WO2009027349A2 - Treatment and prevention of neurodegenerative diseases - Google Patents

Treatment and prevention of neurodegenerative diseases Download PDF

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WO2009027349A2
WO2009027349A2 PCT/EP2008/061036 EP2008061036W WO2009027349A2 WO 2009027349 A2 WO2009027349 A2 WO 2009027349A2 EP 2008061036 W EP2008061036 W EP 2008061036W WO 2009027349 A2 WO2009027349 A2 WO 2009027349A2
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Prior art keywords
uchll
gene
benzyloxy
fluoro
complex
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PCT/EP2008/061036
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French (fr)
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WO2009027349A3 (en
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Tamara Maes
Marta Barrachina Castillo
Isidro Ferrer Abizanda
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Oryzon Genomics Sa
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Priority claimed from GB0716577A external-priority patent/GB0716577D0/en
Priority claimed from GB0719899A external-priority patent/GB0719899D0/en
Priority claimed from GB0724970A external-priority patent/GB0724970D0/en
Application filed by Oryzon Genomics Sa filed Critical Oryzon Genomics Sa
Priority to EP08787434A priority Critical patent/EP2195029A2/en
Publication of WO2009027349A2 publication Critical patent/WO2009027349A2/en
Publication of WO2009027349A3 publication Critical patent/WO2009027349A3/en
Priority to US12/711,938 priority patent/US20100247543A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the invention relates to the treatment and prevention of neurodegenerative diseases.
  • the invention relates to the treatment and prevention of neurodegenerative diseases by repression of the transcriptional complex that represses the promoter of the UCHLl gene.
  • Neurodegenerative diseases are conditions in which cells of the brain and spinal cord are lost. Probably the best known neurodegenerative diseases are Alzheimer's disease, Parkinson's disease and multiple sclerosis which are caused by the gradual deterioration of neurons causing symptoms affecting cognition and/or movement, eventually leading to death.
  • Lewy body disorders is an umbrella term which includes dementia with Lewy bodies (DLB), Parkinson's disease (PD) and PD with dementia (PDD). These disorders are characterised by disorders of alpha-synuclein metabolism, which gives rise to the formation of abnormal neuronal alpha-synuclein inclusions, which are the defining pathologic process common to both PDD and DLB.
  • Synucleinopathies with and without dementia, encompass a wide range of diseases including Parkinson's disease, multiple system atrophy, rapid eye movement (REM), sleep behaviour disorder, and dementia with Lewy bodies (DLB).
  • DLB is a neurodegenerative disorder resulting in slowly progressive and unrelenting dementia until death. Prevalence studies suggest that it is the second most common dementing illness in the elderly.
  • the neuropathologic findings of DLB show a wide anatomic range. Lewy bodies and Lewy-related pathology are found from the brain stem to the cortex and, in many cases, associated with concurrent Alzheimer's disease pathology.
  • PDD and DLB show differing temporal sequences of key symptoms and clinical features. Patients with Parkinson's disease show an increased risk for dementia based on epidemiological studies.
  • DLB dementia with Lewy Bodies
  • Clinical core features of DLB consist of rapid fluctuations in cognition, recurrent visual hallucinations and spontaneous and fluctuating features of Parkinsonism; these are further supported by high sensitivity for extrapyramidal side effects to neuroleptics and rapid eye movement sleep behaviour disorder.
  • Dementia itself describes a syndrome characterised by memory impairment, intellectual deterioration, changes in personality and behavioural abnormalities.
  • Ubiquitin c-terminal hydrolase Ll (UCHLl) is one of the most abundant cytosolic proteins in the brain. In addition to neurons, it is expressed in testes (Wilkinson et al., 1989; Solano et al., 2000). Yet, UCHLl is abnormally over-expressed in non-small-cell lung cancer (Hibi et al., 1999), pancreatic cancer (Tezel et al., 2000), colorectal cancer (Yamazaki et al., 2002) and myeloma cells (Otsuki et al., 2004). UCHLl is an enzyme involved in the hydrolysis of polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS), favouring protein degradation (Liu et al., 2002).
  • UPS ubiquitin proteasome system
  • the invention in a first aspect relates to a method of treating or preventing a neurodegenerative disease in a patient suffering from such a condition which comprises administering to such a patient a therapeutically effective amount of an agent that represses the transcriptional complex that represses the promoter of the UCHLl gene.
  • the agent affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
  • the neurodegenerative disease is a Lewy Body disorder.
  • the agent may be a HDAC inhibitor, such as Trichostatin A (TSA), Suberoylanilide hydroxamic acid (SAHA),
  • TSA Trichostatin A
  • SAHA Suberoylanilide hydroxamic acid
  • the agent may be a small molecule that inhibits the function of REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl in the transcriptional complex that represses the promoter of the UCHLl gene.
  • the agent may be a small molecule that inhibits HDAC6.
  • the inhibition is provided by administering a small double stranded interference RNA (siRNA), a short hairpin RNA (shRNA), a microRNA, an antisense oligonucleotide or monoclonal antibodies directed against at least one of the genes that codes for one of the proteins belonging to the transcriptional repressor complex from UCHLl gene, for example REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl, or a different member of the aforementioned complex, or for a gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene, for example HDAC6.
  • siRNA small double stranded interference RNA
  • shRNA short hairpin RNA
  • microRNA an antisense oligonucleotide or monoclonal antibodies directed
  • a method of screening for molecules that inhibit the transcriptional complex that represses the promoter of the UCHLl gene comprising providing a cell line containing a reporter gene fused with the UCHLl regulatory domains which expresses no or low levels of UCHLl, incubating the cell line with a molecule of interest and screening for expression of the reporter gene, wherein expression of the reporter gene indicates inhibition of the transcriptional complex that represses the promoter of the UCHLl gene by the molecule of interest.
  • Figure 1 shows the schematic representation of UCHLl gene promoter.
  • White boxes represent three putative DNA binding sites for NRSF/REST (NRSE) detected by
  • NRSEl and NRSE3 are located in the complementary DNA chain (-) and the NRSE2 in the positive DNA chain (+). Exon 1 and 2 are indicated as dash boxes and intron 1 corresponds to grey box. The location of NRSEl, NRSE2 and NRSE3 sites is indicated relative to TATA signal located at position 268 of the sequence with GenBank number Xl 7377. The transcription start site is shown with an arrow.
  • A NRSF/REST (121 KDa) and UCHLl (25 KDa) protein levels are 30 detected by Western blot.
  • S- Actin 45 kDa is blotted to control protein loading. The image shows two samples from two different patients and age-matched controls but it is representative of all the samples indicated in the Table I.
  • NRSF/REST protein levels are only seen in the frontal cortex of DLBp and DLBc.
  • B Densitometric analysis of NRSF/REST protein levels normalized with S-actin.
  • AU Arbitrary Units (mean ⁇ SD). *p ⁇ 0.05 compared to control samples (ANOVA with post-hoc LSD test).
  • NRSF/REST expression is compared with UCHLl expression in the same cases to show an inverse relationship between NRSF/REST and UCHLl in every case. The same results were observed in all cases summarized in Table I.
  • Figure 3 shows NRSF/REST and UCHLl expression levels in DMS53, U87-MG and HeLa cell lines.
  • A NRSF/REST and UCHLl mRNA levels normalized with ⁇ - glucuronidase (GUSB). The detection was performed with TaqMan PCR as is indicated in experimental procedures section.
  • B NRSF/REST and UCHLl protein levels detected by Western blotting. The figure shows the densitometric analysis of NRSF/REST and UCHLl protein levels normalized with S-actin.
  • AU Arbitrary Units.
  • Figure 4 shows the effect of NRSF/REST overexpression in DMS53 cell line.
  • A 1 ⁇ g of REEXl vector, which encodes human full-length NRSF cDNA, was transfected in DMS53 cells using lipofectamine 2000.
  • NRSF/REST mRNA and protein levels were increased after 48h of REEXl vector transfection. The protein levels were detected by Western Blot showing two independent transfections. S-Actin (45 kDa) is blotted to control protein loading.
  • S-Actin 45 kDa
  • the over-expression of NRSF/REST transcription factor reduces endogenous UCHLl (B) and synaptophysin (Q mRNA levels.
  • REEXl over- expression was performed in triplicate (6-well plates) in three independent experiments. The mRNA levels of all the analysed genes were detected by TaqMan PCR and the endogenous control used was ⁇ -glucuronidase.
  • Figure 5 shows the effect of NRSF/REST siRNA transfection in U87-MG cell line.
  • A NRSF/REST protein levels are detected by Western Blot after 48h of NRSF/REST siRNA transfection (siRNA#l and siRNA#2). The scramble siRNA transfection does not modify the expression of endogenous NRSF/REST levels. S-Actin (45 kDa) is blotted to control protein loading. Reduction of NRSF/REST transcription factor increases endogenous UCHLl (B) and synaptophysin (Q mRNA levels. siRNA transfection was carried out in triplicate (6-well plates) in three independent experiments.
  • mRNA levels of all the analysed genes were detected by TaqMan PCR and the endogenous control used was ⁇ -glucuronidase (GUSB).
  • AU Arbitrary Units (mean ⁇ SD). */? ⁇ 0.01 and **p ⁇ 0.001 compared with non-transfected cells (ANOVA with post-hoc LSD test).
  • Figure 6 shows a chromatin immunoprecipitation (ChIP) assay using NRSF/REST antibody in HeLa, U87-MG and DMS53 cell lines illustrating that NRSF/REST binds the UCHLl regulatory region.
  • A Schematic representation of minimal UCHLl gene promoter. Grey boxes represent the three putative DNA binding sites for NRSF/REST (NRSE). The transcription start site is indicated as +1.
  • Immunoprecipitated DNA was analysed by PCR using two sets of primers that amplified a 247 bp region of the UCHLl gene promoter which encompasses an NRSE site (NRSEl) and a 214 bp region which contains the other two NRSE sites (NRSE2 and NRSE3).
  • ChIP assay was performed in U87-MG cells using a goat antibody anti-human NRSF/REST and a rabbit polyclonal anti-human acetyl-histone 3 (Lys9) antibody as positive control.
  • the ChIP assay with goat serum was performed as a negative control.
  • Primers set number 1 amplify a 247 bp region of the UCHLl gene promoter and primers set number 2 amplify a 214 bp region as schematically indicated above. The same results were obtained using HeLa cells.
  • C The same ChIP analysis performed but using DMS53 cells. Input refers to DNA chromatin not immunoprecipitated with the specific antibody.
  • ChIP refers to DNA chromatin immunoprecipitated with the specific antibody.
  • FIG. 7 shows the effect of the application of siRNAs and miRNAs directed against the repressor complex on the levels of UCHLl mRNA expression in U87-MG cells.
  • 10OnM of siRNA against HDAC6 indirect inhibition of components the repression complex led to an induction of nearly 7 fold in UCHLl mRNA expression.
  • Figure 8 shows the effect of the application of 24h treatment with 10OmM of the HDAC inhibitor trichostatin A (TSA) on the expression of UCHLl in U87-MG (A) and HeLa (B) cells.
  • TSA trichostatin A
  • Figure 9 shows that no systematic difference in DNA methylation of the minimal UCHLl gene promoter in post-mortem cortical brain samples of patients with Dementia with Lewy Bodies (pure and common form) and age-matched controls can be detected.
  • A Partial consensus sequence of UCHLl gene promoter (GenBank accession number X17377) with nucleotides numbered relative to ATG (+1). Nine CpG islands are indicated with boxes (the completed promoter sequence contains 35 CpG sites).
  • B Chromatogram of a partial PCR product from bisulfite treated UCHLl gene promoter (-248 to -174) of a 79-year-old male.
  • Figure 10 shows the inductory effect of the treatment of HeLa and U87-MG cells with the demethylation agent 5-azacytidine on UCHLl expression.
  • UCHLl The biological role of UCHLl and its role in pathogenesis encouraged the present study focused on the transcriptional regulation of UCHLl.
  • NRSF/REST neuron-restrictive silencer factor/RE-1 silencer transcription factor
  • Parkinson's disease Diffuse Lewy body disease: Dementia with Lewy bodies, pure form (DLBp) and common form (DLBc), and controls.
  • M male
  • F female
  • NFT neurofibrillary tangle.
  • P-m delay post-mortem delay in hours.
  • Braak stages refer to Braak and Braak Alzheimer's disease (AD) changes (Braak and Braak, 1999). Staging of H-synuclein pathology (Lewy bodies and Lewy neurites) related to sporadic Parkinson's disease (PD) was done according to Braak et al., 2003.
  • UCHLl was not identified previously as a target for REST/UCHL1 in genome wide analysis, for example:
  • UCHLl is not included as a target for the REl target database (http://www.bioinformatics.leeds.ac.uk/REldbjiikII/) at any PSSM cutoff level (Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes, Bruce et al, Proc Natl Acad Sci U S A. 2004 July 13; 101(28): 10458-10463).
  • NRSF/REST binds to the promoter of UCHLl and regulates the expression of UCHLl. Together, these findings demonstrate NRSF/REST as a relevant transcription factor that negatively regulates UCHLl expression and causes downregulation of expression in diseases with Lewy bodies, including PD and DLB.
  • a method of treating or preventing a neurodegenerative disease in a patient suffering from such a condition which comprises administering to such a patient a therapeutically effective amount of an agent that represses the transcriptional complex that represses the promoter of the UCHLl gene.
  • Neurodegenerative diseases include Alexander disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe disease, dementia with Lewy bodies, Machado- Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Neuroborreliosis, Parkinson's disease, Parkinson's disease with dementia, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff disease, Schilder's disease, Schizophrenia, Spinocerebellar ataxia (multiple types), Spin
  • treating or preventing we mean symptomatic improvement, which may include enhanced cognition, more autonomy and/or improvement in neuropsychiatric and behavioural dysfunction; and/or disease modification with slowing or arrest of symptom progression of the dementing process; and/or primary prevention of disease by intervention in key pathogenic mechanisms at a pre-symptomatic stage.
  • a Lewy body disorder is defined herein as a condition which is characterised by disorders of alpha- synuclein metabolism, which gives rise to the formation of abnormal neuronal alpha-synuclein inclusions. These are the defining pathologic process common to both PDD and DLB. More particularly Lewy body disorders include dementia with Lewy bodies (DLB), Parkinson's disease (PD) and PD with dementia (PDD).
  • DLB dementia with Lewy bodies
  • PD Parkinson's disease
  • PPD PD with dementia
  • the methods of the invention are particularly suitable for treating a neurodegenerative disease which is characterised by the overexpression of REST.
  • REST can be used as a biomarker for the detection of a neurodegenerative disease which could be treated by the method of the invention.
  • AD is an example of such a condition, where REST is slightly upregulated in the cortex and UCHLl is slightly downregulated.
  • the first model develops Lewy neurites (mouse overexpressing a synuclein with the A53T mutation) and could be envisaged as a model for Lewy Body disease, whilst the second model benefits from recombinant UCHLl protein therapy.
  • the second model (APP/PS1 mouse model) the delivery of an exogenous TAT-UCHLl fusion protein improves the symptoms of the neurodegeneration, supporting the role for UCHLl in therapeutic strategies in neurodegenerative disease.
  • R6/2 mice One transgenic mouse model for Huntington's disease is the R6/2 model (Mangiarini et al. (1996) Cell 87:493-506).
  • the R6/2 mice over-express exon 1 of the human Huntingtin gene which has an expanded CAG/polyglutamine repeat lengths (150 CAG repeats on average).
  • These mice develop a progressive, ultimately fatal, neurological disease with many biochemical and physiological features of human Huntington's disease.
  • abnormal aggregates constituted in part by the N-terminal part of Huntingtin (encoded by HD exon 1), are observed in R6/2 mice, both in the cytoplasm and nuclei of cells (Davies et al. (1997) Cell 90:537-548).
  • transgenic mice are characterized by reduced weight gain, reduced lifespan, and motor impairment characterized by abnormal gait, resting tremor, hindlimb clasping, and hyperactivity from 8 to 10 weeks after birth (for example the R6/2 strain; see Mangiarini et al. (1996) Cell 87:493-506).
  • the phenotype worsens progressively toward hypokinesia.
  • the brains of these transgenic mice demonstrate neurochemical and histological abnormalities, such as changes in neurotransmitter receptors (glutamate, dopaminergic), decreased concentration of N-acetylaspartate (a marker of neuronal integrity), and reduced striatum and brain size.
  • the compounds of the invention can be evaluated in this model by assessing parameters related to neurotransmitter levels, neurotransmitter receptor levels, brain size, striatum size, life-span, biochemical disease evidence (e.g., abnormal aggregates), and motor impairment.
  • the methods of treatment of the invention will be applied to patients that have downregulated expression of the UCHLl protein.
  • the transcriptional complex which is repressed is the NRSF/REST complex.
  • This complex comprises REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, SMARCEl (BAF57), SMARCA4 (BRGl) and SMARCC2 (BAF170) and other components.
  • the transcriptional complex may be repressed directly or indirectly by altering the transcription, translation, subcellular localisation or activity of one or several components of the complex. For example, inhibition of HDAC6 which is involved in the retention of the components of the complex in the cytoplasm can successfully inhibit the transcriptional complex.
  • This complex may be inhibited by various methods.
  • the REST complex may be inhibited by HDAC inhibitors.
  • Histone deacetylase plays a role in transcriptional regulation and catalyses the deacetylation of lysine residues located in the NH(2) terminal tails of histones and non-histone proteins. They play an important role in the regulation of the expression status of genes. Further HDACs are found in the REST transcriptional complex.
  • Histone deacetylases are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8), similar to yeast RPD3 and localized in the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10); homologous to yeast HDAl protein and localized both the nucleus and cytoplasm; and class III HDACs, a structurally distinct class of NAD- dependent enzymes similar yeast SIR2.
  • HDAC inhibitors are small molecules that target histone deacetylases. The application of HDAC inhibitors can reverse the silencing of genes generated by the acetylation of histones; and has been proposed for reactivating silenced tumours suppressor genes in cancer.
  • HDAC inhibitors would be expected to derepress the UCHLl gene and increase the expression of UCHLl. As such, they would be useful in the treatment or prevention of neurodegenerative diseases, and particularly in the treatment of Lewy body disorders.
  • a compound is administered to an individual in need of UCHLl up-regulation, in an amount sufficient to inhibit AOF2 (lysine specific demethylases; LSDl) activity.
  • the AOF2 inhibiting effective amount is an amount sufficient to increase the UCHLl mRNA by 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or 300% or more.
  • the AOF2 inhibiting effective amount is an amount sufficient to increase the UCHLl hydrolase activity by 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or 300% or more.
  • the AOF2 inhibiting effective amount is an amount sufficient to increase the UCHLl protein levels by 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or 300% or more.
  • the individual in need of UCHLl up-regulation is an individual suspected of having Lewy Body Dementia.
  • the individual in need of UCHLl up-regulation is an individual needing or desiring prophylaxis against cognitive decline.
