and Revesz T 2007), composed of filamentous alpha-synuclein. Newly proposed criteria include predominant cerebellar ataxia with predominant motor features and neuropathological evidence of CNS alpha-synuclein-positive glial cytoplasmic inclusions accompanied by neurodegenerative changes in striatonigral or olivopontocerebellar structures (Gilman S 2008). [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Appel-Cresswell S, Vilarino-Guell C, Encarnacion M, Sherman H, Yu I, Shah B, Weir D, Thompson C, Szu-Tu C, Trinh J, Aasly JO, Rajput A, Rajput AH, Jon StoessI A, Farrer MJ. 2013. Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease. Mov. Disord. 28, 811-3. [Non-patent document 2] Angot E, Steiner JA, Lema Tome CM, Ekstrom P, Mattsson B, Bjorklund A, Brundin P. 2012. Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo. PloS ONE 7:e39465.

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[0015] The present invention is based on the surprising discovery that FAF1 increases the accumulation and aggregation of alpha-synuclein in neuronal cells. Following this, the present study confirmed that aminopyrazole derivative FAF1 inhibitors actually reduce alpha-synuclein accumulation and aggregation in neuronal cells. The present invention is also based on the discovery that FAF1 inhibits autophagy through an increase in p62 protein and a decrease in LC3. The inventors' research confirmed that aminopyrazole derivative FAF1 inhibitors actually reverse FAF1 activity and enhance autophagy through a decrease in p62 protein and an increase in LC3. These findings are unexpected because FAF1 is a pro-apoptotic protein that promotes cell death, and FAF1 activity on autophagy is a novel discovery.

[0016] The present invention relates to compositions and methods for modulating alpha-synuclein protein levels via the FAF1 gene and protein, and to inhibitors of FAF1 for modulating alpha-synuclein protein levels, aggregation, or degeneration to treat or prevent alpha-synuclein-mediated synucleinopathies. The disclosure also includes related diagnostic methods and therapeutic methods using compounds with FAF1 level-modulating activity.

[0017] The present disclosure generally relates to compositions and methods that use nucleic acids or small molecules, such as antibodies, peptides, aptamers, or antisense nucleic acids or siRNAs, to alter FAF1 expression or FAF1 protein activity in human cells to prevent or slow the progression of synucleinopathy. Thus, compositions and methods are provided that alter gene sequences and alter protein expression and activity of FAF1, which affect disease progression. The use of such compositions and methods for detecting and quantifying alpha-synuclein in samples and / or characterizing synuclein forms for diagnostic or prognostic purposes is also disclosed.

[0018] Unexpectedly, using a transfected cell-based assay, we found that FAF1 increased alpha-synuclein levels in cells in a dose-dependent manner. Furthermore, increasing FAF1 expression in animals by infection with a recombinant adenovirus expressing FAF1 elevated alpha-synuclein levels. Furthermore, reducing FAF1 by RNAi transfection attenuated alpha-synuclein levels. We also found that FAF1 inhibits autophagy by increasing LC-3II and p62. This strongly suggests that alpha-synuclein is at least partially mediated by the autophagic pathway in vitro and in animal cells.