  • the individual in need of UCHLl up-regulation is an individual needing or desiring a reduction in the rate of cognitive decline.
  • Administration of the compound can reduce the rate of cognitive decline in that patient (or group of patients).
  • HDAC inhibitors that can be used in this first aspect of the invention include inhibitors against any HDAC, including for example inhibitors against HDACl, HDAC2 or
  • HDAC6 examples include:
  • TSA Trichostatin A
  • SAHA Suberoylanilide hydroxamic acid
  • HDAC inhibitors include the carboxylic acid class of HDAC inhibitors and derivatives thereof.
  • the HDAC inhibitor is a short-chain to medium- chain fatty acid or a derivative or analog thereof.
  • short chain fatty acids include, but are not limited to, butyric acid, phenylalkanoic acids, phenylbutyrate (PB), 4-phenylbutyrate (4-PBA), pivaloyloxymethyl butyrate (Pivanex, AN-9), isovalerate, valerate, valproate, valproic acid, propionate, butyramide, isobutyramide, phenylacetate, 3-bromopropionate, or tributyrin.
  • Short-chain fatty acid compounds are described e.g., in U.S. Patent Nos. 4,988,731; 5,212,326; 4,913,906; 6,124,495; 6,110,970; 6,419,953; 6,110,955; 6,043,389; 5,939455; 6,511,678; 6,528,090; 6,528,091; 6,713,086; 6,720,004; U.S. Patent Publication No. 20040087652, Intl. Publication No.
  • Preferred inhibitors are orally administrable and capable of passing the blood brain barrier such as SAHA; and should at least release the repression of the UCHLl promoter; which would be the control for effectiveness.
  • the preferred inhibitors have at least 10%, 20%, or 30% or more blood brain barrier penetration.
  • the REST complex may be inhibited by agents that inhibit the function of the other members of the repression complex, including REST, MeCP2, mSin3a, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl.
  • agents may act by preventing the transcriptional repression complex from binding to the gene promoter or may act by preventing members of the complex from interacting with each other. In either case the end result will be that the complex is prevented from inhibiting gene expression, so the gene, UCHLl will become derepressed.
  • Suitable small molecules include:
  • the hydroxamate group functions as a metal binding group that interacts with the metal ion at the active site of the HDAC enzyme.
  • the A ring system is believed to be at the entrance to the metal ion pocket in the active site.
  • Non- limiting examples of heterocyclic, carbocyclic, and aryl ring systems, along with various linkers are given in the specific exemplified compounds below.
  • the HDAC inhibitor is chosen from N-Hydroxy 2-(5- naphthalen-2-ylmethylhexahydropyrrolo[3,4-c]pyrrol-2[l H]- yl)pyrimidine-5- carboxamide; N-Hydroxy 2- ⁇ 6-[(2-naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]hex-3- yl ⁇ pyrimidine-5-carboxamide trifluoroacetate; N-Hydroxy 2- ⁇ 6-[(6-fluoroquinolin-2- ylmethyl)amino]-3-aza-bicyclo[3.1.0]hex-3-yl ⁇ pyrimidine-5-carboxamide; N-Hydroxy 2-[5-(naphthalene-2-carbonyl)-hexahydropyrrolo[3,4-c]pyrrol-2-yl]pyrimidine-5- carboxamide; (S)-[4-(7-
  • the HDAC inhibitor is a carboline or beta-carboline derivative wherein the carboline or betacarboline ring systems (or analogs thereof) have a hydroxamate or N-hydroxy acylamino metal binding group as a pendant group, and pharmaceutically acceptable salts thereof.
  • the HDAC inhibitor is a benzoimidazole derivative.
  • the benzoimidazole derivative is chosen from 3-[l-(3-Dimethylamino-2,2-dimethyl-propyl)-2-(2,2- dimethyl-propyl)- 1 H-benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(3- Dimethylamino-2,2-dimethyl-propyl)-2-isopropyl-l H-benzoimidazol-5-yl]-N- hydroxy-acrylamide; 3-[2-Butyl-l-(3-dimethylamino-2,2-dimethyl-propyl)-lH- benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(3-Dimethylamino-2,2-dimethyl- propyl)-2-(2- methylsulfanyl-ethyl)-lH
  • the HDAC inhibitor is an imidazo[l,2- A] pyridine derivative.
  • the imidazo[l,2-A] pyridine derivative is chosen from (E)-N-hydroxy-3-(2-phenethyl-3-(3,4,5- trimethoxyphenylamino)imidazo[ 1 ,2-a]pyridin-6- yl)acrylamide; (E)-N-hydroxy-3-(2- phenethyl-3-(3,4,5- trimethoxyphenylamino)imidazo[l,2-a]pyridin-6- yl)acrylamide; (E)-3-(3-(benzo[d][l,3]dioxol-5-ylmethylamino)-2-phenethylimidazo[l,2-a]pyridin-i3- yl)-N- hydroxyacrylamide; N ⁇ Hydroxy-3-[2-phenethyl-3-(4-piperidin
  • the HDAC inhibitor is a benzothiothene derivative.
  • the benzothiothene derivative is chosen from 5-Phenylacetylamino-benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-Benzoylamino-benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-(3-Phenyl-acryloylamino)- benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-[3- Phenyl-2-(toluene-4-sulfonylamino)-propionylamino]-benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-[2-(3,4-Dimethoxy-phenyl)- acetylamino] -benzo [b]thiophene-2- carboxylic acid hydroxyamide; Tetrahydro-furan-2-carboxyl
  • the HDAC inhibitor is a depsipeptide or a derivative or analog of thereof.
  • the depsipeptide is chosen from is FK228 and Spiruchostatin A.
  • the depsipeptide analog or derivative is an amino acid derivative or an analog of FK228 or Spiruchostatin A, and pharmaceutically acceptable salts thereof.
  • the HDAC inhibitor is a stilbene like compound.
  • the stilbene like compound is chosen from (2Z)-3-(3,5-Dimethoxy phenyl)-2-(4-fluorophenyl)-N-[6-(2- hydroxybenzyl amine)-6-oxohexyl] acrylamide; (2Z)-3-(3,5-Dimethoxyphenyl)-2-(4- fluorophenyl)-N-[6-(3-hydroxybenzyl amine) -6-oxohexyl] acrylamide; (2Z)-3-(3,4- Difluorophenyl)-2-(4-fluorophenyl)-N-[6-(2-thiazole amine)-6-oxo hexyl] acrylamide; (2Z)-3-(2,3,4-Trimethoxyphenyl)-2-(4-hydroxyphenyl)-N-[6-(hydroxyamino
  • the HDAC inhibitor is a sulphonylpyrrole derivative.
  • the sulphonylpyrrole derivative is chosen from (E)-N-Hydroxy-3-[l-(toluene-4-sulfonyl)-l-H-pyrrol-3-yl]- acrylamide; (E)-N-(2-Amino-phenyl)-3-[ 1 -(biphenyl-4-sulfonyl)- 1 H-pyrrol-3-yl]- acrylamide; (E)-3-[ 1 -(4-Aminomethyl-benzenesulfonyl)- 1 H-pyrrol-3-yl]-N-hydroxy- acrylamide; (E)-N-Hy droxy-3-[ 1 -(5-pyridin-2-yl-thiophene-2-sulfonyl)- 1 H-pyrrol-3- yl]-acrylamide
  • the HDAC inhibitor is a thiophene or thiazole substituted trifluoroethanone derivative.
  • the thiophene or thiazole substituted trifluoroethanone derivative is chosen from N-(4-Methoxybenzyl)-5-(trifluoroacetyl)thiophene-2-carboxamide; N- Methyl-N-(quinolin-7-ylmethyl)-5-(trifluoroacetyl)thiophene-2-carboxamide; N-Ethyl- 5-(trifluoroacetyl)thiophene-2-carboxamide; N-(4-Chlorobenzyl)-5- (trifluoroacetyl)thiophene-2-carboxamide; N-[2-(3,4-Dihydroquinolin-l(2H)-yl)ethyl]- 5-(trifluoroacetyl)thiophene-2-
  • the HDAC inhibitor is an amino acid derivative.
  • the amino acid derivative is chosen from N 2 - [(5 -Methoxy-2-methyl-lH-indo 1-3 -yl)acetyl] -N'-2-naphthyl-5 -(4-oxopentyl)- Z-cystein amide; N 2 -[(5-Methoxy-2-methyl-lH-indol-3-yl)acetyl]-N'-2-naphthyl-5-[(2- oxopropyl)sulfonyl]-Z- norvalinamide; N-(3-Acetylphenyl)-N 2 -(cyanoacetyl)-5-[(3,3,3- trifluoro-2-oxopropyl)thio]-L-norvalinamide; N-Benzoylglycyl-N-pyridinium-3-
  • the HDAC inhibitor is a benzamide derivative (or analog).
  • the benzamide derivative is chosen from N-(2-Amino-phenyl)-4-[(2-propyl-pentanoylamino)-methyl]-benzamide; N-Hydroxy-4- [(2 -propyl-pentanoylamino)-methyl] -benzamide; N-(2-Amino-phenyl)-4- (2-propyl-pentanoylamino)-benzamide; N-Hydroxy-4-(2-propyl-pentanoylamino)- benzamide; 2-Propyl-pentanoic acid ⁇ 4-[2-amino-phenylcarbamoyl)-methyl]-phenyl ⁇ - amide; 2-Propyl-pentanoic acid (4-hydroxycarbamoyl-methyl-phenyl)-amide; 2- Propyl-p
  • HDAC inhibitors for use in the invention include, but are not limited to those found in international patent applications WO2006/123121 (23.11.2006), WO 2006/117549 (09.11.2006), WO 2006/117548 (09.11.2006), WO 2004/113336 (29.12.2004), WO 2007/058628 (24.05.2007), WO 2006/101456 (28.09.2006), WO 2006/101455 (28.09.2006), WO 2006/101454 (28.09.2006), WO 2005/040161 (06.05.2005) WO 2005/040101 (06.05.2005), WO 2007/061939 (31.05.2007), WO 2007/054776 (18.05.2007), WO 2007/039404 (12.04.2007), WO 2007/039403, (12.04.2007), WO 2007/029036 (15.03.2007), WO 2007/029035 (15.03.2007), WO 2006/129105 (07.12.2006), WO 2006/097460 (21.09.2006), WO 2006/097449 (21.09.2006), WO 2005/108367
  • the compound useful for increasing UCHLl mRNA, UCHLl protein, and/or UCHLl hydrolase activity is selected from the group consisting of halo-N-propargyl-1 -amino indans, indans, indoles, methylproparylamines, 5-substituted 2,4-thiazolidinediones, alkyl and alkylbenzyl ethers of substituted hydroquinones, l,3,4-oxadiazol-2(3H)-one derivatives, 4-(benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazones, N-propargylhydrazines, 4-pyrrolidino derivatives, benzazepine derivatives, 3H-quinazolin-4-one derivatives, N-acylamino aryl derivatives, N-acylamino aryl derivatives, 3-phenyl-propionamidoderivatives, 3- phenyl-propiona
  • the invention provides a method comprising: (1) identifying a patient having Dementia with Lewy Bodies and (2) administering to said patient an amount of a pharmaceutical composition effective to increase UCHLl activity (e.g., mRNA, protein, and/or hydrolase activity) wherein said composition comprises (I) a compound chosen from halo-N-propargyl-1 -amino indans, indans, indoles, methylproparylamines, 5-substituted 2,4-thiazolidinediones, alkyl and alkylbenzyl ether of substituted hydroquinones, l,3,4-oxadiazol-2(3H)-one derivatives, A- (benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazones, N-propargylhydrazines, A- pyrrolidino derivatives, benzazepine derivatives, 3H-quinazolin-4-one derivatives
  • halo-N-propargyl-1 -amino indans include, but are not limited to, 4-fluoro- N-propargyl-1 -amino indan, 5- fluoro-N-propargyl-1 -amino indan, 6-fluoro-N- propargyl-1 -amino indan, optically pure enantiomers thereof, pharmaceutically acceptable salts thereof.
  • the compound is 6-fluoro- N-propargyl-1 -amino indan.
  • the compound is (+)-6-fluoro-N-propargyl- 1 -amino indan.
  • indole compounds include, but are not limited to, N-methyl-N-propargyl- 2-[ 1 -methyl-5-methoxyindolyl]methylamine, N-propargyl-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2-butynyl)-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2-butynyl)-N-methyl-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2,3-butadienyl)-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2,3-butadienyl)-N-methyl-2-[ 1 -methyl-5- methoxyindolyl]methylamine, and 5-methoxyindol-2-ylmethylamine, and pharmaceutically acceptable salts thereof.
  • methylpropargylamine compounds include, but are not limited to, N-(2- Butyl)-N-methylpropargylamine, N-(I -Butyl)-N-methylpropargylamine, N-(2-Propyl)- N-methylpropargylamine, N-(I -Pentyl)-N-methylpropargylamine, N-(2-Pentyl)-N- methylpropargylamine, N-(I -Heptyl)-N-methylpropargylamine, N-(2-Heptyl)-N- methylpropargylamine, N-(2-Decyl)-N-methylpropargylamine, N-(2-Dodecyl)-N- methylpropargylamine, and pharmaceutically acceptable salts thereof.
  • Examples of 5-substitutued 2,4-thiazolidinediones include, but are not limited to, 2,4- Dioxo-5 - [3 -(phenylmethoxy)-phenylmethylene] -4- thiazo lidinebutanenitrile, 2,4- Dioxo-5 - [3 -(phenylmethoxy)-phenylmethylene] -4-thiazo lidinepentanenitrile, and pharmaceutically acceptable salts thereof.
  • alkyl or alkylbenzyl ethers of substituted hydroquinones include, but are not limited to, 4-[2'-Formyl-4'-(m-chlorophenylmethyloxy)phenoxy]butyronitrile, 4-[2'- Methoxymethyl-4'-(m-chlorophenylmethyloxy)phenoxy]butyronitrile, 4-[2'-
  • Examples of l,3,4-oxadiazol-2(3H)-one derivatives include, but are not limited to, 5- [4-(4,4,4-trifluorobutoxy)phenyl]-3-methoxyethyl- 1 ,3,4-oxadiazol-2(3H)-one, 5-[4- (4,4,4-trifluorobutoxy)phenyl]-3-hydroxyethyl-l,3,4-oxadiazol-2(3H)-one, 5-[4-(4,4,4- trifluorobutoxy)phenyl]-3-methylthioethyl-l,3,4-oxadiazol-2(3H)-one, 5-[4-(4,4,4- trifluoro-2-butenyloxy)phenyl]-3-methoxyethyl- 1 ,3 ,4-oxadiazol-2(3H)-one, 5-[4-
  • 4-(benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazones include, but are not limited to, 4-(benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(4- methylbenzyl)oxy]benzaldehyde acetyl (2-cyanoethyl)hydrazone, 4-[(4- nitrobenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(4- chlorobenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(4- methoxybenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(2,4- dichlorobenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)
  • N-propargylhydrazines include, but are not limited to, N 2 - propargylphenelzine, N'-propargylphenelzine, N'-propargyl-N 2 -acetylphenelzine, and pharmaceutically acceptable salts thereof.
  • 4-pyrrolidino derivatives include, but are not limited to, (RS)-l-[4-(3- fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid methylamide, (RS)-I- [4-(3-fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid amide, (RS)-I -[4- (4-fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid amide, (RS)-l-[4-(4- fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid methylamide, (RS)-2- oxo-l-[4-(4-trifluoromethyl-benzyloxy)-phenyl]-pyrrolidine-3-carboxylic acid amide, and (RS)-2-ox
  • benzazepine derivatives include, but are not limited to, l-[7-(3-fluoro- benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-ethanone, 1 -[7-(3-fluoro- benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-2-methoxy-ethanone, 2-[7-(3- fluoro-benzylo xy)-l, 2,4, 5-tetrahydro-benzo[d]azepin-3-yl]-2-oxo-acetamide, 3-[7-(3- fluoro-benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-3-oxo-propionamide, 7-(3- fluoro-benzylo xy)-l, 2,4, 5-tetra
  • 3H-quinazolin-4-one derivatives include, but are not limited to, 2-[7-(3- fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-acetamide, 2-[7-(3-fluoro-benzyloxy)-4- oxo-4H-quinazolin-3-yl]-propionamide, 2-[7-(4-fluoro-benzyloxy)-4-oxo-4H- quinazolin-3-yl]-acetamide, 2-[7-(4-fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]- propionamide, 2-[7-(3-fluoro-benzyloxy)-2-methyl-4-oxo-4H-quinazolin-3-yl]- acetamide, 2-[2-cyclopropyl-7-(3-fluoro-benzyloxy)-4-oxo-4H-quina
  • N-acylamino aryl derivatives include, but are not limited to, N-[4-(3- fluoro-benzyloxy)-phenyl]-malonamide, N-[4-(3-fluoro-benzyloxy)-phenyl]- malonamic acid methyl ester, N-[4-(3-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[3-fluoro-4-(3-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[4-(4-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[2-fluoro-4-(3- fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[4-(2,4-difluoro- benzyloxy)-phenyl]-malonamic acid methyl
  • 3-phenyl-propionamidos, 3-phenyl-acrylamidos, or 3-phenyl- propynamidos include, but are not limited to, N-methyl-3-[4-(4-methyl-benzyloxy)- phenylj-acrylamide, 3-[4-(3-methoxy-benzyloxy)-phenyl]-N-methyl-acrylamide, 3-[4- (3-fluoro-benzyloxy)-phenyl]-2,N-dimethyl-acrylamide, 3-[4-(3-fluoro-benzyloxy)- phenyl] -N-methyl-acrylamide, N-methyl-3 - [4-(4-trifluoromethyl-benzyloxy)-phenyl] - acrylamide, 3-[4-(3,4-difluoro-benzyloxy)-phenyl]-N-methyl-acrylamide, 3-[4-(4- fluoro-benzyloxy)-phenyl]-N-methyl-acrylamide, and pharmaceutical
  • fluorobenzamide derivatives include, but are not limited to, (S)-N-(I- carbamoyl-ethyl)-2-fluoro-4-(3-fluoro-benzyloxy)-benzamide, 2-[4-(3- fluorobenzyloxy)-2-fluoro-benzamido]acetamide, (S)-N-(I -carbamoyl-2-hydroxy- ethyl)-2-fluoro-4-(3-fluoro-benzyloxy)-benzamide, (R)-N-(I -carbamoyl-ethyl)-2- fluoro-4-(3-fluoro-benzyloxy)-benzamide, 2-[4-(4-fluorobenzyloxy)-2-fluoro- benzamido]acetamide, (S)-N-( 1 -carbamoyl-ethyl)-2-fluoro-4-(4-fluoro-benzyloxy
  • Examples of a 2,3-Dihydro-isoindol-l-one derivatives include, but are not limited to, 2- [5-(3-fluoro-benzyloxy)-l-oxo-l,3-dihydro-isoindol-2-yl]-acetamide, 2-[5-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (S)-2-[6-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (R)-2-[6-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide , (S)-2-[ 1 -oxo
  • fluoroallylamines include, but are not limited to, 2-isobutyl-3- fluoroallylamine, 2-isopropyl-3-fluoroallylamine, 2-(9-octadecenyl)-3- fluoroallylamine, 2-(3 -methyl-3 -butenyl)-3 -fluorallylamine, 2-(4-methoxy-2-butenyl)- 3 -fluoroallylamine, 2-isobutylsulfonylmethyl-3 -fluoroallylamine, 2-sec-butyl-3 - fluoroallylamine, 2-butyl-3-fluoroallylamine, 2-hexyl-3-fluoroallylamine, 2-heptyl-3- fluoroallylamine, 2-ethoxymethyl-3-fluoroallylamine, and 2-thioethoxymethyl-3- fluoroallylamine, 2-(2'-chlorophenoxy)methyl-3-fluoroallylamine, 2-(4'-ch
  • pyridine-2-carboxamides examples include but are not limited to, N-(2- aminoethyl)-5-chloropyridine-2-carboxamide and pharmaceutically acceptable salts thereof.
  • silyl alkylene amines include, but are not limited to, ⁇ -(benzyldimethylsilyl)ethanamine.hydrochloride, ⁇ -(dimethyl-2-phenylethylsilyl)ethanamine. hydrochloride, ethyl-4- fluorobenzylmethylsilylmethanamine.hydrochloride, dimethyl-4-fluorobenzylsilylmethanamine. hydrochloride, dimethyl-3-fluorobenzylsilylmethanamine. hydrochloride, 3,4-difluorobenzyldimethylsilylmethanamine.
  • hydrochloride 2,6-difluorobenzyldimethylsilylmethanamine, hydrochloride, 2,4-difluorobenzyl)dimethylsilylmethanamine. hydrochloride, dimethyl-2-fluorobenzylsilylmethanamine. hydrochloride, cyclohexylmethyldimethylsilylmethanamine. hydrochloride, ⁇ -(benzyldimethylsilyl)ethanamine.hydrochloride, ⁇ -(dimethyl-2-phenylethylsilyl)ethanamine.
  • hydrochloride ethyl-4-fluorobenzylsilylmethanamine.hydrochloride, dimethyl-4-fluorobenzylsilylmethanamine. hydrochloride, dimethyl-3-fluoroenzylsilylmethanamine. hydrochloride, 3,4-difluorobenzyldimethylsilylmethanamine. hydrochloride, 2, 6-difluorobenzyldimethylsilylmethanamine. hydrochloride, 2,4-difluorobenzyldimethylsilylmethanamine. hydrochloride, dimethyl-2-fluoroenzylsilylmethanamine.
  • phthalimido derivatives include, but are not limited to, 2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl]-acetamide, (S)-2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (S)-2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-iso indol-2-yl]-3-hydroxy- propionamide,
  • isoquinolino derivatives examples include, but are not limited to,
  • pyridine amidos examples include but are not limited to
  • oxazolo[3,4-a]quinolin-l-ones include, but are not limited to, 3-methoxymethyl-7-(4,4,4-trifluoro-3-hydroxybutoxy)-3,3a,4,5-tetrahydro-lH-oxazolo [3,4-a]quinolin-l-one, 3-methoxymethyl-7-(4,4,4-trifluorobutoxy)-3,3a,4,5-tetrahydro- lH-oxazolo[3,4-a]quinolin-l-one, 7-(4,4,4-trifluorobutoxy)-3,3a,4,5-tetrahydro-lH- oxazolo[3,4-a]quinolin-l-one, 7-(3-hydroxy-4,4,4-trifluorobutoxy)-3,3a,4,5-tetrahydro- 1 H-oxazolo [3 ,4-a]quino lin- 1 -one, 3 -meth
  • Examples of a 3,4-Dihydro-2H-pyrimido(2,l-b)benzothiazoles include, but are not limited to, N-(l-ethylpropyl)-3,4-dihydro-2H-pyrimido[2,l-b]benzothiazol-7-amine, the pharmaceutically acceptable acid addition salts thereof, and the pyrimido[2,l- b]benzothiazolium salts thereof.
  • thioxanthen-9-ones include, but are not limited to, 7-isopropyl-3-(2- methyl)-2H-tetrazol-5-yl)thioxanthen-9-one 10,10-dioxide, 3-(2-methyl-lH-tetrazol-5- yl)thioxanthen-9-one 10,10-dioxide, and 3-(l-methyl-lH-tetrazol-5yl)thioxanthen-9- one 10,10-dioxide, and pharmaceutically acceptable salts thereof.
  • ethylenediamine monoamides examples include, but are not limited to,
  • Examples of l,2,3,4-tetrahydrocyclopent[b]indoles and l,2,3,3a,4,8a- hexahydrocyclopent[B]indoles include, but are not limited to, 4-methyl-3- phenylmethylamino-l,2,3,4-tetrahydrocyclopent[b]indol7-yl methylcarbamate, 3-(N- cyclopropyl)amino-4-methyl- 1 ,2,3 ,4-tetrahydrocyclopent[b]indol7-yl methylcarbamate, l,2,3,3a,4,8-hexahydro-4-methyl-3-(N-phenylmethyloxycarbonyl)amino-cyclopent[b] indol-7-yl methylcarbamate, 1 ,2,3,3a,4,8a-hexahydro-4-methyl-3-(N-phenylmethyl-N- methylaminocarbonyl
  • arylethynylphenylcyclopropylamines include, but are not limited to,
  • cyclopent[b]indoles include, but are not limited to, 4-Methyl-3- (2propynyl)amino-l,2,3,4-tetrahydrocyclopent[b]-indol-7-yl-methylcarbamate, l,2,3,3a,4,8b-Hexahydro-4-methyl-3-(N-phenylmethoxycarbonyl)aminocyclopent[b] indol-7-yl methylcarbamate, 1 ,2,3,3a,4,8b-Hexahydro-4-methyl-3-(N-phenylmethyl-N- methylaminocarbonyl)aminocyclopent[b] indo 1-7-yl-phenylmethylcarbamate, and pharmaceutically acceptable salts thereof.
  • benzamides include, but are not limited to, N-(2-Aminoethyl)-p- chlorobenzamide, N-(2-aminoethyl)-p-fluorobenzamide, N-(2-aminoethyl)-p- bromobenzamide, N-(2-aminoethyl)-3,4-dichlorobenzamide, N-(2-aminoethyl)-2,4- dichlorobenzamide and N-(2-aminoethyl)benzamide, and pharmaceutically acceptable salts thereof.
  • 1,2,4-oxadiazoles include, but are not limited to, 3-[4-[3-(lH-imidazol- lyl)propoxy]phenyl]-5-ethyl-l,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-trichloromethyl-l,2,4-oxadiazo Ie, 3-[4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-propyl-l,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-cyclopropyl-l,2,4-oxadiazole, 3- [4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-phenyl-l,2,4-oxadiazole, 3-
  • oxazolidones include, but are not limited to, 3-[2-(l-hydroxy-3- cyanopropyl)benzothiazo 1-6-yl] -5 -methoxymethyl-2-oxazo lidone; 3 - [2-( 1 (S)-hydroxy- 3 -cyanopropyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone; 3 - [2-( 1 (R)- hydroxy-3 -cyanopropyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone; 3 - [2- (3 -cyanopropyl)benzothiazo 1-6-yl] -5 -methoxymethyl-2-oxazo lidone; 3-[2-(3- cyanopropyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone; 3-[2-(3- cyanopropyl)benzothi
  • 3-(aminoalkylamino)-l,2-benzisoxazoles examples include, but are not limited to, 6-
  • the compound useful for increasing UCHLl activity is a polyamine, or an analog or derivative thereof.
  • polyamine is a guanidine or biguanides analog or derivative.
  • the polyamine compound (or analog or derivative thereof) useful for increasing UCHLl activity is of the formula E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B- NH-E.
  • A is independently chosen from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl;
  • B is independently chosen from the group consistingof a single bond, C1-C6 alkyl, and C2-C6 alkenyl;
  • E is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, C3-C6 cycloalkenyl; with the proviso that either at least one A moiety is chosen from the group consisting of C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycl
  • the polyamine compound (or analog or derivative thereof) useful for increasing UCHLl activity is of the formula E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A- B-NH)x-E.
  • A is independently chosen from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl;
  • B is independently chosen from the group consisting of a single bond, C1-C6 alkyl, and C2-C6 alkenyl;
  • E is independently chosen from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl; and
  • x is an integer from 2 to 16; with the proviso that either at least one A moiety is chosen from the group consisting of C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-
  • Specific polyamine compounds include l,l l-bis ⁇ / 2 , ⁇ / 3 -dimethyl-N 1 -guanidino ⁇ -4,8- diazaundecane; l,15-bis ⁇ / 5 -[3,3-(diphenyl)propyl]- ⁇ f 1 -biguanido ⁇ -4,12- diazapentadecane; BENSpm; N 1 ,N 11 -bis(ethyl)norspermine; CPENSpm, N'-ethyk/V 11 - [(cyclopropyl)methyl]-4,8,-diazaundecane; CHENSpm; N 1 StKyI-N 11 -
  • Inhibitors of the function of AOF2 include Spermine; N-Acetyl-D-Glucosamine; Mdl72527 (N,N'-bis(2,3-butadienyl)-l,4-butane-diamine); alpha-D-mannose; alpha-D- fucose; Flavin- Adenine Dinucleotide; octane 1,8-diamine; L-deprenyl and tranylcypromine .
  • Demethylation agents such as 5-azacytidine and 5-aza-2'-deoxycytidine may also be useful in the method of the invention.
  • any of these small molecule chemical agents would be useful in the treatment or prevention of neurodegenerative diseases, and particularly in the treatment of Lewy body disorders.
  • a cell based screen for molecules that inhibit the UCHLl inhibitory complex may be done by using a cell line expressing REST with low/no levels of UCHLl in which a reporter cassette (e.g. GFP or luciferin) was inserted in its original genomic environment or in which a reporter construct fusing the UCHLl regulatory domains with a reporter cassette was inserted randomly into the genome; and the screening for reactivation and expression of the reporter gene.
  • a reporter cassette e.g. GFP or luciferin
  • the invention provides a method of treating a patient by administering a combination of two or more inhibitors; for example the first may inhibit one protein belonging to the transcriptional repressor complex from the UCHLl gene such as REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl, and the second may inhibit a different member of the aforementioned complex.
  • the inhibitor may indirectly inhibit the complex by altering the transcription, translation, subcellular localisation or activity of one or several components of the complex. Any combination of inhibitors may be used and is not restricted to the example above.
  • the invention uses an RNAi approach to directly or indirectly inhibit proteins in the transcriptional repressor complex, as would be apparent to the person skilled in the art.
  • miRNAs are small double stranded RNA molecules that are encoded in miRNA precursor genes. miRNA precursors mRNAs are transcribed; fold and are processed by the proteins Drosha and DICER to 20-25 base pair double-stranded RNA molecules. miRs negatively regulate expression of their target genes at the posttranscriptional level.
  • computer algorithms were used to identify REST/NRSF target sequences (UCHLl was not among the targets identified).
  • Wu and Xi (2006) have used these algorithms to identify a set of miRNAs (hsa-miR-124a, hsa-miR-132, hsa-miR-135a , hsa-miR-153, hsa-miR-218, hsa-miR-29b, hsa-miR-9, hsa-miR-9*) whose expression may be regulated by the REST complex.
  • the targets of the identified miRNAs are the different components of the REST complex (a regulation feedback loop).
  • siRNAs small double stranded interference RNAs
  • shRNAs short hairpin RNAs
  • siRNAs examples include siRNAs, siRNAs, and siRNAs.
  • a further approach is to use antisense technology to inhibit proteins in the transcriptional repressor complex, as would be apparent to the person skilled in the art.
  • the antibodies employed are preferably designed to be able to target and act in the cell nucleus (e.g. by fusion to the monoclonal antibody 3E10 Fv fragment (Hansen JE et al. Antibody-mediated p53 protein therapy prevents liver metastasis in vivo. Cancer Res. 2007 Feb 15;67(4): 1769-74).
  • the antibodies may be designed to sequester proteins of the complex in the cytosol.
  • Monoclonal antibodies have been described in the literature that would be suitable for use according to the present invention (e.g. Battaglioni et al.
  • Antibodies directed against the members of the complex regulating the UCHLl promoter can be generated by standard methods involving the fusion of antibody secreting B cells with cell lines selected for their ability to confer in vitro immortality on the antibody secreting cells.
  • DNA encoding monoclonal antibodies, antigen binding chains or domains can be cloned and expressed using standard methods of recombinant DNA technology. Recombinant antigen binding molecules can be manipulated to improve therapeutic properties such as specificity, affinity, half-life and lack of immunogenicity.
  • Rodent (e.g. rat or mouse) or other non-human animal (e.g. horse) antibodies can be used according to the invention. However, for use in man it is preferred that the antibody has been engineered to limit the anti-globulin response.
  • antibodies engineered in this way are chimeric antibodies (where the constant regions of a non-human antibody are replaced by human constant regions) and humanised antibodies where the antibody is engineered to appear human to the immune system of the recipient.
  • humanised antibodies are CDR-grafted antibodies where as well as replacing the constant regions of a non-human antibody with a human constant region, the framework regions of the variable regions are also replaced by human variable regions.
  • the agent that represses the transcriptional complex that represses the promoter of the UCHLl gene may be administered to the patient in a number of ways. For example use could be made of liposomes, nanoparticles, viral vectors, and the like. In particular it is preferred to use a delivery method that will allow the agent to cross the blood-brain barrier.
  • Example 1 Weak potential NRSF/REST binding sites are present in the UCHLl regulatory region
  • NRSE neuron-restrictive silencer elements
  • NRSE2 (0.71) and NRSE3 (0.67) were located in the intron 1, in the coding and complementary chains, respectively. Both NRSE elements were located very close together, being separated only by 11 bp. The sequence of all NRSE elements and the consensus sequence are shown in Figure IB.
  • NRSF/REST is a putative regulator of UCHLl, and its expression level is inversely related to UCHLl in the frontal cortex in PD and DLB.
  • UCHLl protein levels are reduced in the cerebral cortex in DLB but remain unchanged in the cerebral cortex of PD samples (Barrachina et al., 2006, Neurobiol. Dis. 22, 265-273).
  • NRSF is a repressor transcription factor
  • NRSF levels were not detected in control and PD samples but were increased in DLBp and DLBc samples ( Figure 2).
  • increased NRSF protein levels occurred, in the cerebral cortex, in parallel with reduced UCHLl protein levels (Barrachina et al., 2006).
  • Example 3 NRSF and UCHLl expression levels are inversely related in cell lines.
  • NRSF human lung carcinoma cell line
  • U87-MG human glioblastoma cell line
  • HeLa human cervical cancer cell line
  • NRSF mRNA levels were absent in DMS53 cells but higher in U87-MG and, particularly, in HeLa cells ( Figure 3A).
  • UCHLl mRNA levels were very high in NRSF-negative DMS53 cells, lower in U87-MG and undetectable in NRSF-positive HeLa cells. The same situation was found in relation to NRSF and UCHLl protein ( Figure 3B).
  • the precipitated DNA fragments were amplified with two sets of primers: set 1 spanned a 247 bp region covering the NRSEl and set 2 spanned a 214 bp region covering the NRSE2 and NRSE3 of the UCHLl gene promoter ( Figure 6A).
  • Figure 6A ChIP PCR products were detected with the NRSF and acetyl-histone 3 antibodies but not with a goat serum used as a negative control ( Figure 6B).
  • Example 7 Identification of NRSF/REST repressor complex components required for negative regulation of UCHLl.
  • NRSF/REST repressor complex that regulates UCHLl
  • Example 8 Identification of factors NRSF/REST repressor complex components required for negative regulation of UCHLl.
  • Example 9 Inhibition of the REST/NRSF complex and expression of UCHLl by application of miRs targeting components of the REST/NRSF complex.
  • NRSF has been known to recruit histone deacetylases (HDAC) to act as repressors through chromatin remodelling (Naruse et al., 1999; Huang et al., 1999).
  • HDAC histone deacetylases
  • TSA a specific inhibitor of HDAC in U87-MG, HeLa and DMS53 cells.
  • HDAC inhibitors M344 selective for HDAC6 over HDACl
  • MOCPAC selective for HDAcI over HDAC6
  • BATCP selective for HDAC6 over HDACl
  • Example 11 UCHLl promoter methylation is not consistently increased in Lewy body diseases.
  • cytosines represent, after sequence analysis, methylated cytosines (compare Figure 9A and B).
  • Figure 9C the vast majority of the 35 CpG islands described in the UCHLl gene promoter were non-methylated in age-matched controls and in DLB samples.
  • Sample 9C the sample number 3 presented only three methylated positions (CpG islands 3, 24 and 29) in two of three clones analysed.
  • Sample 4 presented three methylated positions in one of the four clones examined (CpG islands 1, 2 and 8).
  • Sample 9 presented three methylated positions in one of the four clones analysed (positions 10-12). Only position 1 was methylated in one of the two analysed clones of sample 11.
  • Example 12 Application of demethylating agents increases UCHLl expression in U87-MG and HeLa cells
  • the proclivity for UCHLl gene de-repression by a demethylating agent was also tested in U87-MG and HeLa cells.
  • the treatment with 5-azacitidine 5 ⁇ M for 72 h up- regulated the expression of UCHLl in U87-MG cells and induced its expression in HeLa cells (p ⁇ 0.05, ANOVA with post-hoc LSD test) (Figure 10A) without affecting the NRSF mRNA levels in either cell line ( Figure 10B).
  • the induction was especially clear in HeLa cells; in which the UCHLl promoter is known to be methylated.
  • Example 13 Cell-based screening method for molecules that inhibit the UCHLl inhibitory complex
  • Human reporter lines for example, can be produced through plasmid or recombinant adeno associated viral vectors (rAAV) delivery of knock in constructs and homologous recombination with the endogenous UCHLl gene; or by transient or stable transfection with promoter reporter fusion constructs.
  • rAAV adeno associated viral vectors
  • Regions of homology at the UCHLl locus can be amplified from genomic DNA obtained from U87-MG; HeLa cells or other cells in which UCHLl expression is downregulated, using a High Fidelity DNA Polymerase (e.g. Pfu DNA Polymerase).
  • a High Fidelity DNA Polymerase e.g. Pfu DNA Polymerase
  • 5 to 7kb fragments are amplified from the upstream homology arm.
  • a DNA fragment covering 1.5kb of the promoter of UCHLl, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3 and exon 4 can be amplified for the upstream homology region; which includes all the known regulatory elements for expression of the UCHLl gene.
  • shorter fragments of 2 to 3 kb are sufficient for the downstream homology arm; for example the DNA fragment covering exon 5, intron 5, exon 6, intron 6 and exon 7 of the UCHLl gene.
  • Targeting plasmids are constructed by ligating the homology arms, and targeting/reporter cassette in the MCS of pBR322.