[0019] The present inventors have developed the FAF1 inhibitor KM-819 (KR-88493, U.S. Patent No. 7,939,550, Yoo SE et al. 2016, incorporated herein by reference in its entirety and for all purposes). The present inventors disclose herein that the aminopyrazole compound KM-819, whose chemical name is 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid, is a FAF1 inhibitor. The present inventors disclose herein that the aminopyrazole compound KM-819 reverses the effects of FAF1 overexpression and reduces alpha-synuclein in cells and animals. In summary, the experiments in the following examples demonstrate that FAF1 siRNA or antisense or related gene constructs, peptides, antibodies, and small molecules that regulate FAF1 can be used to regulate the amount or phosphorylation state of alpha-synuclein and treat alpha-synuclein-induced synucleinopathies. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 depicts overexpression of the transfected FAF1-FLAG transgene in SH-SY5Y cells. [Figure 2] FIG. 2 shows synuclein aggregation in SH-SY5Y overexpressing the FAF1-FLAG transgene. [Figure 3A] Figures 3A and 3B show the formation of Lewy body-like morphology in SH-SY5Y cells overexpressing the transfected FAF1-FLAG transgene. Figure 3A shows cells transfected with a vector control. Figure 3B shows cells transfected with the FAF1-FLAG transgene. DAPI-DAPI staining; α-syn-anti-α-synuclein antibody staining; Ps129-α-syn staining with anti-phosphorylated α-synuclein staining; overlay of three images. [Figure 3B]Figures 3A and 3B show the formation of Lewy body-like morphology in SH-SY5Y cells overexpressing the transfected FAF1-FLAG transgene. Figure 3A shows cells transfected with a vector control. Figure 3B shows cells transfected with the FAF1-FLAG transgene. DAPI-DAPI staining; α-syn-anti-α-synuclein antibody staining; Ps129-α-syn staining with anti-phosphorylated α-synuclein staining; overlay of three images. [Figure 4] FIG. 4 depicts overexpression of FAF1 in the midbrain of mice with a FAF1-expressing adenoviral construct microinjected into the midbrain tissue. [Figure 5] FIG. 5 shows the attenuation of FAF1 expression in SH-SY5Y cells co-transfected with the α-synuclein gene and the corresponding siRNA. [Figure 6] FIG. 6 shows changes in p62 and LC3 protein expression in SH-SY5Y cells transfected with the FAF1 gene. [Figure 7] FIG. 7 shows the binding of KM-819 to FAF1 in cells transfected with the FAF1-FLAG construct. [Figure 8] FIG. 8 shows the inhibition of the formation of FAF1-caspase 8 and FAF1-JNK complexes by KM-819 in cell extracts. [Figure 9] FIG. 9 shows inhibition of caspase 3 cleavage, caspase 8 activity and JNK phosphorylation by KM-819 in cell extracts. [Figure 10] FIG. 10 shows a decrease in the amount of p62 protein and an increase in the amount of LC3 protein in SH-SY5Y cells overexpressing FAF1 and treated with KM-819. [Figure 11] FIG. 11 shows the reduction of α-synuclein protein in cells co-transfected with a FAF1-FLAG expression construct and an α-synuclein-GFP (green fluorescent protein) expression construct. [Figure 12]FIG. 12 shows the reduction in the amount of α-synuclein and S129-phosphorylated α-synuclein in the midbrain tissue of transgenic mice overexpressing FAF1 after administration of KM-819. [Figure 13] FIG. 13 shows the reduction in α-synuclein levels in the hippocampus of transgenic mice overexpressing FAF1 after KM-819 administration. [Figure 14] FIG. 14 shows the reduction in α-synuclein levels in the cerebral cortex of transgenic mice overexpressing FAF1 after administration of KM-819. [Figure 15] FIG. 15 shows the reduction in α-synuclein levels in the cerebellum of transgenic mice overexpressing FAF1 after KM-819 administration. [Figure 16] FIG. 16 shows the reduction in α-synuclein levels in the striatum of transgenic mice overexpressing FAF1 after KM-819 administration. DETAILED DESCRIPTION OF THE INVENTION

[0021] One embodiment of the present disclosure provides a method for treating a disease or condition resulting from synucleinopathy, comprising administering to a subject exhibiting symptoms of or at risk for synucleinopathy an inhibitor of FAF1 activity that increases the amount of alpha-synuclein or modulates the amount of LC-3 and p62 in the subject's cells, wherein the effective amount administered is an amount that decreases the amount of alpha-synuclein, or an amount that increases the amount of LC-3 and / or decreases the amount of p62 in the subject's cells, respectively.

[0022] In some implementations of this method, the inhibitor of FAF1 activity has the formula 1:

[0023] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0024] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched or cyclic C1-C3 alkyl; A is CH or N. or a pharmaceutically acceptable salt thereof.

[0025] In some cases, the aminopyrazole compound can be 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid or an ester or amide thereof.

[0026] Another aspect of the present disclosure provides a method for treating a disease or condition resulting from synucleinopathy, the method comprising administering to a subject exhibiting symptoms of or at risk for synucleinopathy a compound that binds to FAF1 protein in an amount effective to reduce the amount of alpha-synuclein phosphorylated at amino acid S129 in the subject's cells.

[0027] In some implementations of this embodiment, the inhibitor of FAF1 activity has the formula 1:

[0028] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0029] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched or cyclic C1-C3 alkyl; A is CH or N. or a pharmaceutically acceptable salt thereof.

[0030] In some cases, the aminopyrazole compound can be 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid or an ester or amide thereof.

[0031] In any of the therapeutic aspects of the present disclosure, the FAF1 inhibitor can be administered orally, or by injection into the brain, cerebrospinal fluid, or blood, or through the skin, or by other common practical methods such as minipump implantation. The most convenient route is to administer the FAF1-binding compound orally.

[0032] The aminopyrazole compound 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid has an EC50 of 1 nM for regulating the level of LC-3 and p62 in cells, and an EC50 of 20 μM for reducing the amount of alpha-synuclein in cells.Therefore, the effective amount of this compound for regulating the amount of LC-3 and p62 in brain tissue cells is an amount that provides a concentration of at least 0.5 nM, preferably at least 1 nM, at least 2 nM, at least 5 nM or at least 10 nM in the cells of brain tissue, and the effective amount of this compound for reducing the amount of alpha-synuclein in brain tissue cells is an amount that provides a concentration of at least 10 nM, preferably at least 20 nM, at least 40 nM, at least 200 nM or in brain tissue.

[0033] Another aspect of the present disclosure provides a method for diagnosing synucleinopathy or assessing the risk of synucleinopathy in a subject, comprising measuring the amount of FAF1 protein or the level of FAF1 activity, which inhibits autophagy mediated by LC-3 and p62 proteins, in cells of the subject or a sample taken from the subject, wherein a higher FAF1 activity or protein level than in a normal human population indicates the development of synucleinopathy or the subject's risk of developing synucleinopathy.

[0034] Another aspect of the present disclosure provides a method for treating a disease or symptom resulting from a synucleiopathy in a subject by combining the diagnosis and treatment, the method comprising administering to a subject who exhibits a higher amount of FAF1 protein or a level of FAF1 activity in the subject's cells that inhibits autophagy mediated by LC-3 and p62 protein in the subject's cells than in a normal human population, an inhibitor of FAF1 activity that regulates the amount of LC3 and p62 in the subject's cells, in an amount effective to increase the amount of LC3 and / or decrease the amount of p62, or a compound that binds to FAF1 protein, in an amount effective to decrease the amount of alpha-synuclein phosphorylated at amino acid S129 in the subject's cells.

[0035] In some implementations of this aspect of the disclosure, the compound that binds to FAF1 or inhibits the activity of FAF1 in regulating the amount of LC-3 and p62 is represented by Formula 1:

[0036] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0037] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched or cyclic C1-C3 alkyl; A is CH or N. or a pharmaceutically acceptable salt thereof.

[0038] Cerebrospinal fluid (CSF) can be collected to measure the level of FAF1 and alpha-synuclein in the subject.For example, as described by TH Langenickel et al., Br. J. Clin Pharmacol., vol. 81, pp. 878-890 (2016) (the entirety of which is incorporated herein by reference), CSF samples can be collected for the assay of FAF1 or alpha-synuclein using time-sampling by an indwelling spinal catheter. [Example]

[0039] 1. Increased alpha-synuclein levels in cells due to FAF1 FAF1 was overexpressed in SH-SY5Y cells by transfection with a Flag-tagged FAF1 gene. Overexpression was confirmed by Western blot of cell extracts using anti-Flag and anti-FAF1 antibodies. Rabbit anti-FAF1 antibody was purchased from Proteintech, Chicago, IL, USA, Cat. No. 10271-1-AP. These cells were cotransfected with the alpha-synuclein gene. Alpha-synuclein levels were monitored by Western blot using anti-alpha-synuclein antibodies. Clearly and unexpectedly, alpha-synuclein levels increased with FAF1 overexpression (Figure 1). Note that beta-actin levels were used as an internal control. Note that a certain level of endogenous FAF1 protein is present in the cells.

[0040] 2. Increase in aggregated forms of alpha-synuclein by FAF1 in cells FAF1 was overexpressed in SH-SY5Y cells by transfection with a Flag-tagged FAF1 gene. These cells were then cotransfected with the alpha-synuclein gene. The levels of aggregated forms of alpha-synuclein were monitored by Western blot using an anti-alpha-synuclein antibody with prolonged film exposure. Clearly and unexpectedly, the levels of aggregated forms of alpha-synuclein increased with FAF1 overexpression (Figure 2).