  • an adequate targeting/reporting cassette contains a hybrid 5 '-regulatory element containing a short length of intron sequence followed by a splice acceptor site, an IRES, which permits the translation of the open reading frame (ORF) of the reporter gene (preferentially Luciferase; the synthetic firefly Iuc2 (Photinus pyralis) and Renilla hRluc ⁇ Renilla reniformis) included in the pGL4 vectors from Promega) from RNA transcripts initiating from upstream exons (Topaloglu et al. Nucleic Acids Res. 2005; 33(18): el58), followed by a polyadenylation site.
  • the components of the targeting/reporter cassette have been codon optimized and engineered to reduce the number of consensus transcription factor binding sites to reduce the risk of anomalous transcription.
  • the targeting/reporting cassette contains resistance gene (e.g. neo) expressed from its own promoter and provided with its own polyadenylation site for selection of stably transformed cells.
  • rAAV targeting constructs are assembled by ligation of homology arms and selectable marker cassettes, amplified using a high fidelity DNA Polymerase (e.g. DNA polymerase) from the targeting plasmid vector using oligonucleotide primers with embedded unique restriction sites allow inserted between the two Notl sites of p AAV- MCS, an AAV shuttle vector that carries the two inverted terminal repeat (ITR) sequences necessary for viral packaging (Stratagene).
  • DNA Polymerase e.g. DNA polymerase
  • ITR inverted terminal repeat
  • homology arms in rAAV targeting constructs need not be as long those for plasmid targeting vectors, lkb being sufficient.
  • Infectious rAAV stocks can be produced with the AAV Helper-Free System (Stratagene) according to the manufacturer's protocols. Briefly, ITR-containing targeting constructs are co-transfected with the plasmids pAAV-RC and pHELPER. Approximately 5 x 10 6 AAV-293 cells are transfected with a mixture of 2.5 ⁇ g of each of the above three plasmids, using 54 ⁇ l of Lipofectamine (Invitrogen) as described by the manufacturer. Two days after transfection, cells are scraped into 1 ml of phosphate- buffered saline and frozen and thawed three times. The crude lysate is clarified by centrifugation.
  • AAV Helper-Free System (Stratagene) according to the manufacturer's protocols. Briefly, ITR-containing targeting constructs are co-transfected with the plasmids pAAV-RC and pHELPER. Approximately 5 x 10 6 AAV-293 cells are transfected
  • Approximately 5 x 10 6 cells are transfected with a mixture of 2.5 ⁇ g of the targeting/reporter plasmid, using 54 ⁇ l of Lipofectamine (Invitrogen) as described by the manufacturer. Cells are replated in eight 96-well plates in medium containing geneticin (Invitrogen) at a final concentration of 0.4 mg/ml. Drug resistant colonies are grown for 3-4 weeks.
  • Locus-specific integration of the targeting/reporting constructs is assessed by PCR using primers outside the homology arms in combination with targeting cassette specific primers.
  • DNA polymerases fit for long PCR reactions (e.g. Pfu DNA polymerase) are employed.
  • the UCHLl regulatory sequences including the promoter sequences and the first intron of the UCHLl gene, inserted in a pGL4 vector (Promega), transfected in transient or stable manner.
  • the low expression level of the reporter gene in the cell lines and its induction by the application of the agents previously identified to effectively induce UCHLl expression are verified, and cell lines with the correct response are selected.
  • GIo Lysis Buffer equilibrated at 22 0 C, is added to the cells, equilibrated to room temperature; and incubated for 5 minutes at room temperature to allow lysis to occur.
  • the lysates are transferred to lumino meter tubes or plates and a volume of Bright
  • GloTM Assay Reagent equal of GIo Lysis Buffer is added and luminescence is measured with a luminometer.
  • a cell line with correct expression response selected above is then employed to perform high throughput evaluation of compounds, extracts or biologicals (siRNA, miRNA, antibodies, and proteins) to assess their effectiveness in inducing UCHLl expression.
  • Assays can be performed either in plate format or using reverse transfected/treated cell arrays.
  • PD and DLB are considered ⁇ -synucleinopathies because abnormal ⁇ -synuclein is aggregated into Lewy bodies (LBs) and Lewy neurites in selected nuclei of the brain stem, spinal cord and autonomic ganglia.
  • LBs Lewy bodies
  • DLB is characterized by the widespread distribution of LBs and Lewy neurites in the cerebral cortex (Forno, 1996; Ince et al, 1998; Spillantini et al, 1998; Ince and McKeith, 2003; Jellinger and Mizuno, 2003).
  • DLB is often accompanied by Alzheimer's disease (AD); this is considered the common form (DLBc).
  • DLB DLB
  • the pure form of DLB is characterized by minimal ⁇ A4-amyloid deposits and no tau pathology (Kosaka, 1993).
  • the brains of six patients with PD, six DLBp, seven DLBc, and five aged-matched controls were obtained at autopsy, following informed consent of the patients or their relatives and the approval of the local ethics committees. Cases with prolonged agonal state, pyrexia, hypoxia, seizures or coma were excluded from the present study. Age range was between 57 and 91 years (mean age 75 years), and the average time between death and tissue processing was 6 h (between 2 and 13 h). pH range was between 6 and 7.
  • AD stages were further established depending on the amyloid deposition burden and neurofibrillary pathology, following the nomenclature of Braak and Braak (Braak and Braak, 1999). Stages of amyloid deposition refer to initial deposits in the basal neocortex (stage A), deposits extended to the association areas of the neocortex (stage B), and heavy deposition throughout the entire cortex (stage C). Stages of neurofibrillary pathology correspond to transentorhinal (I-II), limbic (III-IV) and neo cortical (V and VI).
  • stages 1 and 2 affect the medulla oblongata plus the pontine tegmentum; stage 3, the midbrain; stage 4, the basal prosencephalon and mesocortex; and stages 5 and 6, the neocortex.
  • stage 3 the midbrain
  • stage 4 the basal prosencephalon and mesocortex
  • stages 5 and 6 the neocortex.
  • all cases of PD had suffered from classical PD lasting from 8 to 15 years, and none of them had cognitive impairment.
  • Cases with DLB fulfilled the clinical criteria proposed by the consortium on DLB international workshop (McKeith et al, 1996, 2000).
  • Control cases were considered in the absence of neurological symptoms and signs, and no abnormalities in the neuropathological study.
  • the main neuropathological data in the present series are summarized in Table I.
  • Biochemical studies were carried out in frozen samples of the frontal cortex (area 8). Control and diseased brains were processed in parallel.
  • genomic DNA isolated from human frozen brain homogenate was re- suspended in 50 ⁇ l of water and denatured, adding 5.7 ⁇ l of 3M NaOH for 10 min at 37 0 C. Then 33 ⁇ l of 20 mM hydroquinone (Sigma) and 530 ⁇ l of 4.3 M sodium bisulfite (Sigma) at pH 5.0 were added. The DNA solution was incubated for 16 h at 5O 0 C. After that, DNA samples were desalted through a column (Wizard DNA Clean- Up System, Promega) and eluted with 50 ⁇ l of water. Then, the eluted DNA was treated with 5.7 ⁇ l of 3M NaOH for 20 min at 37 0 C.
  • DNA was precipitated, adding 1 ⁇ l of 10 mg/ml glycogen, 17 ⁇ l of 1OM ammonium acetate and 450 ⁇ l of ethanol overnight at -8O 0 C.
  • the bisulfite-modified genomic DNA was re-suspended in 50 ⁇ l of water.
  • the conditions used in PCR amplification of bisulfite-modified genomic DNA have been previously described (Bittencourt-Rosas et al., 2001).
  • the primers used were the CPGP9.5-Fow: 5 ' -TT AAAAgg ATTgTTTT AT ATATTT AAggAAT-3' and CPGP9.5- Rev: 5 '-CACTCACTTTATTCAACATCTAAAAAAC-S ' .
  • the PCR product (473 bp) was cloned in TA pCRII vector (Invitrogen) and transformed in OneShot TOPlO chemically competent bacteria (Invitrogen).
  • HeLa cells were maintained in Dulbecco's minimal essential medium (DMEM, Gibco, Invitrogen) supplemented with 10% foetal bovine serum.
  • DMEM Dulbecco's minimal essential medium
  • U87-MG cells ATCC® number: HTB- 14
  • DMS53 cells ATCC® number: CRL-2062
  • GEBCO Waymouth's MB 752/1 medium
  • TSA was dissolved in ethanol and 5-azacytidine in water:acetic acid (1 :1).
  • acetic acid 1 :1
  • TSA treatment cells were plated in 6-well dishes at a concentration of 105 cell/well and cultured overnight before activation. Cells were plated at a concentration of 50,000 cell/well for 5-azacytidine and also cultured overnight before treatment.
  • DMS53 cells were plated in 6-well dishes at a concentration of 105 cells/well and cultured overnight before transfection. 1 ⁇ g of REEXl vector (kindly provided by Dr. Gail Mandel) was trans fected using lipofectamineTM 2000 (Invitrogen) following the instructions of the manufacturer. After 5 hours of post-transfection the medium was replaced by fresh medium. The efficiency of transfection was around 40% using the pEGFP-Cl vector (BD Biosciences Clontech).
  • U87-MG cells were plated in 6-well dishes at a concentration of 50,000 cells/well and cultured overnight before transfection. 100 nM of siRNA, a mix of miRs at 1OnM each or scramble siRNA (Ambion, Cat. N°4611) were transfected using lipofectamineTM 2000 (Invitrogen) following the instructions of the manufacturer. After 5 hours of post- transfection the medium was replaced by fresh medium. The analysis of the siRNA or miR transfection was performed 48 hours later. All siRNAs and miRs used were from Ambion (Applied Biosystems): NRSF/REST; 5'-GCUUAUUAUGCUGGCAAAUTT-S'; Ambion, Cat.
  • HDAC6 HDAC6; ID # 120452 (NM_006044); AM51320
  • Frozen frontal cortex (area 8; 100 mg) was directly homogenized in 1 ml lysis buffer (20 mM Hepes, 10 mM KCl, 1.5 mM MgC12, 1 mM EDTA, 1 mM EGTA, 1 mM DDT, 2 mM PMSF, 1 ⁇ g/ml aprotinin, leupeptin and pepstatin) and then sonicated. Cell lines grown in 10 ml-plates were homogenized in the same way without sonication. Lysates were centrifuged at 2650 g for 10 minutes at 4 0 C, and protein concentration was determined with BCA (Pierce) method. 30 ⁇ g of total protein was boiled at Q5°C for 3 min and loaded in SDS-polyacrylamide gels with Tris-glycine running buffer.
  • 1 ml lysis buffer (20 mM Hepes, 10 mM KCl, 1.5 mM MgC12, 1 mM ED
  • Proteins were electrophoresed using a mini-protean system (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad) with a Mini Trans-Blot electrophoresis transfer cell (Bio-Rad) for 1 h at 100 V. Nitrocellulose membranes were blocked with Tween 20 TBS (TBST), containing 5% skimmed milk, for 30 min. Subsequently, the membranes were incubated at 4°C overnight with one of the primary antibodies in TBST containing 3% BSA.
  • TBS TBS
  • anti-REST Abeam
  • anti- UCHLl AB5937, Chemicon
  • anti-S- actin clone AC- 74, Sigma
  • the membranes were washed three times with TBST for 5 min at room temperature, and then incubated with the corresponding anti-rabbit, anti-goat or anti-mouse IgG antibody labelled with horseradish peroxidase (Dako) at a dilution of 1 :1,000 (1 :10,000 for S- actin) for 1 h at room temperature.
  • the membranes were washed five times, 5 min each, with TBST at room temperature, and developed with the chemiluminescence ECL Western blotting system (Amersham/Pharmacia), followed by apposition of the membranes to autoradiographic films (Hyperf ⁇ lm ECL, Amersham).
  • RNA from cell lines was purified with RNeasy Midi kit (Qiagen) following the protocol provided by the manufacturer. The concentration of each sample was obtained from A260 measurements. RNA integrity was tested using the Agilent 2100 Bio Analyzer (Agilent).
  • the retrotranscriptase reaction (100 ng RNA/ ⁇ l) was performed using the High capacity cDNA Archive kit (Applied Biosystems) following the protocol provided by the supplier. Parallel reactions for each RNA sample were run in the absence of MultiScribe Reverse Transcriptase to assess the degree of contaminating genomic DNA.
  • TaqMan PCR
  • the NRSF/REST TaqMan assay (HsOO 194498 ml, TaqMan probe 5 ' -AGGAAGGCCGAATACAGTTATGGCC-S ' ) (Applied Biosystems) generates an amplicon of 79 bp and is located at position 341 between 1 and 2 exon boundary of NM_005612.3 transcript sequence.
  • the TaqMan assay for UCHLl (HsOOl 88233_ml, TaqMan probe 5'- CCTGCTGAAGGACGCTGCCAAGGTC-3 ) (Applied Biosystems) is located at position 648 between 8 and 9 exon boundary of NM 004181.3 transcript sequence. It generates an amplicon of 100 bp.
  • the TaqMan assay for Synaptophysin (Hs00300531_ml, TaqMan probe 5 - CGAGTACCCCTTCAGGCTGCACCAA-3 ') (Applied Biosystems), generates an amplicon of 63 bp and is located at position 241 of NM_003179.2 transcript sequence.
  • TaqMan PCR assays for NRSF/REST, UCHLl and synaptophysin were performed in duplicate on cDNA samples in 96-well optical plates using an ABI Prism 7700 Sequence Detection system (Applied Biosystems). The plates were capped using optical caps (Applied Biosystems). For each 20 ⁇ l TaqMan reaction, 9 ⁇ l cDNA (diluted 1/50) was mixed with 1 ⁇ l 2Ox TaqMan® Gene Expression Assays and 10 ⁇ l of 2x TaqMan Universal PCR Master Mix (Applied Biosystems).
  • the amount of targets (NRSF/REST, UCHLl and synaptophysin) and endogenous reference (GUSB) was determined for each experimental sample from the appropriate standard curve, which was plotted showing the cycle threshold, Ct (y), versus log ng total control RNA (x). The amount of each target was divided by the endogenous reference amount to obtain a normalized target value (arbitrary units). Chromatin immunoprecipitation (ChIP assay)
  • ChIP assay was performed according to the manufacturer's protocol (Upstate) using 106 U87-MG, HeLa and DMS53 cells. 10 ⁇ g Anti-NRSF/REST (P-18X, SC-15118X Santa Cruz) and 10 ⁇ g antiacetylated H3 (residue Lys9, Cell Signalling) were used for immunoprecipitation. Purified DNA was resuspended in 20 ⁇ l of DNAse-free water and 1 ⁇ l was used as template in 25 ⁇ l of PCR reaction using GoTaq Flexi DNA Polymerase (Promega). Primer concentration was 200 nM.
  • PCR primers were 5'- ACAAATCCCgTCTCCACAAC-3' and 5'-gCCTAgggAAgACgAAAAACA-3' for the amplification of NRSEl sequence of UCHLl gene promoter.
  • the NRS E2 and NRSE3 sequences were amplified with 5'-gCTCCgTAgCTgTTTTTCgT-3' and 5 '-gCC ACTC ACTTTgTTC AgC A-3'.
  • the reaction was carried out using the following parameters: 95 0 C for 2 min and 35 cycles of 95 0 C for 30 sec, 65 0 C for 30 sec and 72 0 C for 30 sec. Finally, a last hold of 72 0 C for 5 min was performed.

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Abstract

The treatment and prevention of neurodegenerative diseases by repression of the transcriptional complex that silences the promoter of the UCHL1 gene.

Description

Treatment and Prevention of Neurodegenerative Diseases
Introduction
The invention relates to the treatment and prevention of neurodegenerative diseases. In particular the invention relates to the treatment and prevention of neurodegenerative diseases by repression of the transcriptional complex that represses the promoter of the UCHLl gene.
Background
Neurodegenerative diseases are conditions in which cells of the brain and spinal cord are lost. Probably the best known neurodegenerative diseases are Alzheimer's disease, Parkinson's disease and multiple sclerosis which are caused by the gradual deterioration of neurons causing symptoms affecting cognition and/or movement, eventually leading to death.
"Lewy body disorders" is an umbrella term which includes dementia with Lewy bodies (DLB), Parkinson's disease (PD) and PD with dementia (PDD). These disorders are characterised by disorders of alpha-synuclein metabolism, which gives rise to the formation of abnormal neuronal alpha-synuclein inclusions, which are the defining pathologic process common to both PDD and DLB.
Synucleinopathies, with and without dementia, encompass a wide range of diseases including Parkinson's disease, multiple system atrophy, rapid eye movement (REM), sleep behaviour disorder, and dementia with Lewy bodies (DLB). DLB is a neurodegenerative disorder resulting in slowly progressive and unrelenting dementia until death. Prevalence studies suggest that it is the second most common dementing illness in the elderly. The neuropathologic findings of DLB show a wide anatomic range. Lewy bodies and Lewy-related pathology are found from the brain stem to the cortex and, in many cases, associated with concurrent Alzheimer's disease pathology. PDD and DLB show differing temporal sequences of key symptoms and clinical features. Patients with Parkinson's disease show an increased risk for dementia based on epidemiological studies. The criteria by McKeith et al. (2005) (Neurology, 65, 1863-72) have become a standard for studies in dementia with Lewy Bodies (DLB), which show a very high specificity but low sensitivity. Clinical core features of DLB consist of rapid fluctuations in cognition, recurrent visual hallucinations and spontaneous and fluctuating features of Parkinsonism; these are further supported by high sensitivity for extrapyramidal side effects to neuroleptics and rapid eye movement sleep behaviour disorder. Dementia itself describes a syndrome characterised by memory impairment, intellectual deterioration, changes in personality and behavioural abnormalities.
Ubiquitin c-terminal hydrolase Ll (UCHLl) is one of the most abundant cytosolic proteins in the brain. In addition to neurons, it is expressed in testes (Wilkinson et al., 1989; Solano et al., 2000). Yet, UCHLl is abnormally over-expressed in non-small-cell lung cancer (Hibi et al., 1999), pancreatic cancer (Tezel et al., 2000), colorectal cancer (Yamazaki et al., 2002) and myeloma cells (Otsuki et al., 2004). UCHLl is an enzyme involved in the hydrolysis of polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS), favouring protein degradation (Liu et al., 2002).