[0041] 3. Generation of Lewy body-like forms of alpha-synuclein by FAF1 in cells FAF1 was overexpressed in SH-SY5Y cells by transfection with a Flag-tagged FAF1 gene. These cells were then cotransfected with the alpha-synuclein gene. The LB-like morphology of alpha-synuclein was monitored by immunofluorescence staining using an anti-alpha-synuclein antibody. Clearly and unexpectedly, the LB-like morphology of alpha-synuclein was observed when FAF1 was overexpressed (Figure 3B, top, arrow). However, the LB-like morphology was not observed in the vector control (Figure 3A, top), demonstrating that FAF1 induces the LB-like morphology.

[0042] Alpha-synuclein is known to be phosphorylated at serine 129, a potential pathological process that leads to the formation of Lewy bodies. The phosphorylated form of alpha-synuclein at position 129 was monitored by immunofluorescence staining using an anti-Ps129-alpha-synuclein antibody. A distinct LB-like form of alpha-synuclein was observed when FAF1 was overexpressed (Figure 3B, top, arrow). A distinct S129-phosphorylated form of alpha-synuclein was observed when FAF1 was overexpressed in an LM-like form (Figure 3B, bottom, arrow). However, this phosphorylation was not observed in the vector control (Figure 3A, bottom), indicating that FAF1 induces phosphorylation of alpha-synuclein at serine 129.

[0043] 4. FAF1-induced increase in alpha-synuclein in mice FAF1 was overexpressed in mice by stereotypic injection of recombinant adenovirus containing the FAF1 gene into the midbrain region. Overexpression was confirmed by Western blot of midbrain tissue extracts using anti-FAF1 antibodies. The midbrain contains nigral dopaminergic neurons, which are known to affect locomotion in synucleinopathies. Two mice were injected with the FAF1 adenovirus, and two uninjected mice served as controls. Alpha-synuclein levels were monitored by Western blot using anti-alpha-synuclein antibodies. Clearly, elevated alpha-synuclein levels correlated with FAF1 overexpression (Figure 4).

[0044] 5. Reduction of alpha-synuclein by FAF1 siRNA in cells FAF1 expression was attenuated by treating SH-SY5Y cells with siRNA (sequence: GUGUUGUGGCACAAACCAU), and the reduction in FAF levels was confirmed by Western blot of cell extracts using anti-FAF1 antibody. Cells were co-transfected with the alpha-synuclein gene. The level of alpha-synuclein was monitored by Western blot using anti-alpha-synuclein antibody. Clearly, the level of alpha-synuclein was reduced by FAF1 siRNA treatment (Figure 5). In contrast, treatment with control siRNA (scRNA, degenerate sequence) did not reduce alpha-synuclein levels.

[0045] 6. FAF1 is an inhibitor of autophagy Alpha-synuclein is known to be degraded by various intracellular pathways, including proteasome, autophagy, and chaperone-mediated autophagy (Sampaio-Marques B and Ludovico P 2015). FAF1 was overexpressed by transfection in SH-SY5Y cells, and various proteins involved in the proteolytic pathway were analyzed by Western blotting (Figure 6).

[0046] As shown in Figure 6, overexpression of FAF1 increases p62 protein and decreases LC3, indicating that FAF1 inhibits autophagy. Note that all other proteins remain unchanged. This change mediates autophagy, as previously described (Sampaio-Marques B and Ludovico P 2015).

[0047] 7.KM-819 (KR-33493) is a FAF1 inhibitor KM-819 (KR-33493, 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid) is an aminopyrazole derivative described in the prior art as an ischemic cell death inhibitor (U.S. Patent No. 7,939,550, Yoo SE et al. 2016). Inhibition of FAF1 by KM-819 is confirmed by its binding to FAF1, as shown in Figure 7. Inhibition of FAF1 by KM-819 is also confirmed by its inhibition of downstream targets in cells, including the apoptosis pathway proteins caspase 8 and JAK (Figure 8). The compound showed 70% inhibition of apoptosis at 10 μM in an ischemic cell death assay, and deoxyglucose induced hypoxia-mediated apoptosis (U.S. Patent No. 7,939,550).

[0048] KM-819 binding to FAF1 was confirmed by pull-down analysis in SH-SY6H cell extracts using biotin-labeled KM-819 as described (Yoo SE et al. 2016). Cells were transfected with Flag-tagged FAF1. The resulting FAF1-KM-819 complex was precipitated using avidin-coated agarose. The precipitated FAF1 in the complex was measured by Western blot using an anti-Flag antibody. Apparently, FAF1 binds to KM-819 when cells are stressed by hydrogen peroxide, activating FAF1. Note that total FAF1 levels in whole-cell extracts (WCL) were measured as a control.

[0049] FAF1 activates apoptotic stress through various stimuli via activation of the caspase cascade and necrosis, as described (Yu C et al. 2016). The direct targets of FAF1 are caspase-8 and JNK in these two pathways, respectively. This activation step involves the formation of complexes that subsequently activate downstream targets.

[0050] Inhibition of FAF1-caspase 8 and FAF1-JNK complex formation by KM-819 was confirmed by standard immunoprecipitation assays using caspase 8 DED and JNK antibodies, and bound and precipitated FAF1 was measured by Western blot analysis using anti-FAF1 antibodies. Clearly, KM-819 effectively inhibited complex formation, achieving approximately 80% inhibition at 0.1 μM (Figure 8).

[0051] Inhibition of caspase-8 and JNK activation by KM-819 is confirmed by assaying caspase-8 substrate, caspase-3 cleavage, and JNK autophosphorylation, respectively (FIG. 9). Cells were treated with different concentrations of KM-819 as indicated, and the levels of cleaved caspase-3 and autophosphorylated JNK were measured by Western blot using anti-caspase-3 and anti-phospho-JNK antibodies, respectively. Clearly, KM-819 inhibits the activity of caspase-8 and JNK activation (Figure 9). The IC50 of KM-819 for caspase-8 activation is 0.73 μM, and the IC50 of KM-819 for JNK activation is 0.25 μM.

[0052] 8. KM-819 promotes autophagy. Alpha-synuclein is known to be degraded by various intracellular pathways, including proteasome, autophagy, and chaperone-mediated autophagy (Sampaio-Marques B and Ludovico P 2015). FAF1 inhibits autophagy.

[0053] To confirm that FAF1 inhibitors inhibit FAF1-mediated autophagy suppression, SH-SY5Y cells overexpressing FAF1 were treated with KM-819 as indicated, and LC3 and p62 levels were measured by Western blot (Figure 10). KM-819 reduced p62 protein and increased LC3, indicating that KM-819 promotes the autophagy pathway. The EC50 for KM-819 to promote autophagy is 1 nM.

[0054] 9. Reduction of alpha-synuclein by FAF1 inhibitors in cells To confirm whether FAF1 inhibitors reduce alpha-synuclein levels in cells, SH-SY5Y cells were treated with the FAF1 inhibitor KM-819 (KR-88493, U.S. Patent No. 7,939,550, Yoo SE et al. 2016). Cells were co-transfected with genes encoding Flag-tagged FAF1 and GFP-tagged alpha-synuclein. Alpha-synuclein levels were measured by Western blot of cell extracts using an anti-GFP antibody. FAF1 levels were measured by Western blot of cell extracts using an anti-Flag antibody. Clearly, alpha-synuclein levels were reduced by treatment with the FAF1 inhibitor (Figure 11). The IC50 was 20 nM, and inhibition was greater than 95% at 10 uM. This is significantly higher potency than the apoptosis inhibitory activity measured in the prior art, which was 70% inhibition at 10 uM. Note that FAF1 levels themselves are not altered by treatment with FAF1 inhibitors.

[0055] 10. FAF1 inhibitor reduces alpha-synuclein in the midbrain of mice To examine the effect of FAF1 inhibitors on alpha-synuclein levels in animals, we treated alpha-synuclein-overexpressing transgenic mice with the FAF1 inhibitor KM-819. The transgenic mice are a mouse strain carrying the human alpha-synuclein mutant gene A53T under the control of the prion gene promoter (Lee MK et al. 2002).

[0056] Three-month-old mice were orally administered KM-819 at doses of 1, 5, and 10 mg / kg / day for 9 days. Two non-transgenic mice (non-Tg) served as controls, and three transgenic mice (alpha-syn A53T Tg) were administered vehicle alone without KM-819. Two mice were used per KM-819 administration. Animals were sacrificed, and midbrain tissue was isolated and extracted. Alpha-synuclein, the S129 phosphorylated form of alpha-synuclein, and FAF1 were measured by Western blot using anti-alpha-synuclein, P-alpha-syn, and FAF1 antibodies, respectively. Significantly, the levels of both alpha-synuclein and its phosphorylated form were reduced by treatment with the FAF1 inhibitor KM-819, indicating that the FAF1 inhibitor reduces alpha-synuclein accumulation in the animals (Figure 12). Note that FAF1 levels themselves are not altered by treatment with FAF1 inhibitors, and beta-actin levels are used as an internal control.