Several studies support the existence of a link between UCHLl and certain degenerative disorders of the nervous system. An I93M mutation was described in UCHLl gene in a German family with autosomal dominant Parkinson's disease (PD) (Leroy et al., 1998). This mutation leads to a 50% reduction of its hydro lytic activity in vitro (Lansbury and Brice, 2002). Moreover, UCHL 1I93M transgenic mice show loss of dopaminergic neurons in the substantia nigra and reduced dopamine content in the striatum at 20 weeks of age (Setsuie et al., 2007). Recent studies have shown classical Lewy pathology in a deceased sibling of a family affected by the I93M UCHLl mutation who developed, in addition to DOPA-responsive Parkinsonism, marked cognitive deficits (Auburger et al., 2005). A possible link of UCHLl with Alpha- synuclein pathology is supported by the observation that inhibition of UCHLl activity in foetal rat ventral mesencephalic cultures is associated with Alpha-synuclein aggregates (McNaught et al, 2002). Increased intracellular aggregates containing ubiquitinated proteins have been found after UCHLl inhibition by prostaglandins in human SK-N-SH cells (Li et al., 2004). These findings suggest that reduced UCHLl activity impairs UPS function and protein degradation, thus facilitating, under appropriate conditions, the accumulation of abnormal protein aggregates. In line with this, reduced UCHLl mRNA and protein is found in PD and in DLB, but only in brain regions in which aggregated proteins occur in the form of Lewy bodies and Lewy neurites (Barrachina et al., 2006).
It is an object of the invention to provide a method for treating or preventing neurodegenerative diseases.
Summary of the invention
In a first aspect the invention relates to a method of treating or preventing a neurodegenerative disease in a patient suffering from such a condition which comprises administering to such a patient a therapeutically effective amount of an agent that represses the transcriptional complex that represses the promoter of the UCHLl gene.
In one embodiment, the agent affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
In one embodiment, the neurodegenerative disease is a Lewy Body disorder.
The agent may be a HDAC inhibitor, such as Trichostatin A (TSA), Suberoylanilide hydroxamic acid (SAHA),
N-Hydroxy-4-(Methyl{[5-(2-Pyridinyl)-2-Thienyl]Sulfonyl}Amino)Benzamide,
4-Dimethylamino-N-(6-Hydroxycarbamoyethyl)Benzamide-N-Hydroxy-7-(4-
Dimethylaminobenzoyl)Aminoheptanamide,
7-[4-(Dimethylamino)Phenyl]-N-Hydroxy-4,6-Dimethyl-7-Oxo-2,4-Heptadienamide, and Docosanol. The agent may be a small molecule that inhibits the function of REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl in the transcriptional complex that represses the promoter of the UCHLl gene. Alternatively the agent may be a small molecule that inhibits HDAC6.
In further embodiments the inhibition is provided by administering a small double stranded interference RNA (siRNA), a short hairpin RNA (shRNA), a microRNA, an antisense oligonucleotide or monoclonal antibodies directed against at least one of the genes that codes for one of the proteins belonging to the transcriptional repressor complex from UCHLl gene, for example REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl, or a different member of the aforementioned complex, or for a gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene, for example HDAC6.
In one embodiment there is provided a method of screening for molecules that inhibit the transcriptional complex that represses the promoter of the UCHLl gene comprising providing a cell line containing a reporter gene fused with the UCHLl regulatory domains which expresses no or low levels of UCHLl, incubating the cell line with a molecule of interest and screening for expression of the reporter gene, wherein expression of the reporter gene indicates inhibition of the transcriptional complex that represses the promoter of the UCHLl gene by the molecule of interest.
Brief description of drawings
The present invention will now be described with reference to the accompanying drawings:
Figure 1 shows the schematic representation of UCHLl gene promoter. White boxes represent three putative DNA binding sites for NRSF/REST (NRSE) detected by
Matlnspector software. NRSEl and NRSE3 are located in the complementary DNA chain (-) and the NRSE2 in the positive DNA chain (+). Exon 1 and 2 are indicated as dash boxes and intron 1 corresponds to grey box. The location of NRSEl, NRSE2 and NRSE3 sites is indicated relative to TATA signal located at position 268 of the sequence with GenBank number Xl 7377. The transcription start site is shown with an arrow.
Figure 2 shows NRSF/REST protein levels in frontal cortex homogenates in age- matched control cases (C, n=5), Parkinson's disease (PD, n=6), Dementia with Lewy Bodies, pure form (DLBp, n=6), and DLB common form (DLBc, n=7). A, NRSF/REST (121 KDa) and UCHLl (25 KDa) protein levels are 30 detected by Western blot. S- Actin (45 kDa) is blotted to control protein loading. The image shows two samples from two different patients and age-matched controls but it is representative of all the samples indicated in the Table I. NRSF/REST protein levels are only seen in the frontal cortex of DLBp and DLBc. B, Densitometric analysis of NRSF/REST protein levels normalized with S-actin. AU: Arbitrary Units (mean ± SD). *p<0.05 compared to control samples (ANOVA with post-hoc LSD test). NRSF/REST expression is compared with UCHLl expression in the same cases to show an inverse relationship between NRSF/REST and UCHLl in every case. The same results were observed in all cases summarized in Table I.
Figure 3 shows NRSF/REST and UCHLl expression levels in DMS53, U87-MG and HeLa cell lines. A, NRSF/REST and UCHLl mRNA levels normalized with β- glucuronidase (GUSB). The detection was performed with TaqMan PCR as is indicated in experimental procedures section. B, NRSF/REST and UCHLl protein levels detected by Western blotting. The figure shows the densitometric analysis of NRSF/REST and UCHLl protein levels normalized with S-actin. AU: Arbitrary Units.
Figure 4 shows the effect of NRSF/REST overexpression in DMS53 cell line. A, 1 μg of REEXl vector, which encodes human full-length NRSF cDNA, was transfected in DMS53 cells using lipofectamine 2000. NRSF/REST mRNA and protein levels were increased after 48h of REEXl vector transfection. The protein levels were detected by Western Blot showing two independent transfections. S-Actin (45 kDa) is blotted to control protein loading. The over-expression of NRSF/REST transcription factor reduces endogenous UCHLl (B) and synaptophysin (Q mRNA levels. REEXl over- expression was performed in triplicate (6-well plates) in three independent experiments. The mRNA levels of all the analysed genes were detected by TaqMan PCR and the endogenous control used was β-glucuronidase.
Figure 5 shows the effect of NRSF/REST siRNA transfection in U87-MG cell line. A, NRSF/REST protein levels are detected by Western Blot after 48h of NRSF/REST siRNA transfection (siRNA#l and siRNA#2). The scramble siRNA transfection does not modify the expression of endogenous NRSF/REST levels. S-Actin (45 kDa) is blotted to control protein loading. Reduction of NRSF/REST transcription factor increases endogenous UCHLl (B) and synaptophysin (Q mRNA levels. siRNA transfection was carried out in triplicate (6-well plates) in three independent experiments. The mRNA levels of all the analysed genes were detected by TaqMan PCR and the endogenous control used was β-glucuronidase (GUSB). AU: Arbitrary Units (mean ± SD). */?<0.01 and **p<0.001 compared with non-transfected cells (ANOVA with post-hoc LSD test).
Figure 6 shows a chromatin immunoprecipitation (ChIP) assay using NRSF/REST antibody in HeLa, U87-MG and DMS53 cell lines illustrating that NRSF/REST binds the UCHLl regulatory region. A, Schematic representation of minimal UCHLl gene promoter. Grey boxes represent the three putative DNA binding sites for NRSF/REST (NRSE). The transcription start site is indicated as +1. Immunoprecipitated DNA was analysed by PCR using two sets of primers that amplified a 247 bp region of the UCHLl gene promoter which encompasses an NRSE site (NRSEl) and a 214 bp region which contains the other two NRSE sites (NRSE2 and NRSE3). B, ChIP assay was performed in U87-MG cells using a goat antibody anti-human NRSF/REST and a rabbit polyclonal anti-human acetyl-histone 3 (Lys9) antibody as positive control. The ChIP assay with goat serum was performed as a negative control. Primers set number 1 amplify a 247 bp region of the UCHLl gene promoter and primers set number 2 amplify a 214 bp region as schematically indicated above. The same results were obtained using HeLa cells. C, The same ChIP analysis performed but using DMS53 cells. Input refers to DNA chromatin not immunoprecipitated with the specific antibody. ChIP refers to DNA chromatin immunoprecipitated with the specific antibody. M: 100 bp DNA ladder marker. Figure 7 shows the effect of the application of siRNAs and miRNAs directed against the repressor complex on the levels of UCHLl mRNA expression in U87-MG cells. Treatment with 10OnM of total siRNA or miRNA concentration during 48h led to over 2 fold induction of expression was when siRNAs directed against MeCP2, RCORl, sin3A, HDACl, HDAC2, and AOF2 were employed (direct inhibition of components the repression complex). In this experiment, treatment with 10OnM of siRNA against HDAC6 (indirect inhibition of components the repression complex) led to an induction of nearly 7 fold in UCHLl mRNA expression. Lower inductions (25%) were observed with siRNAs directed against REST and JARIDlC and the pool of miRNAs
Figure 8 shows the effect of the application of 24h treatment with 10OmM of the HDAC inhibitor trichostatin A (TSA) on the expression of UCHLl in U87-MG (A) and HeLa (B) cells.
Figure 9 shows that no systematic difference in DNA methylation of the minimal UCHLl gene promoter in post-mortem cortical brain samples of patients with Dementia with Lewy Bodies (pure and common form) and age-matched controls can be detected. A, Partial consensus sequence of UCHLl gene promoter (GenBank accession number X17377) with nucleotides numbered relative to ATG (+1). Nine CpG islands are indicated with boxes (the completed promoter sequence contains 35 CpG sites). B, Chromatogram of a partial PCR product from bisulfite treated UCHLl gene promoter (-248 to -174) of a 79-year-old male. C, Summary of all clones determined by bisulfite sequencing analysis of UCHLl gene promoter from age-matched controls (n=5), dementia with Lewy bodies pure form (DLBp, n=6) and common form (DLBc, n=7). Every circle corresponds to the 35 CpG sites described in UCHLl gene promoter (Bittencourt-Rosas et al., 2001). Non-methylated cytosines (white circles) and methylated cytosines (black circles) are indicated. The number of clones with methylated cytosines is indicated with respect to the total number of clones analysed for each sample. Sample number corresponds to that found in Table 1.
Figure 10 shows the inductory effect of the treatment of HeLa and U87-MG cells with the demethylation agent 5-azacytidine on UCHLl expression. Detailed description of the invention
The biological role of UCHLl and its role in pathogenesis encouraged the present study focused on the transcriptional regulation of UCHLl. One report has demonstrated that the UCHLl gene promoter contains 35 CpG islands which are fully methylated in UCHLl non-expressing HeLa cells (Bittencourt-Rosas et al., 2001). For this reason, we have analysed the methylation status of the UCHLl gene promoter in human postmortem frontal cortex samples of patients with DLB. Because of the negative results, we then carried out a bioinformatic search employing the Genomatix Matlnspector software to identify putative transcription factors binding sites in the UCHLl gene promoter. Among the promoter elements, we identified 1 weak binding site for REST neuron-restrictive silencer factor/RE-1 silencer transcription factor (NRSF/REST) in the promoter and 2 weak binding sites in the first intron. NRSF/REST has been defined as a neuronal gene repressor in non-neuronal cells (Chong et al., 1995; Schoenherr and Anderson, 1995).
However, the potential NRSF/REST binding sites detected in the UCHLl gene promoter and first intron had low matrix similarity scores (0,69; 0,71 and 0,67 respectively) and the "Explanations of Scores" for the Matlnspector software define a "good" match as one having a matrix similarity of >0.80 (Table I). Therefore it was not predicted from this data that REST would bind to the UCHLl gene promoter.
Table 1. Summary of the main clinical and neuropathological findings in the present series.
Parkinson's disease (PD), Diffuse Lewy body disease: Dementia with Lewy bodies, pure form (DLBp) and common form (DLBc), and controls. M: male, F: female. NFT: neurofibrillary tangle. P-m delay: post-mortem delay in hours. Braak stages refer to Braak and Braak Alzheimer's disease (AD) changes (Braak and Braak, 1999). Staging of H-synuclein pathology (Lewy bodies and Lewy neurites) related to sporadic Parkinson's disease (PD) was done according to Braak et al., 2003.
Figure imgf000011_0001
In addition UCHLl was not identified previously as a target for REST/UCHL1 in genome wide analysis, for example:
• UCHLl is not included as a target for the REl target database (http://www.bioinformatics.leeds.ac.uk/REldbjiikII/) at any PSSM cutoff level (Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes, Bruce et al, Proc Natl Acad Sci U S A. 2004 July 13; 101(28): 10458-10463).
• UCHLl is not included as a target in the supplementary tables in Otto SJ et al. A new binding motif for the transcriptional repressor REST uncovers large gene networks devoted to neuronal functions. J Neurosci. 2007 Jun 20;27(25):6729- • UCHLl is not included in the supplementary tables of Johnson DS et al. Genome-wide mapping of in vivo protein-DNA interactions. Science. 2007 Jun 8;316(5830): 1497-502. Epub 2007 May 31.
We evaluated REST expression in PD and DLB and found that REST was slightly overexpressed. Its expression is inversely related to the UCHLl expression levels in the frontal cortex of pure and common DLB cases. In addition; we reviewed data published on incipient AD (Blalock EM et al. Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc Natl Acad Sci U S A. 17 Feb 2004;101(7):2173-8. Epub 9 Feb 2004). Although not specifically cited in Table 4, dedicated to transcription factors associated with incipient AD; we found a 35% increase was for the expression level of REST and a 35% decrease in the UCHLl level of severe AD cases in Supporting Table 5 of the cited article; indicating that the relationship between NRSF/REST may also exist in AD.
Figure imgf000012_0001
After performing several functional analyses with three cell lines, we confirmed that NRSF/REST binds to the promoter of UCHLl and regulates the expression of UCHLl. Together, these findings demonstrate NRSF/REST as a relevant transcription factor that negatively regulates UCHLl expression and causes downregulation of expression in diseases with Lewy bodies, including PD and DLB.
As a result of this new understanding of the role of NRSF/REST in the down regulation of UCHLl gene expression and the known importance of UCHL gene expression in DLB, other Lewy body disorders and other neurodegenerative diseases, we are able to propose several new and inventive approaches to the treatment and prevention of neurodegenerative diseases.
According to the invention there is provided a method of treating or preventing a neurodegenerative disease in a patient suffering from such a condition which comprises administering to such a patient a therapeutically effective amount of an agent that represses the transcriptional complex that represses the promoter of the UCHLl gene.
Specific neurodegenerative diseases include Alexander disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe disease, dementia with Lewy bodies, Machado- Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Neuroborreliosis, Parkinson's disease, Parkinson's disease with dementia, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoff disease, Schilder's disease, Schizophrenia, Spinocerebellar ataxia (multiple types), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, and Tabes dorsalis. The method of the invention is particularly suitable for treating Alzheimer's disease, Huntington's disease and Lewy Body disorders, such as dementia with Lewy bodies, Parkinson's disease, Parkinson's disease with dementia.
By "treating or preventing" we mean symptomatic improvement, which may include enhanced cognition, more autonomy and/or improvement in neuropsychiatric and behavioural dysfunction; and/or disease modification with slowing or arrest of symptom progression of the dementing process; and/or primary prevention of disease by intervention in key pathogenic mechanisms at a pre-symptomatic stage.
A Lewy body disorder is defined herein as a condition which is characterised by disorders of alpha- synuclein metabolism, which gives rise to the formation of abnormal neuronal alpha-synuclein inclusions. These are the defining pathologic process common to both PDD and DLB. More particularly Lewy body disorders include dementia with Lewy bodies (DLB), Parkinson's disease (PD) and PD with dementia (PDD).
The methods of the invention are particularly suitable for treating a neurodegenerative disease which is characterised by the overexpression of REST. In other words, REST can be used as a biomarker for the detection of a neurodegenerative disease which could be treated by the method of the invention. AD is an example of such a condition, where REST is slightly upregulated in the cortex and UCHLl is slightly downregulated.
In this respect we have identified two models which may benefit from the treatments proposed herein. The first model develops Lewy neurites (mouse overexpressing a synuclein with the A53T mutation) and could be envisaged as a model for Lewy Body disease, whilst the second model benefits from recombinant UCHLl protein therapy. In the second model (APP/PS1 mouse model), the delivery of an exogenous TAT-UCHLl fusion protein improves the symptoms of the neurodegeneration, supporting the role for UCHLl in therapeutic strategies in neurodegenerative disease.
Further, a number of animal model systems for Huntington's disease are available. See, e.g., Brouillet (2000) Functional Neurology 15(4):239-251; Ona et al. (1999) Nature 399:263-267; Bates et al. (1997) Hum. MoI. Genet. 6(10): 1633-1637; Hansson et al. (2001) J. Neurochem. 78:694-703; and Rubinsztein (2002) Trends Genet. 18:202- 209 (a review on various animal and non-human models of HD).
One transgenic mouse model for Huntington's disease is the R6/2 model (Mangiarini et al. (1996) Cell 87:493-506). The R6/2 mice over-express exon 1 of the human Huntingtin gene which has an expanded CAG/polyglutamine repeat lengths (150 CAG repeats on average). These mice develop a progressive, ultimately fatal, neurological disease with many biochemical and physiological features of human Huntington's disease. For example, abnormal aggregates, constituted in part by the N-terminal part of Huntingtin (encoded by HD exon 1), are observed in R6/2 mice, both in the cytoplasm and nuclei of cells (Davies et al. (1997) Cell 90:537-548). These transgenic mice are characterized by reduced weight gain, reduced lifespan, and motor impairment characterized by abnormal gait, resting tremor, hindlimb clasping, and hyperactivity from 8 to 10 weeks after birth (for example the R6/2 strain; see Mangiarini et al. (1996) Cell 87:493-506). The phenotype worsens progressively toward hypokinesia. The brains of these transgenic mice demonstrate neurochemical and histological abnormalities, such as changes in neurotransmitter receptors (glutamate, dopaminergic), decreased concentration of N-acetylaspartate (a marker of neuronal integrity), and reduced striatum and brain size. Thus, the compounds of the invention can be evaluated in this model by assessing parameters related to neurotransmitter levels, neurotransmitter receptor levels, brain size, striatum size, life-span, biochemical disease evidence (e.g., abnormal aggregates), and motor impairment.
In one embodiment, the methods of treatment of the invention will be applied to patients that have downregulated expression of the UCHLl protein. In the disease conditions specified above, it may be preferable to verify an intact UCHLl CDS prior to treatment, since overexpression of deficient UCHLl enzymes, like the I93M allele may be contraindicated. In other words, patient stratification may be advantageous.
In one embodiment the transcriptional complex which is repressed is the NRSF/REST complex. This complex comprises REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, SMARCEl (BAF57), SMARCA4 (BRGl) and SMARCC2 (BAF170) and other components. The transcriptional complex may be repressed directly or indirectly by altering the transcription, translation, subcellular localisation or activity of one or several components of the complex. For example, inhibition of HDAC6 which is involved in the retention of the components of the complex in the cytoplasm can successfully inhibit the transcriptional complex.
This complex may be inhibited by various methods. For example in one embodiment the REST complex may be inhibited by HDAC inhibitors. Histone deacetylase (HDAC) plays a role in transcriptional regulation and catalyses the deacetylation of lysine residues located in the NH(2) terminal tails of histones and non-histone proteins. They play an important role in the regulation of the expression status of genes. Further HDACs are found in the REST transcriptional complex.
Histone deacetylases (HDACs) are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8), similar to yeast RPD3 and localized in the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10); homologous to yeast HDAl protein and localized both the nucleus and cytoplasm; and class III HDACs, a structurally distinct class of NAD- dependent enzymes similar yeast SIR2. HDAC inhibitors are small molecules that target histone deacetylases. The application of HDAC inhibitors can reverse the silencing of genes generated by the acetylation of histones; and has been proposed for reactivating silenced tumours suppressor genes in cancer.
Accordingly, with this new understanding of the regulation of the UCHLl promoter and the relevance of UCHLl expression in the development of the disease, HDAC inhibitors would be expected to derepress the UCHLl gene and increase the expression of UCHLl. As such, they would be useful in the treatment or prevention of neurodegenerative diseases, and particularly in the treatment of Lewy body disorders.
In one embodiment of the invention, a compound is administered to an individual in need of UCHLl up-regulation, in an amount sufficient to inhibit AOF2 (lysine specific demethylases; LSDl) activity. In a more specific aspect of this embodiment, the AOF2 inhibiting effective amount is an amount sufficient to increase the UCHLl mRNA by 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or 300% or more. In another specific aspect of this embodiment, the AOF2 inhibiting effective amount is an amount sufficient to increase the UCHLl hydrolase activity by 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or 300% or more. In another specific aspect of this embodiment, the AOF2 inhibiting effective amount is an amount sufficient to increase the UCHLl protein levels by 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or 300% or more. In a specific aspect of this embodiment, the individual in need of UCHLl up-regulation is an individual suspected of having Lewy Body Dementia. In another specific aspect of this embodiment, the individual in need of UCHLl up-regulation is an individual needing or desiring prophylaxis against cognitive decline. In another specific aspect of this embodiment, the individual in need of UCHLl up-regulation is an individual needing or desiring a reduction in the rate of cognitive decline. Administration of the compound can reduce the rate of cognitive decline in that patient (or group of patients). HDAC inhibitors that can be used in this first aspect of the invention include inhibitors against any HDAC, including for example inhibitors against HDACl, HDAC2 or
HDAC6. Examples of such inhibitors include:
Trichostatin A (TSA),
Suberoylanilide hydroxamic acid (SAHA),
N-Hydroxy-4-(Methyl{[5-(2-Pyridinyl)-2-Thienyl]Sulfonyl}Amino)Benzamide,
4-Dimethylamino-N-(6-Hydroxycarbamoyethyl)Benzamide-N-Hydroxy-7-(4-
Dimethylaminobenzoyl)Aminoheptanamide,
7-[4-(Dimethylamino)Phenyl]-N-Hydroxy-4,6-Dimethyl-7-Oxo-2,4-Heptadienamide,
Docosanol,
(5)-[5-Acetylamino-l-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl) pentyljcarbamic acid tert-butyi ester (BATCP),
Benzyl ((S)-[ 1 -(4-methyl-2-oxo-2Η-chromen-7-ylcarbamoyl)-5-propionyl aminopentyljcarbamate (MOCPAC), and
4-(Dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-benzamide (M344).
Preferred HDAC inhibitors include the carboxylic acid class of HDAC inhibitors and derivatives thereof. In one aspect, the HDAC inhibitor is a short-chain to medium- chain fatty acid or a derivative or analog thereof. Examples of short chain fatty acids include, but are not limited to, butyric acid, phenylalkanoic acids, phenylbutyrate (PB), 4-phenylbutyrate (4-PBA), pivaloyloxymethyl butyrate (Pivanex, AN-9), isovalerate, valerate, valproate, valproic acid, propionate, butyramide, isobutyramide, phenylacetate, 3-bromopropionate, or tributyrin. Short-chain fatty acid compounds are described e.g., in U.S. Patent Nos. 4,988,731; 5,212,326; 4,913,906; 6,124,495; 6,110,970; 6,419,953; 6,110,955; 6,043,389; 5,939455; 6,511,678; 6,528,090; 6,528,091; 6,713,086; 6,720,004; U.S. Patent Publication No. 20040087652, Intl. Publication No. WO 02/007722, and in Phiel et al, J Biol Chem., 276(39):36734-41 (2001), Rephaeli et al., Int J Cancer.,116(2):226-35 (2005), Reid et al., Lung Cancer., 45(3):381-6 (2004), Gottlicher et al., 2001, EMBO J., 22(13):341 1-20 (2003), and Vaisburg et al., Bioorg Med Chem Lett., 14(l):283-7 (2004). Other short to medium chain carboxylic acids include, but are not limited to, those disclosed in e.g., US patent 7,176,240; WO 98/22436; and WO 2004/110974. Preferred inhibitors are orally administrable and capable of passing the blood brain barrier such as SAHA; and should at least release the repression of the UCHLl promoter; which would be the control for effectiveness. In some embodiments, the preferred inhibitors have at least 10%, 20%, or 30% or more blood brain barrier penetration.
In another embodiment the REST complex may be inhibited by agents that inhibit the function of the other members of the repression complex, including REST, MeCP2, mSin3a, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl. Such agents may act by preventing the transcriptional repression complex from binding to the gene promoter or may act by preventing members of the complex from interacting with each other. In either case the end result will be that the complex is prevented from inhibiting gene expression, so the gene, UCHLl will become derepressed.
Examples of suitable small molecules include:
Benzyl ((S)-[ 1 -(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5- propionylaminopentyl] carbamate; Molecular Formula: C27H31N3O6;
(iS)-[5-Acetylamino- 1 -(2-oxo-4-trifluoromethyl-2H-chromen-7- ylcarbamoyl)pentyl]carbamic acid tert-butyi ester; Molecular Formula: C23Η28F3N3O6;
4-(Dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-benzamide;
N-Hydroxy-7-(4-dimethylaminobenzoyl)-aminoheptanamide. (all available from Sigma)
In one embodiment of the invention, the histone deacetylase inhibitor has a structure of (R)-(A)-(L)-C=(O)NHOH wherein (A) is a carbocyclic, aryl, or heterocyclic ring system substituted with one or more R groups, and L is a linker group. In compounds having this general formula it is believed that the hydroxamate group functions as a metal binding group that interacts with the metal ion at the active site of the HDAC enzyme. The A ring system is believed to be at the entrance to the metal ion pocket in the active site. Non- limiting examples of heterocyclic, carbocyclic, and aryl ring systems, along with various linkers are given in the specific exemplified compounds below.
In one preferred embodiment the HDAC inhibitor is chosen from N-Hydroxy 2-(5- naphthalen-2-ylmethylhexahydropyrrolo[3,4-c]pyrrol-2[l H]- yl)pyrimidine-5- carboxamide; N-Hydroxy 2-{6-[(2-naphthylsulfonyl)amino]-3-azabicyclo[3.1.0]hex-3- yl}pyrimidine-5-carboxamide trifluoroacetate; N-Hydroxy 2-{6-[(6-fluoroquinolin-2- ylmethyl)amino]-3-aza-bicyclo[3.1.0]hex-3-yl}pyrimidine-5-carboxamide; N-Hydroxy 2-[5-(naphthalene-2-carbonyl)-hexahydropyrrolo[3,4-c]pyrrol-2-yl]pyrimidine-5- carboxamide; (S)-[4-(7-Hydroxycarbamoyl-heptanoylamino)-benzylamino]-phenyl- acetic acid cyclopentyl ester; (S)-2-[3-(7-Hydroxycarbamoyl-heptanoylamino)- benzylamino]-4-phenyl-butyric acid cyclopentyl ester; (S)-[3-(7-Hydroxycarbamoyl- heptanoylamino)-benzylamino]-phenyl-acetic acid cyclopentyl ester; (4-{[(2- Hydroxycarbamoyl-benzo[b]thiophen-6-ylmethyl)-amino]-methyl}-benzylamino)- acetic acid cyclopentyl ester, (S)-2-Amino-4-(4-{[(2-hydroxycarbamoyl- benzo[b]thiophen-6-ylmethyl)-amino]-methyl} -phenoxy)-butyric acid cyclopentyl ester; (S)-2-Amino-5-(4-{[(2-hydroxycarbamoyl-benzo[b]thiophen-6-ylmethyl)- amino]-methyl}-phenoxy)-pentanoic acid cyclopentyl ester; (R)-2-Amino-4-(4-{[(2- hydroxycarbamoyl-benzo[b]thiophen-6-ylmethyl)-amino]-methyl}-phenoxy)-butyric acid cyclopentyl ester; 2-(S)-Amino-3-[4-(4-{[(2-hydroxycarbamoyl-benzo[b]thiophen- 6-ylmethyl)-amino]-methyl}-phenoxy)-cyclohexyl]-propionic acid cyclopentyl ester, and pharmaceutically acceptable salts thereof.
In a further preferred embodiment of the invention the HDAC inhibitor is a carboline or beta-carboline derivative wherein the carboline or betacarboline ring systems (or analogs thereof) have a hydroxamate or N-hydroxy acylamino metal binding group as a pendant group, and pharmaceutically acceptable salts thereof.
In a yet further preferred embodiment of the invention, the HDAC inhibitor is a benzoimidazole derivative. In one aspect of this embodiment, the benzoimidazole derivative is chosen from 3-[l-(3-Dimethylamino-2,2-dimethyl-propyl)-2-(2,2- dimethyl-propyl)- 1 H-benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(3- Dimethylamino-2,2-dimethyl-propyl)-2-isopropyl-l H-benzoimidazol-5-yl]-N- hydroxy-acrylamide; 3-[2-Butyl-l-(3-dimethylamino-2,2-dimethyl-propyl)-lH- benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(3-Dimethylamino-2,2-dimethyl- propyl)-2-(2- methylsulfanyl-ethyl)-lH-benzoimidazol-5-yl]-N- hydroxyl-acrylamide; 3-[2-(2,2-Dimethyl-propyl)- 1 -(2-isopropylamino-ethyl)- 1 -H-benzoimidazo l-5-yl]-N- hydroxy-acrylamide; 3-[ 1 -(2-Diisopropylamino-ethyl)-2-(2,2-dimethyl-propyl)- 1 -H- benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(2-Diisopropylamino-ethyl)-2- isobutyl-lH-benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[l-(3-Dimethylamino-2,2- dimethyl-propyl)-2-hex-3-enyl- 1 -H-benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 - (3-Dimethylamino-2,2-dimethyl-propyl)-2-(2,4,4-trimethyl-pentyl)-l H- benzoimidazol-5-yl]- N-hydroxy-acrylamide; 3-[2-Cyclohexyl- 1 -(3-dimethylamino- 2,2- dimethyl-propyl)-l H-benzoimidazol-5-yl]-N-hydroxyacrylamide; 3-[2- Bicyclo[2.2.1 ]hept-5-en-2-yl- 1 -(3- dimethylamino-2,2-dimethyl-propyl)- 1 H- benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(2-Diethylamino-ethyl)-2-hex-3- enyl- 1 H-benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(2-Diisopropylamino- ethyl)-2-hex-3-enyl- 1 H-benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[2-Hex-3-enyl- 1 -(2-isopropylamino-ethyl)- 1 H-benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[2- Hex-3-enyl- 1 -(3-isopropylamino-propyl)- 1 H-benzoimidazo 1-5 -yl] -N-hydroxy- acrylamide; 3-[l-(2-Ethylamino-ethyl)-2-hex-3-enyl-l H- benzoimidazol-5-yl]-N- hydroxya-crylamide; 3-[l-(2-Diethylamino-ethyl)-2-hexyl-lH- benzoimidazol-5-yl]-N- hydroxy-acrylamide; 3-[2-(3,3-Dimethyl-butyl)-l-(2-Dimethylamino-ethyl)-lH- benzoimidazol-5-yl]-N-hydroxy-acrylamide; 3-[ 1 -(2-Dimethylamino-ethyl)-2-pentyl- 1 H-benzoimidazo 1-5 -yl] -N-hydroxy-acrylamide; 3-[ 1 -(2-Dimethylamino-ethyl)-2-(2,2,2- trifluoro-ethyl)- 1 H-benzoimidazo 1-5 -yl] -N-hydroxy- acrylamide; N-Hydroxy-3 - [ 1 -(5 - methyl- 1 -H-pyrazol-3-yl)-2-(2,4,4-trimethyl-pentyl)- 1 H-benzoimidazo 1-5-yl]- acrylamide; 3-[ 1 -(2-Ethylamino-ethyl)-2-pentyl- 1 H-benzoimidazol-5-yl]-N-hydroxy- acrylamide 3 -(2-Butyl- 1 -pyrrolidin-3 -yl- 1 H-benzoimidazo 1-5 -yl)-N-hydroxy- acrylamide; 3-(2-Butyl-l-piperidin-4-yl-lH-benzoimidazol-5-yl)-N-hydroxy- acrylamide; 3-[2-(4-Cyano-butyl)-l-(2-diethylamino-ethyl)-lH-benzoimidazol-5-yl]-N- hydroxy-acrylamide; (E)-3-(l-(l-butylpiperidin-3-yl)-lH-benzo[d]imidazol-5-yl)-N- hydroxy-acrylamide; (E)-N-hydroxy-3-(l-(l-(pent-4-enyl)piperidin-3- yl)-l H- benzo [d]imidazo 1-5 -yl)acrylamide; (E)-3 -( 1 -( 1 -(3 -dimethylbutyl)piperidin-4-yl)- 1 H- ben2o[d]imidazol-5-yl)-N-hydroxy-acrylamide; 3-[ 1 -(2-Diethylamino-ethyl)-2- propylamino-1 H- benzoimidazol-5-yl]-N-hydroxy-acrylamide; (E)-N-hydroxy-3-(l-(2- (isopropyl(propyl)amino)ethyl)-! H-benzo[d] imidazol-5-yl)acrylamide; 3-{l-[2- (Butyl-isopropyl-amino)-ethyl]-l H- benzoimidazol-5-yl}-N-hydroxy-acrylamide; 3- (l-{2-[(2-Ethyl-butyl)-methyl-amino]-ethyl}-l H- benzoimidazol-5-yl)-N-hydroxy- acrylamide; (E)-3-(l-(2-(bis(3,3-dimethylbutyl)amino)ethyl)- 1 H-benzo[d]imidazol-5- yl)-N-hydroxy-acrylamide; (E)-3-(l-(2-(diisobutylamino)ethyl)-l H-benzo[d]imidazol- 5 -yl)-N-hydroxy-acrylamide; 3 - { 1 - [2-(3 ,3 -Dimethyl-butylamino)-ethyl] - 1 H- benzoimidazol-5-yl} -N-hydroxy-acrylamide; N-Hydroxy-3- { 1 -[2-(methyl-pent-4-enyl- amino)- ethyl]-l H-benzoimidazol-5-yl}-acrylamide; 3-(l-{2-[(2,2-Dimethyl-propyl)- propyl-amino]- ethyl}-l H-benzoimidazol-5-yl)-N-hydroxy-acrylamide; 3-{l-[2-(3,3- Dimethyl-butylamino)-ethyl]-2-ethyl- lH-benzoimidazol-5-yl}-N-hydroxy-acrylamide; 3-(l-{2-[(3,3-Dimethyl-butyl)-methyl-amino]-ethyl}-2-propyl-lH-benzoimidazol-5- yl)-N- hydroxy-acrylamide; 3-(l-{2-[(3,3-Dimethyl-butyl)-(2,2,2-trifluoro- ethyl)- amino]-ethyl}-l H-benzoimidazol-5-yl)-N-hydroxy-acrylamide; 3-(l-{2-[Butyl-(2,2,2- trifluoro-ethyl)-amino]-ethyl} - 1 H-benzoimidazol-5-yl)-N-hydroxy-acrylamide; and pharmaceutically acceptable salts thereof.
In a yet further preferred aspect of the invention the HDAC inhibitor is an imidazo[l,2- A] pyridine derivative. In one aspect of this embodiment, the imidazo[l,2-A] pyridine derivative is chosen from (E)-N-hydroxy-3-(2-phenethyl-3-(3,4,5- trimethoxyphenylamino)imidazo[ 1 ,2-a]pyridin-6- yl)acrylamide; (E)-N-hydroxy-3-(2- phenethyl-3-(3,4,5- trimethoxyphenylamino)imidazo[l,2-a]pyridin-6- yl)acrylamide; (E)-3-(3-(benzo[d][l,3]dioxol-5-ylmethylamino)-2-phenethylimidazo[l,2-a]pyridin-i3- yl)-N- hydroxyacrylamide; N~Hydroxy-3-[2-phenethyl-3-(4-piperidin- 1 -yl- phenylamino)-imidazo[ 1 ,2-a]pyridin-6-yl]- acrylamide;3-(2-Hexyl-3- {2-[(2-hydroxy- ethyl)-propyl- carbamoyl]-ethylamino} -imidazo[ 1 ,2-a]pyridin-7- yl)-N-hydroxy- acrylamide; N-Hydroxy-3 -(2 -phenyl- imidazo[l,2-a]pyridin-7- yl)-acrylamide ; 3-(3- Butylaminomethyl-2-phenyl-imidazo[ 1 ,2- a]pyridin-7-yl)-N-hydroxy-acrylamide; N- Hydroxy-3- {3-[(methyl-propyl-amino)-methyl]-2-phenyl-imidazo[ 1 ,2-a]pyridin-7-yl} - acrylamide; N-Hydroxy-3 -(2 -methyl- imidazo[l,2-a]pyridin-7- yl)-acrylamide; 3-(3- Butylaminomethyl-2-methyl-imidazo[ 1 ,2- a]pyridin-7-yl)-N-hydroxy-acrylamide; 3- {2-tert-Butyl-3-[(2-diethylamino-ethylamino)- methyl] -imidazo[ 1 ,2-a]pyridin-7-yl} -N- hydroxy-acrylamide; 3-(3-{[(2-Dimethylamino-ethyl)-ethyl-amino]- methyl} -2-phenyl- imidazo[ 1 ,2-a]pyridin-7-yl)-N- hydroxy-acrylamide; N-Hydroxy-3 -(2 -phenyl-3- pyrrolidin- 1 -ylmethyl- imidazo[ 1 ,2-a]pyridin-7-yl)-acrylamide; 3- {3- [(Cyclopropylmethyl-amino)-methyl]-2- phenyl- imidazo[ 1 ,2-a]pyridin-7-yl} -N- hydroxy- acrylamide; 3-(3-Cyclopropylaminomethyl-2-phenyl- imidazo[l,2-a]pyridin- 7-yl)-N-hydroxy-acrylamide; 3 - [3 -Butylaminomethyl-2-(4-fluoro-phenyl)- imidazo[ 1 ,2-a]pyridin-7-yl]-N-hydroxy- acrylamide; 3-[3-(tert-Butylamino-methyl)-2- (4-fluoro- phenyl)-imidazo[ 1 ,2-a]pyridin-7-yl]-N-hydroxy-acrylamide, and pharmaceutically acceptable salts thereof.