[0057] 11. FAF1 inhibitor reduces alpha-synuclein in the mouse hippocampus To examine the effect of FAF1 inhibitors on alpha-synuclein levels in animals, we treated alpha-synuclein-overexpressing transgenic mice with the FAF1 inhibitor KM-819. The transgenic mice are a mouse strain carrying the human alpha-synuclein mutant gene A53T under the prion gene promoter (Human-synuclein-harboring familial Parkinson's syndrome, Lee MK et al. 2002).

[0058] Three-month-old mice were orally administered KM-819 at doses of 1, 5, and 10 mg / kg / day for 9 days. Three non-transgenic (non-Tg) mice served as controls, and two transgenic mice (alpha-syn A53T Tg) were administered vehicle alone without KM-819. Two mice were used for each KM-819 administration. The animals were sacrificed, and hippocampal tissue was isolated and extracted. Alpha-synuclein and FAF1 were measured by Western blot using anti-alpha-synuclein and anti-FAF1 antibodies, respectively. Significantly, alpha-synuclein levels were reduced by treatment with the FAF1 inhibitor KM-819, indicating that the FAF1 inhibitor reduces alpha-synuclein accumulation in the animals (Figure 13).

[0059] 12. FAF1 inhibitor reduces alpha-synuclein in mouse cortex To examine the effect of FAF1 inhibitors on alpha-synuclein levels in animals, we treated alpha-synuclein-overexpressing transgenic mice with the FAF1 inhibitor KM-819. The transgenic mice are a mouse strain carrying the human alpha-synuclein mutant gene A53T under the prion gene promoter (Human-synuclein-harboring familial Parkinson's syndrome, Lee MK et al. 2002).

[0060] Three-month-old mice were orally administered KM-819 at doses of 1, 5, and 10 mg / kg / day for 9 days. Two non-transgenic mice (non-Tg) served as controls, and two transgenic mice (alpha-syn A53T Tg) were administered vehicle alone without KM-819. Two mice were used per KM-819 administration. Animals were sacrificed, and cortical tissue was isolated and extracted. Alpha-synuclein was measured by Western blot using an anti-alpha-synuclein antibody. Alpha-synuclein levels were significantly reduced by treatment with the FAF1 inhibitor KM-819, indicating that FAF1 inhibitors reduce alpha-synuclein accumulation in the animals (Figure 14).

[0061] 13. FAF1 inhibitor reduces alpha-synuclein in mouse cerebellum To examine the effect of FAF1 inhibitors on alpha-synuclein levels in animals, we treated alpha-synuclein-overexpressing transgenic mice with the FAF1 inhibitor KM-819. The transgenic mice are a mouse strain carrying the human alpha-synuclein mutant gene A53T under the prion gene promoter (Human-synuclein-harboring familial Parkinson's syndrome, Lee MK et al. 2002).

[0062] Three-month-old mice were orally administered KM-819 at doses of 1, 5, and 10 mg / kg / day for 9 days. Three non-transgenic mice (non-Tg) served as controls, and two transgenic mice (alpha-syn A53T Tg) were administered vehicle alone without KM-819. Two mice were used per KM-819 administration. The animals were sacrificed, and cerebellar tissue was isolated and extracted. Alpha-synuclein and FAF1 were measured by Western blot using anti-alpha-synuclein and FAF1 antibodies, respectively. Significantly, both alpha-synuclein levels were reduced by treatment with the FAF1 inhibitor KM-819, indicating that FAF1 inhibitors reduce alpha-synuclein accumulation in the animals (Figure 15).

[0063] 14. FAF1 inhibitor reduces alpha-synuclein in the mouse striatum To examine the effect of FAF1 inhibitors on alpha-synuclein levels in animals, we treated alpha-synuclein-overexpressing transgenic mice with the FAF1 inhibitor KM-819. The transgenic mice are a mouse strain carrying the human alpha-synuclein mutant gene A53T under the prion gene promoter (Human-synuclein-harboring familial Parkinson's syndrome, Lee MK et al. 2002).