In a yet further preferred embodiment of the invention, the HDAC inhibitor is a benzothiothene derivative. In one aspect of the invention the benzothiothene derivative is chosen from 5-Phenylacetylamino-benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-Benzoylamino-benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-(3-Phenyl-acryloylamino)- benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-[3- Phenyl-2-(toluene-4-sulfonylamino)-propionylamino]-benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-[2-(3,4-Dimethoxy-phenyl)- acetylamino] -benzo [b]thiophene-2- carboxylic acid hydroxyamide; Tetrahydro-furan-2-carboxylic acid [1- (2- hydroxycarbamoyl-benzo [b]thiophen-5 -ylcarbamoy l)-2- phenyl-ethyl] -amide; Tetrahydro-furan-3-carboxylic acid [ 1 -(2-hydroxycarbamoyl-benzo[b]thiophen-5- ylcarbamoyl)-2-phenyl-ethyl]-amide; 5- (2-[3-(2,5-Dimethoxy-benzyl)- ureido]-3- phenyl-propionylamino}-benzo[b]thiophene-2-carboxylic acid hydroxyamide; 5-[3- Phenyl-2-(3 -thiophen-2-ylmethyl-ureido)-propionylamino] -benzo [b]thiophene-2- carboxylic acid hydroxyamide; 5-[(3,3-Dimethyl-butyryl)-(2-isopropylamino-ethyl)- amino] -benzo [b]thiophene-2-carboxylic acid hydroxyamide, and pharmaceutically acceptable salts thereof.
In a yet further preferred embodiment of the invention, the HDAC inhibitor is a depsipeptide or a derivative or analog of thereof. In one aspect of this embodiment, the depsipeptide is chosen from is FK228 and Spiruchostatin A. In one aspect of this embodiment, the depsipeptide analog or derivative is an amino acid derivative or an analog of FK228 or Spiruchostatin A, and pharmaceutically acceptable salts thereof.
In a yet further preferred embodiment of the invention, the HDAC inhibitor is a stilbene like compound. In one aspect of this embodiment, the stilbene like compound is chosen from (2Z)-3-(3,5-Dimethoxy phenyl)-2-(4-fluorophenyl)-N-[6-(2- hydroxybenzyl amine)-6-oxohexyl] acrylamide; (2Z)-3-(3,5-Dimethoxyphenyl)-2-(4- fluorophenyl)-N-[6-(3-hydroxybenzyl amine) -6-oxohexyl] acrylamide; (2Z)-3-(3,4- Difluorophenyl)-2-(4-fluorophenyl)-N-[6-(2-thiazole amine)-6-oxo hexyl] acrylamide; (2Z)-3-(2,3,4-Trimethoxyphenyl)-2-(4-hydroxyphenyl)-N-[6-(hydroxyamino)-6- oxohexyl] acrylamide; N-hydroxy-2- {[(2Z)-3-(3,4 difluorophenyl)-2-(4-fluorophenyl)- acrylamide]6-oxohexyl]amino} -3-(4-hydroxyphenyl)propanamide, and pharmaceutically acceptable salts thereof.
In a yet further preferred embodiment of the invention, the HDAC inhibitor is a sulphonylpyrrole derivative. In one aspect of this embodiment, the sulphonylpyrrole derivative is chosen from (E)-N-Hydroxy-3-[l-(toluene-4-sulfonyl)-l-H-pyrrol-3-yl]- acrylamide; (E)-N-(2-Amino-phenyl)-3-[ 1 -(biphenyl-4-sulfonyl)- 1 H-pyrrol-3-yl]- acrylamide; (E)-3-[ 1 -(4-Aminomethyl-benzenesulfonyl)- 1 H-pyrrol-3-yl]-N-hydroxy- acrylamide; (E)-N-Hy droxy-3-[ 1 -(5-pyridin-2-yl-thiophene-2-sulfonyl)- 1 H-pyrrol-3- yl]-acrylamide; (E)-N-(2-Amino-phenyl)-3- { 1 -[3-(I H-pyrazol-4-yl)-benzenesulfonyl]- 1 H-pyrro 1-3 -yl} -acrylamide, and pharmaceutically acceptable salts thereof.
In a yet further preferred embodiment of the invention, the HDAC inhibitor is a thiophene or thiazole substituted trifluoroethanone derivative. In one aspect of this embodiment, the thiophene or thiazole substituted trifluoroethanone derivative is chosen from N-(4-Methoxybenzyl)-5-(trifluoroacetyl)thiophene-2-carboxamide; N- Methyl-N-(quinolin-7-ylmethyl)-5-(trifluoroacetyl)thiophene-2-carboxamide; N-Ethyl- 5-(trifluoroacetyl)thiophene-2-carboxamide; N-(4-Chlorobenzyl)-5- (trifluoroacetyl)thiophene-2-carboxamide; N-[2-(3,4-Dihydroquinolin-l(2H)-yl)ethyl]- 5-(trifluoroacetyl)thiophene-2-carboxamide; 2,2,2-Trifluoro-l-(5-{[4-(3-pyridin-3-yl- 1 ,2,4-oxadiazol-5-yl)piperidin- 1 -yljcarbonyl} -2-thienyl)ethanone; 1 -(5- {[4-(l ,3- Benzothiazol-2-yl)piperazin-l-yl]carbonyl}-2-thienyl)-2,2,2-trifluoroethanone; 1 - {5- [(4-Benzoylpiperidin- 1 -yl)carbonyl]-2-thienyl} -2,2,2-trifluoroethanone; N-[2-(4- Phenyl-l,3-thiazol-2-yl)ethyl]-5-(trifluoroacetyl)thiophene-2-carboxamide; 1 -(5- {[4-(l ,3-Benzothiazol-2-yl)piperazin- 1 -yljcarbonyl} -2-thienyl)-2,2,2-trifluoroethanone; N- [4-(2-Chlorophenyl)-l,3-thiazol-2-yl]-5-(trifluoroacetyl)thiophene-2-carboxamide; N- [(3-chlorobenzyl)sulfonyl]-5-(trifluoroacetyl)thiophene-2-carboxamide, and pharmaceutically acceptable salts thereof. In a yet further preferred embodiment of the invention, the HDAC inhibitor is an amino acid derivative. In one aspect of this embodiment, the amino acid derivative is chosen from N2- [(5 -Methoxy-2-methyl-lH-indo 1-3 -yl)acetyl] -N'-2-naphthyl-5 -(4-oxopentyl)- Z-cystein amide; N2-[(5-Methoxy-2-methyl-lH-indol-3-yl)acetyl]-N'-2-naphthyl-5-[(2- oxopropyl)sulfonyl]-Z- norvalinamide; N-(3-Acetylphenyl)-N2-(cyanoacetyl)-5-[(3,3,3- trifluoro-2-oxopropyl)thio]-L-norvalinamide; N-Benzoylglycyl-N-pyridinium-3-yl-5- [(3,3,3-trifluoro-2-oxopropyl)thio]-L-norvalinamide trifluoroacetate; N-Benzoylglycyl- N-(3,5-dichlorophenyl)-5-[(3,3,3-trifluoro-2-oxopropyl)thio]-L-norvalinamide, and pharmaceutically acceptable salts thereof.
In a yet further embodiment of the invention, the HDAC inhibitor is a benzamide derivative (or analog). In one aspect of the invention, the benzamide derivative is chosen from N-(2-Amino-phenyl)-4-[(2-propyl-pentanoylamino)-methyl]-benzamide; N-Hydroxy-4- [(2 -propyl-pentanoylamino)-methyl] -benzamide; N-(2-Amino-phenyl)-4- (2-propyl-pentanoylamino)-benzamide; N-Hydroxy-4-(2-propyl-pentanoylamino)- benzamide; 2-Propyl-pentanoic acid {4-[2-amino-phenylcarbamoyl)-methyl]-phenyl}- amide; 2-Propyl-pentanoic acid (4-hydroxycarbamoyl-methyl-phenyl)-amide; 2- Propyl-pentanoic acid {4-[2-amino-phenylcarbamoyl)-ethyl]-phenyl} -amide; 2-Propyl- pentanoic acid [4-(2-hydroxycarbamoyl-ethyl)-phenyl]-amide; 2-Propyl-pentanoic acid {4-2-(2-amino-phenylcarbamoyl)-vinyl]-phenyl} -amide; and 2-Propyl-pentanoic acid [4-(2-hydroxycarbamoyl-vinyl)-phenyl]-amide; N-(2-aminophenyl)-4-(3-chloro-5- {[(3,4-dimethoxybenzyl)amino]methyl}pyridin-2- yl)benzamide; N-(2-aminophenyl)- 4-(3-chloro-5-{[(4- methoxybenzyl)amino]methyl}pyridin-2-yl)benzamide;N-(2- aminophenyl)-4-(3-chloro-5-{[(tetrahydrofuran-2-ylmethyl)amino]methyl}pyridin-2- yl)benzamide; N-(2-aminophenyl)-4-[3-chloro-5-({[(5 -methylisoxazol-3- yl)methyl] amino } methyl)pyridin-2-yl]benzamide; 5 N-(2-aminophenyl)-4- [5 -( { [(1,5 - dimethyl- lH-pyrazo 1-3 -yl)methyl] amino } methyl)-3 -methylpyridin-2-yl]benzamide; N- (2-aminophenyl)-4-(3-methyl-5-{[(tetrahydrofuran-2-ylmethyl)amino]methyl}pyridin- 2-yl)benzamide, and pharmaceutically acceptable salts thereof.
Examples of HDAC inhibitors for use in the invention include, but are not limited to those found in international patent applications WO2006/123121 (23.11.2006), WO 2006/117549 (09.11.2006), WO 2006/117548 (09.11.2006), WO 2004/113336 (29.12.2004), WO 2007/058628 (24.05.2007), WO 2006/101456 (28.09.2006), WO 2006/101455 (28.09.2006), WO 2006/101454 (28.09.2006), WO 2005/040161 (06.05.2005) WO 2005/040101 (06.05.2005), WO 2007/061939 (31.05.2007), WO 2007/054776 (18.05.2007), WO 2007/039404 (12.04.2007), WO 2007/039403, (12.04.2007), WO 2007/029036 (15.03.2007), WO 2007/029035 (15.03.2007), WO 2006/129105 (07.12.2006), WO 2006/097460 (21.09.2006), WO 2006/097449 (21.09.2006), WO 2005/108367 (17.11.2005), WO 2004/067480 (12.08.2004), WO 2007/084390 (26.07.2007), WO 2007/071961 (28.06.2007), WO 2007/071956 (28.06.2007), WO 2007/067995 (14.06.2007), WO 2007/067994 (14.06.2007), WO 2007/061978 (31.05.2007), and WO 2005/028447 (31.03.2005); and those found in US patent applications 20060199829 (September 7, 2006), 20050250784 (November 10, 2005), 20050234033 (October 20, 2005), 20050197336 (September 8, 2005), 20050137232 (June 23, 2005), 20040266769 (December 30, 2004), 20070225373 (September 27, 2007) and 20040254220 (December 16, 2004), each of which is hereby incorporated by reference in its entirety.
In one embodiment of the invention, the compound useful for increasing UCHLl mRNA, UCHLl protein, and/or UCHLl hydrolase activity is selected from the group consisting of halo-N-propargyl-1 -amino indans, indans, indoles, methylproparylamines, 5-substituted 2,4-thiazolidinediones, alkyl and alkylbenzyl ethers of substituted hydroquinones, l,3,4-oxadiazol-2(3H)-one derivatives, 4-(benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazones, N-propargylhydrazines, 4-pyrrolidino derivatives, benzazepine derivatives, 3H-quinazolin-4-one derivatives, N-acylamino aryl derivatives, N-acylamino aryl derivatives, 3-phenyl-propionamidoderivatives, 3- phenyl-acrylamido derivatives, 3-phenyl-propynamido derivatives, fluorobenzamide derivatives, 2,3-dihydro-isoindol-l-one derivatives, fluoroallylamines, pyridine-2- carboxamides, pyridine amidos, silyl alkylene amines, phthalimido derivatives, N-aryl 5-aminomethyl oxazolidine-2-one, isoquinolino derivatives, oxazolo[3,4-a]quinolin-l- ones, 2,3-dihydro-imidazo[2,l-b]benzothiazoles, 5H-furanones, 3H-dihydrofuranones, (2-benzofuranyl)-l,2,3,6-tetrahydropyridines, (2-benzofuranyl)-piperidines, 4-(2- benzofuranyl)-piperidines, 2-(5,6-dimethyl-2-benzofuranyl)-piperidines, 3,4-Dihydro- 2H-pyrimido(2,l-b)benzothiazoles, thioxanthen-9-ones, a 3-N-phenylacetylamino-2,6- piperdinediones, β-(Fluoromethylene)-5-hydroxytryptophans, ethylenediamine monoamides, 1 ,2,3,4-tetrahydrocyclopent[b]indoles, 1 ,2,3,3a,4,8a- hexahydrocyclopent[B]indoles, 4-(2-benzofuranyl)-piperidines, 2-(5,6-dimethyl-2- benzofuranyl)-piperidines, arylethynylphenylcyclopropylamines, cyclopent[b]indoles, benzamides, 1,2,4-oxadiazoles, oxazolidones, 3-(aminoalkylamino)-l,2- benzisoxazoles, analogs thereof, and derivatives thereof.
In one embodiment, the invention provides a method comprising: (1) identifying a patient having Dementia with Lewy Bodies and (2) administering to said patient an amount of a pharmaceutical composition effective to increase UCHLl activity (e.g., mRNA, protein, and/or hydrolase activity) wherein said composition comprises (I) a compound chosen from halo-N-propargyl-1 -amino indans, indans, indoles, methylproparylamines, 5-substituted 2,4-thiazolidinediones, alkyl and alkylbenzyl ether of substituted hydroquinones, l,3,4-oxadiazol-2(3H)-one derivatives, A- (benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazones, N-propargylhydrazines, A- pyrrolidino derivatives, benzazepine derivatives, 3H-quinazolin-4-one derivatives, N- acylamino aryl derivatives, N-acylamino aryl derivatives, 3-phenyl- propionamidoderivatives, 3-phenyl-acrylamido derivatives, 3-phenyl-propynamido derivatives, fluorobenzamide derivatives, 2,3-dihydro-isoindol-l-one derivatives, fluoroallylamines, pyridine-2-carboxamides, pyridine amidos, silyl alkylene amines, phthalimido derivatives, N-aryl 5-aminomethyl oxazolidine-2-one, isoquinolino derivatives, oxazolo[3,4-a]quinolin-l-ones, 2,3-dihydro-imidazo[2,l-b]benzothiazoles, 5H-furanones, 3H-dihydrofuranones, (2-benzofuranyl)-l,2,3,6-tetrahydropyridines, (2- benzofuranyl)-piperidines, 4-(2-benzofuranyl)-piperidines, 2-(5,6-dimethyl-2- benzofuranyl)-piperidines, 3,4-Dihydro-2H-pyrimido(2,l-b)benzothiazoles, thioxanthen-9-ones, a 3-N-phenylacetylamino-2,6-piperdinediones, β-
(Fluoromethylene)-5-hydroxytryptophans, ethylenediamine monoamides, 1,2,3,4- tetrahydrocyclopent[b]indoles, 1 ,2,3,3a,4,8a-hexahydrocyclopent[B]indoles, 4-(2- benzofuranyl)-piperidines, 2-(5,6-dimethyl-2-benzofuranyl)-piperidines, arylethynylphenylcyclopropylamines, cyclopent[b]indoles, benzamides, 1,2,4- oxadiazoles, oxazolidones, 3-(aminoalkylamino)-l,2-benzisoxazoles, analogs thereof, and derivatives thereof, and (II) a pharmaceutically acceptable excipient. Examples of halo-N-propargyl-1 -amino indans include, but are not limited to, 4-fluoro- N-propargyl-1 -amino indan, 5- fluoro-N-propargyl-1 -amino indan, 6-fluoro-N- propargyl-1 -amino indan, optically pure enantiomers thereof, pharmaceutically acceptable salts thereof. In one aspect of this embodiment, the compound is 6-fluoro- N-propargyl-1 -amino indan. In another aspect of this embodiment, the compound is (+)-6-fluoro-N-propargyl- 1 -amino indan.
Examples of indole compounds include, but are not limited to, N-methyl-N-propargyl- 2-[ 1 -methyl-5-methoxyindolyl]methylamine, N-propargyl-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2-butynyl)-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2-butynyl)-N-methyl-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2,3-butadienyl)-2-[ 1 -methyl-5- methoxyindolyl]methylamine, N-(2,3-butadienyl)-N-methyl-2-[ 1 -methyl-5- methoxyindolyl]methylamine, and 5-methoxyindol-2-ylmethylamine, and pharmaceutically acceptable salts thereof.
Examples of methylpropargylamine compounds include, but are not limited to, N-(2- Butyl)-N-methylpropargylamine, N-(I -Butyl)-N-methylpropargylamine, N-(2-Propyl)- N-methylpropargylamine, N-(I -Pentyl)-N-methylpropargylamine, N-(2-Pentyl)-N- methylpropargylamine, N-(I -Heptyl)-N-methylpropargylamine, N-(2-Heptyl)-N- methylpropargylamine, N-(2-Decyl)-N-methylpropargylamine, N-(2-Dodecyl)-N- methylpropargylamine, and pharmaceutically acceptable salts thereof.
Examples of 5-substitutued 2,4-thiazolidinediones include, but are not limited to, 2,4- Dioxo-5 - [3 -(phenylmethoxy)-phenylmethylene] -4- thiazo lidinebutanenitrile, 2,4- Dioxo-5 - [3 -(phenylmethoxy)-phenylmethylene] -4-thiazo lidinepentanenitrile, and pharmaceutically acceptable salts thereof.
Examples of alkyl or alkylbenzyl ethers of substituted hydroquinones include, but are not limited to, 4-[2'-Formyl-4'-(m-chlorophenylmethyloxy)phenoxy]butyronitrile, 4-[2'- Methoxymethyl-4'-(m-chlorophenylmethyloxy)phenoxy]butyronitrile, 4-[2'-
Carbomethoxy-4'-(m-chlorophenylmethyloxy)phenoxy]butyronitrile, 4-[2'-Acyl-4'-(m- chlorophenylmethyloxy)phenoxy]butyronitrile, 4-[3'-Acyl-4'-(m- chlorophenylmethyloxy)phenoxy]butyronitrile, 4-{2'-Acetyl-4'-[(3",5"-bis- trifluoromethylphenyl)-methyloxy]phenoxy}butyronitrile, 3-[2'-Acyl-4'-(m- trifluoromethylphenylmethyloxy)phenoxy]propanol, N-2-[3'-Acyl-4'-(m- chlorophenylmethyloxy)phenoxy]- 1 -methylethyl-N-methylpropargylamine, 1 - [T- Acyl-4'-(m-trifluoromethylphenylmethyloxy)phenoxy]-3-methoxy-2-propanol, S(+)- 1 - [2'-Acyl-4'-(m-trifluoromethylphenylmethyloxy)phenoxy]-3-methoxy-2-propanol, (S)- l-[2'-Acyl-4'-(m-trifluoromethylphenylmethyloxy)phenoxy]-3-methoxy-2-propyl acetate; (S)-l-[2'-Acyl-4'-(m-trifluoromethylphenylmethyloxy)phenoxy]-3-methoxy-2- propyl methylcarbonate, and pharmaceutically acceptable salts thereof.