[0064] Three-month-old mice were orally administered KM-819 at doses of 1, 5, and 10 mg / kg / day for 9 days. Three non-transgenic (non-Tg) mice served as controls, and two transgenic mice (alpha-syn A53T Tg) were administered vehicle alone without KM-819. Two mice were used per KM-819 administration. The animals were sacrificed, and striatal tissue was isolated and extracted. Alpha-synuclein and FAF1 were measured by Western blot using anti-alpha-synuclein and anti-FAF1 antibodies, respectively. Significantly, alpha-synuclein levels were reduced by treatment with the FAF1 inhibitor KM-819, indicating that FAF1 inhibitors reduce alpha-synuclein accumulation in the animals (Figure 16).

[0065] [Embodiment] Embodiment 1: A method of treating a disease or condition resulting from synucleinopathy, comprising administering to a subject exhibiting symptoms of or at risk for synucleinopathy an effective amount of an inhibitor of FAF1 activity that increases the amount of alpha-synuclein, or an effective amount of an inhibitor of FAF1 activity that modulates the amount of LC-3 and p62.

[0066] Embodiment 2: An inhibitor of FAF1 activity is represented by Formula 1:

[0067] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0068] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched or cyclic C1-C3 alkyl; A is CH or N. or a pharmaceutically acceptable salt thereof.

[0069] Embodiment 3: The method of embodiment 1, wherein the aminopyrazole compound is 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid or an ester or amide thereof.

[0070] Embodiment 4: A method for treating a disease or condition resulting from synucleinopathy, comprising administering to a subject exhibiting symptoms of or at risk for synucleinopathy an effective amount of a compound that binds to FAF1 protein, the amount of alpha-synuclein phosphorylated at amino acid S129 in the subject's cells.

[0071] Embodiment 5: An inhibitor of FAF1 activity is represented by Formula 1:

[0072] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0073] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched or cyclic C1-C3 alkyl; A is CH or N. or a pharmaceutically acceptable salt thereof.

[0074] Embodiment 6: The method of embodiment 4, wherein the aminopyrazole compound is 4-[2-(4-bromophenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid or an ester or amide thereof.

[0075] Example 7: The method of any one of embodiments 1 to 3, wherein the FAF1 inhibitor is administered orally, or by injection into the brain or blood, or through the skin.

[0076] Embodiment 8: The method of any one of embodiments 4 to 6, wherein the compound that binds to the FAF1 protein is administered orally, or by injection into the brain or blood, or through the skin, or by other commonly used methods, wherein the FAF1-binding compound is administered orally.

[0077] Embodiment 9: A method for diagnosing synucleinopathy or assessing the risk of synucleinopathy in a subject, comprising measuring the amount of FAF1 protein or the level of FAF1 activity in the subject's cells, which inhibits autophagy mediated by LC-3 and p62 proteins, wherein a higher FAF1 activity or protein level than in a normal human population indicates the development of synucleinopathy or the subject's risk of developing synucleinopathy.

[0078] Embodiment 10: A method of treating a disease or condition resulting from synucleinopathy in a subject, comprising administering to a subject exhibiting FAF1 activity or FAF1 levels in the subject's cells that inhibit autophagy involving LC-3 and p62 proteins in the subject's cells, an amount of a compound that binds to FAF1 protein effective to decrease the amount of alpha-synuclein phosphorylated at amino acid S129 in the subject's cells, wherein a higher FAF1 activity or protein level than in a normal human indicates development of a synucleinopathy or the subject's risk of developing a synucleopathy.

[0079] Embodiment 11: An inhibitor of FAF1 activity is represented by Formula 1:

[0080] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0081] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched or cyclic C1-C3 alkyl; A is CH or N. or a pharmaceutically acceptable salt thereof.

[0082] Embodiment 12: Formula 1:

[0083] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0084] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched or cyclic C1-C3 alkyl; A is CH or N. or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier.

[0085] Embodiment 13: The method of embodiment 12, wherein the medicament is for treating a disease or condition resulting from a synucleinopathy in a subject.

[0086] Embodiment 14: A compound of Formula 1:

[0087] [ka] [In the formula, R1 is -CO2R3, -CH2OR3, -CONR3R4 or

[0088] [ka] wherein R3 and R4 are, independently of each other, H or a linear, branched or cyclic C1-C6 alkyl; R2 is -(CH2)2-phenyl; B is H, phenyl, or C1-C3 alkyl or halogen substituted phenyl; n is an integer from 0 to 2; Y is S, O, CH2, SO, SO2 or NR3R4, where R3 and R4, independently of each other, are H or linear, branched or cyclic C1-C6 alkyl; Z is H, halogen, OCH3, NO2, NH2, or linear, branched, or cyclic C1-C3 alkyl. 10. Use of an aminopyrazole compound represented by the formula: or a pharmaceutically acceptable salt thereof.

[0089] Embodiment 15: The use of embodiment 14, wherein the aminopyrazole compound is effective in inhibiting FAF1 activity to increase the amount of alpha-synuclein in the cells of a subject or in cells of a sample derived from a subject, or is effective in increasing autophagy mediated by LC-3 and p62 proteins in the cells of a subject or in cells of a sample derived from a subject, or is effective in decreasing the amount of alpha-synuclein phosphorylated at amino acid S129 in the cells of a subject or in cells of a sample derived from a subject.

Claims

1. A medicament for treating a disease or condition resulting from synucleinopathy, comprising: A medicament comprising an aminopyrazole, 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid, or an ester or amide thereof, in an amount that provides a concentration of at least 10 nM in cells of a subject.

2. The pharmaceutical of claim 1, wherein the amount provides a concentration of 20 nM to 10 μM in the cells of the subject.

3. The method of claim 1, wherein the amount provides a dose of 1 to 10 mg / kg / day to a subject.

4. A disease or condition resulting from a synucleinopathy, A group of neurodegenerative diseases caused by the accumulation and aggregation of misfolded alpha-synuclein; or caused by mutations in one or more of the alpha-synuclein gene (SNCA), glucocerebrosidase gene (GBA), parkin gene, LRRK2 gene, DJ-gene 1, and PINK1 gene; or mediated by FAF1 activity regulating the amount of LC-3 and p62, or by FAF1 activity regulating the amount of alpha-synuclein; or mediated by the amount of aggregated alpha-synuclein; or mediated by the amount of alpha-synuclein phosphorylated at amino acid S129 in the subject's cells; or resulting from the accumulation of misfolded alpha-synuclein or the accumulation of alpha-synuclein aggregates in the subject's cells The pharmaceutical composition according to any one of claims 1 to 3,

5. The pharmaceutical agent according to any one of claims 1 to 4, which is administered orally, by injection or infusion into the brain, or by injection or infusion into the blood.

6. 1. A method of dispensing a medicament for treating a disease or condition resulting from a synucleinopathy in a subject, comprising:

1. A method comprising combining an amount of 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid, or an ester or amide thereof, with a pharmaceutically acceptable carrier, wherein said amount of 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid, or an ester or amide thereof, provides a concentration of at least 10 nM in cells of the subject.

7. 7. The method of claim 6, wherein the amount of 4-[2-(4-bromo-phenylsulfanyl)-acetylamino]-1-phenethyl-1H-pyrazole-3-carboxylic acid or its ester or amide provides a concentration of 20 nM to 10 μM in the cells of the subject.

8. 8. The method of claim 6 or 7, wherein the amount provides a dose of 1 to 10 mg / kg / day to the subject.

9. A disease or condition resulting from a synucleinopathy, A group of neurodegenerative diseases caused by the accumulation and aggregation of misfolded alpha-synuclein; or caused by mutations in one or more of the alpha-synuclein gene (SNCA), glucocerebrosidase gene (GBA), parkin gene, LRRK2 gene, DJ-gene 1, and PINK1 gene; or mediated by FAF1 activity regulating the amount of LC-3 and p62, or by FAF1 activity regulating the amount of alpha-synuclein; or mediated by the amount of aggregated alpha-synuclein; or mediated by the amount of alpha-synuclein phosphorylated at amino acid S129 in the subject's cells; or resulting from the accumulation of misfolded alpha-synuclein or the accumulation of alpha-synuclein aggregates in the subject's cells The method according to any one of claims 6 to 8, wherein

10. The method according to any one of claims 6 to 9, wherein the medicament is formulated for oral administration, or for administration by injection or infusion into the brain, or for administration by injection or infusion into the blood.

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