Examples of l,3,4-oxadiazol-2(3H)-one derivatives include, but are not limited to, 5- [4-(4,4,4-trifluorobutoxy)phenyl]-3-methoxyethyl- 1 ,3,4-oxadiazol-2(3H)-one, 5-[4- (4,4,4-trifluorobutoxy)phenyl]-3-hydroxyethyl-l,3,4-oxadiazol-2(3H)-one, 5-[4-(4,4,4- trifluorobutoxy)phenyl]-3-methylthioethyl-l,3,4-oxadiazol-2(3H)-one, 5-[4-(4,4,4- trifluoro-2-butenyloxy)phenyl]-3-methoxyethyl- 1 ,3 ,4-oxadiazol-2(3H)-one, 5-[4-
(4,4,4-trifluoro-3(R)-hydroxybutoxy)phenyl]-3-methoxyethyl-l,3,4-oxadiazol-2(3H)- one, 5 - [4-(tetrahydropyran-3 -ylmethoxy)phenyl] -3 -methoxy ethyl- 1 ,3 ,4-oxadiazo 1-
2(3H)-one, and pharmaceutically acceptable salts thereof.
Examples of 4-(benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazones, include, but are not limited to, 4-(benzyloxy)benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(4- methylbenzyl)oxy]benzaldehyde acetyl (2-cyanoethyl)hydrazone, 4-[(4- nitrobenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(4- chlorobenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(4- methoxybenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[(2,4- dichlorobenzyl)oxy]benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-[2- chlorobenzyl)oxy]benzaldehyde acetyl (2-cyanoethyl)hydrazone, 4-
(benzyloxy)benzaldehyde acetyl(2-hydroxyethyl)hydrazone, 4-
(benzyloxy)benzaldehyde acetyl (2-methoxycarbonyl-ethyl)hydrazone, 4-(2- phenylethoxy)benzaldehyde acetyl(2-cyanoethyl)hydrazone, 4-
(benzyloxy)benzaldehyde acetyl(2-cyanopropyl)hydrazone, A-
(benzyloxy)benzaldehyde acetyl(cyanomethyl)hydrazone, 4-(benzyloxy)benzaldehyde acetyl (3-cyanopropyl)hydrazone, 4-(benzyloxy)benzaldehyde acetylpropargylhydrazone, 4-(benzyloxy)benzaldehyde(2-cyanoethyl)
(ethoxycarbonyl)hydrazone, 4-(benzyloxy)benzaldehyde(2-hydroxyethyl)
(ethoxycarbonyl)hydrazone, benzaldehyde(2-cyanoethyl)(N- methylcarbamoyl)hydrazone, 4-(benzyloxy)benzaldehyde(2-cyanoethyl)(N- methylcarbamoyl)hydrazone, benzaldehyde(2-cyanoethyl)(N- phenylcarbamoyl)hydrazone, 4-(benzyloxy)benzaldehyde(2-cyanoethyl)(N- phenylcarbamoyl)hydrazone, 4-(benzyloxy)acetophenone acetyl(2- cyanoethyl)hydrazone, and pharmaceutically acceptable salts thereof.
Examples of N-propargylhydrazines include, but are not limited to, N2- propargylphenelzine, N'-propargylphenelzine, N'-propargyl-N2 -acetylphenelzine, and pharmaceutically acceptable salts thereof.
Examples of 4-pyrrolidino derivatives include, but are not limited to, (RS)-l-[4-(3- fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid methylamide, (RS)-I- [4-(3-fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid amide, (RS)-I -[4- (4-fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid amide, (RS)-l-[4-(4- fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidine-3-carboxylic acid methylamide, (RS)-2- oxo-l-[4-(4-trifluoromethyl-benzyloxy)-phenyl]-pyrrolidine-3-carboxylic acid amide, and (RS)-2-oxo- 1 -[4-(4-trifluoromethyl-benzyloxy)-phenyl]-pyrrolidine-3-carboxylic acid methylamide, (S)-N-[l-(4-benzyloxy-phenyl)-2-oxo-pyrrolidin-3-yl]-acetamide, (S)-N-[ 1 -(4-benzyloxy-phenyl)-2-oxo-pyrrolidin-3-yl]-methanesulfonamide, (S)-N- { 1 - [4-(3-fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidin-3-yl} -acetamide, (R)-N- { 1 -[4-(3- fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidin-3-yl}-acetamide, (R)-N- {l-[4-(3-fluoro- benzyloxy)-phenyl]-2-oxo-pyrrolidin-3-yl} -methanesulfonamide, (S)-N- { 1 -[4-(3- fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidin-3-yl} -methanesulfonamide, and (S)- { 1 -[4- (3-fluoro-benzyloxy)-phenyl]-2-oxo-pyrrolidin-3-yl}-carbamic acid methyl ester, and pharmaceutically acceptable salts thereof.
Examples of benzazepine derivatives include, but are not limited to, l-[7-(3-fluoro- benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-ethanone, 1 -[7-(3-fluoro- benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-2-methoxy-ethanone, 2-[7-(3- fluoro-benzylo xy)-l, 2,4, 5-tetrahydro-benzo[d]azepin-3-yl]-2-oxo-acetamide, 3-[7-(3- fluoro-benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-3-oxo-propionamide, 7-(3- fluoro-benzylo xy)-l, 2,4, 5-tetrahydro-benzo[d]azepine-3-carboxylic acid methyl ester, 7-(3-fluoro-benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepine-3-carbaldehyde, 7-(3- fluoro-benzyloxy)-3-methanesulfonyl-2,3,4,5-tetrahydro-lH-benzo[d]azepine, 7-(3- fluoro-benzylo xy)-l, 2,4, 5-tetrahydro-benzo[d]azepine-3-carboxylic acid amide, 7-(3- fluoro-benzylo xy)-l, 2,4, 5-tetrahydro-benzo[d]azepine-3-carboxylic acid ethylamide, 2- [7-(3-fluoro-benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-acetamide, (RS)-2-[7- (3-fluoro-benzyloxy)- 1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-propionamide, and pharmaceutically acceptable salts thereof.
Examples of 3H-quinazolin-4-one derivatives, include, but are not limited to, 2-[7-(3- fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-acetamide, 2-[7-(3-fluoro-benzyloxy)-4- oxo-4H-quinazolin-3-yl]-propionamide, 2-[7-(4-fluoro-benzyloxy)-4-oxo-4H- quinazolin-3-yl]-acetamide, 2-[7-(4-fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]- propionamide, 2-[7-(3-fluoro-benzyloxy)-2-methyl-4-oxo-4H-quinazolin-3-yl]- acetamide, 2-[2-cyclopropyl-7-(3-fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]- acetamide, 7-(3-fluoro-benzyloxy)-3-(2-methoxy-ethyl)-3H-quinazolin-4-one, 7-(4- fluoro-benzyloxy)-3-(2-methoxy-ethyl)-3H-quinazolin-4-one, 7-(3-fluoro-benzyloxy)- 3-(2-methoxy-ethyl)-2-methyl-3H-quinazolin-4-one, 3-(2-amino-ethyl)-7-(3-fluoro- benzyloxy)-3H-quinazolin-4-one, 3-(3-amino-propyl)-7-(3-fluoro-benzyloxy)-3H- quinazolin-4-one, 3-(2-amino-ethyl)-7-(4-fluoro-benzyloxy)-3H-quinazolin-4-one, 2- [7-(3-fluoro-benzyloxy)-2-methyl-4-oxo-4H-quinazolin-3-yl]-ethyl-ammonium chloride, [7-(3-fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-acetic acid ethyl ester, fluoro-[7-(3-fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-acetic acid ethyl ester, 2-[7- (3-fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-propionic acid ethyl ester, [7-(3- fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-acetic acid tert-butyl ester, 2-[7-(3- fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-propionic acid tert-butyl ester, [7-(4- fluoro-benzyloxy)-4-oxo-4H-quinazolin-3-yl]-acetic acid ethyl ester, 2-[7-(4-fluoro- benzyloxy)-4-oxo-4H-quinazolin-3-yl]-propionic acid ethyl ester, and pharmaceutically acceptable salts thereof.
Examples of N-acylamino aryl derivatives include, but are not limited to, N-[4-(3- fluoro-benzyloxy)-phenyl]-malonamide, N-[4-(3-fluoro-benzyloxy)-phenyl]- malonamic acid methyl ester, N-[4-(3-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[3-fluoro-4-(3-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[4-(4-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[2-fluoro-4-(3- fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[4-(2,4-difluoro- benzyloxy)-phenyl]-malonamic acid methyl ester, N-[4-(2-fluoro-benzyloxy)-phenyl]- malonamic acid methyl ester, N-[4-(2,4,5-trifluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[2-fluoro-4-(4-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, N-[4-(3,5-bis-trifluoromethyl-benzyloxy)-2-fluoro-phenyl]-malonamic acid methyl ester, N-[4-(3-fluoro-benzyloxy)-3-methyl-phenyl]-malonamic acid methyl ester, N-[3- chloro-4-(3-fluoro-benzyloxy)-phenyl]-malonamic acid methyl ester, cyclopropane- 1,1-dicarboxylic acid amide [4-(3-fluoro-benzyloxy)-phenyl]-amide, N-[4-(3-fluoro- benzyloxy)-phenyl]-malonamide, N-[4-(3-fluoro-benzyloxy)-phenyl]-2-methyl- malonamide, N-[3-fluoro-4-(3-fluoro-benzyloxy)-phenyl]-malonamide, N-[4-(4-fluoro- benzyloxy)-phenyl]-malonamide, N-[4-(2,4-difluoro-benzyloxy)-phenyl]-malonamide, N-[4-(2,4,5-trifluoro-benzyloxy)-phenyl]-malonamide, N-[4-(2-fluoro-benzyloxy)- phenylj-malonamide, N-(4-benzyloxy-phenyl)-malonamide, N-[4-(4-chloro- benzyloxy)-phenyl]-malonamide, N-[4-(3-fluoro-benzyloxy)-2-hydroxy-phenyl]- malonamide, N-[2-fluoro-4-(4-fluoro-benzyloxy)-phenyl]-malonamide, N-[4-(3-fluoro- benzyloxy)-3-methyl-phenyl]-malonamide, N-[3-chloro-4-(3-fluoro-benzyloxy)- phenylj-malonamide, cyclopropane- 1,1-dicarboxylic acid amide [2-fluoro-4-(4-fluoro- benzyloxy)-phenyl]-amide, 2-Acetylamino-N-[2-fluoro-4-(4-fluoro-benzyloxy)- phenylj-acetamide, 2-Acetylamino-N-[2-fluoro-4-(3-fluoro-benzyloxy)-phenyl]- acetamide, N-[2-Fluoro-4-(4-fluoro-benzyloxy)-phenyl]-2-formylamino-acetamide, and N-[2-Fluoro-4-(3-fluoro-benzyloxy)-phenyl]-2-formylamino-acetamide, and pharmaceutically acceptable salts thereof.
Examples of 3-phenyl-propionamidos, 3-phenyl-acrylamidos, or 3-phenyl- propynamidos include, but are not limited to, N-methyl-3-[4-(4-methyl-benzyloxy)- phenylj-acrylamide, 3-[4-(3-methoxy-benzyloxy)-phenyl]-N-methyl-acrylamide, 3-[4- (3-fluoro-benzyloxy)-phenyl]-2,N-dimethyl-acrylamide, 3-[4-(3-fluoro-benzyloxy)- phenyl] -N-methyl-acrylamide, N-methyl-3 - [4-(4-trifluoromethyl-benzyloxy)-phenyl] - acrylamide, 3-[4-(3,4-difluoro-benzyloxy)-phenyl]-N-methyl-acrylamide, 3-[4-(4- fluoro-benzyloxy)-phenyl]-N-methyl-acrylamide, and pharmaceutically acceptable salts thereof.
Examples of fluorobenzamide derivatives include, but are not limited to, (S)-N-(I- carbamoyl-ethyl)-2-fluoro-4-(3-fluoro-benzyloxy)-benzamide, 2-[4-(3- fluorobenzyloxy)-2-fluoro-benzamido]acetamide, (S)-N-(I -carbamoyl-2-hydroxy- ethyl)-2-fluoro-4-(3-fluoro-benzyloxy)-benzamide, (R)-N-(I -carbamoyl-ethyl)-2- fluoro-4-(3-fluoro-benzyloxy)-benzamide, 2-[4-(4-fluorobenzyloxy)-2-fluoro- benzamido]acetamide, (S)-N-( 1 -carbamoyl-ethyl)-2-fluoro-4-(4-fluoro-benzyloxy)- benzamide, (S)-N-( 1 -carbamoyl-ethyl)-2-fluoro-4-(4-trifluoromethyl-benzyloxy)- benzamide, (S)-4-(3,5-bis-trifluoromethyl-benzyloxy)-N-(l-carbamoyl-ethyl)-2-fluoro- benzamide, (S)-N-(l-Carbamoyl-ethyl)-3-fluoro-4-(4-trifluoromethyl-benzyloxy)- benzamide, (R)-N-(l-Carbamoyl-ethyl)-3-fluoro-4-(4-trifluoromethyl-benzyloxy)- benzamide, N-Cyanomethyl-3-fluoro-4-(4-trifluoromethyl-benzyloxy)-benzamide, N- (2-Amino-ethyl)-3-fluoro-4-(4trifluoromethyl-benzyloxy)-benzamide 1 : 1 hydrochloride; (R)-N-( 1 -Carbamoyl-ethyl)-2,6-difluoro-4-(4-fluoro-benzyloxy)- benzamide (S)-N-( 1 -Carbamoyl-2-hydroxy-ethyl)-2,6difluoro-4-(4-fluoro-benzyloxy)- benzamide, N-(2-Amino-ethyl)-2,6-difluoro-4-(4-fluoro-benzyloxy)-benzamide 1 : 1 hydrochloride, 2,6-Difluoro-4-(4-fluoro-benzyloxy)-N-(2-hydroxy-ethyl)-benzamide, (R)-2,6-Difluoro-4-(4-fluoro-benzyloxy)-N-(2-hydroxy-l-methyl-ethyl)-benzamide, (S)-N-(I -Carbamoyl-ethyl)-2,6-difluoro-4-(3-fluoro-benzyloxy)-benzamide, (R)-N-(I- Carbamoyl-ethyl)-2,6-fluoro-4-(3-fluoro-benzyloxy)-benzamide, and pharmaceutically acceptable salts thereof.
Examples of a 2,3-Dihydro-isoindol-l-one derivatives include, but are not limited to, 2- [5-(3-fluoro-benzyloxy)-l-oxo-l,3-dihydro-isoindol-2-yl]-acetamide, 2-[5-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (S)-2-[6-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide,, (R)-2-[6-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide , (S)-2-[ 1 -oxo-6-(4- trifluoromethyl-benzyloxy)- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (R)-2-[ 1 -oxo-6- (4-trifluoromethyl-benzyloxy)- 1 ,3-dihydro-isoindol-2-yl]-propionamide, [-[6-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-acetamide, (R)-2-[6-(3-fluoro- benzyloxy)- 1 -oxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide , (S)-2-[ 1 -oxo-6-(4- trifluoromethyl-benzyloxy)- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (R)-2-[ 1 -oxo-6- (4-trifluoromethyl-benzyloxy)- 1 ,3-dihydro-isoindol-2-yl]-propionamide, 2-(2-
Methoxy-ethyl)-6-(3-fluoro-benzyloxy)-2,3-dihydro-isoindol- 1 -one, 2-(2-methoxy- ethyl)-6-(4-trifluoromethyl-benzyloxy)-2,3-dihydro-isoindol- 1 -one, 2-(2-amino-ethyl)- 6-(4-trifluoromethyl-benzyloxy)-2,3-dihydro-isoindol-l-one 1 :1 hydrochloride, 2-(2- amino-ethyl)-6-(4-trifluoromethyl-benzyloxy)-2,3-dihydro-isoindol- 1 -one 1 : 1 hydrochloride, and pharmaceutically acceptable salts thereof.
Examples of fluoroallylamines include, but are not limited to, 2-isobutyl-3- fluoroallylamine, 2-isopropyl-3-fluoroallylamine, 2-(9-octadecenyl)-3- fluoroallylamine, 2-(3 -methyl-3 -butenyl)-3 -fluorallylamine, 2-(4-methoxy-2-butenyl)- 3 -fluoroallylamine, 2-isobutylsulfonylmethyl-3 -fluoroallylamine, 2-sec-butyl-3 - fluoroallylamine, 2-butyl-3-fluoroallylamine, 2-hexyl-3-fluoroallylamine, 2-heptyl-3- fluoroallylamine, 2-ethoxymethyl-3-fluoroallylamine, and 2-thioethoxymethyl-3- fluoroallylamine, 2-(2'-chlorophenoxy)methyl-3-fluoroallylamine, 2-(4'- chlorophenoxy)methyl-3-fluoroallylamine, 2-(4'-fluorophenoxy)methyl-3- fluoroallylamine, 2-thiophenoxymethyl-3-fluoroallylamine, 2-(2',4'- dichlorophenoxy)methyl-3-fluoroallylamine, 2-(2',4'-dichlorothiophenoxy)methyl-3- fluoroallylamine, 2-(5'-chloro-3'-fluorophenoxy)methyl-3-fluoroallylamine, 2- (2'chlorothiophenoxy)methyl-3-fluoroallylamine, 2-(4'-fluorothiophenoxy)methyl-3- fluoroallylamine, 2-phenoxymethyl-3-fluoroallylamine, and 2-(2'-chloro-4'- fluorothiophenoxy)methyl-3-fluoroallylamine, and pharmaceutically acceptable salts thereof.
Examples of a pyridine-2-carboxamides include but are not limited to, N-(2- aminoethyl)-5-chloropyridine-2-carboxamide and pharmaceutically acceptable salts thereof.
Examples of silyl alkylene amines include, but are not limited to, β-(benzyldimethylsilyl)ethanamine.hydrochloride, β-(dimethyl-2-phenylethylsilyl)ethanamine. hydrochloride, ethyl-4- fluorobenzylmethylsilylmethanamine.hydrochloride, dimethyl-4-fluorobenzylsilylmethanamine. hydrochloride, dimethyl-3-fluorobenzylsilylmethanamine. hydrochloride, 3,4-difluorobenzyldimethylsilylmethanamine. hydrochloride, 2,6-difluorobenzyldimethylsilylmethanamine, hydrochloride, 2,4-difluorobenzyl)dimethylsilylmethanamine. hydrochloride, dimethyl-2-fluorobenzylsilylmethanamine. hydrochloride, cyclohexylmethyldimethylsilylmethanamine. hydrochloride, β-(benzyldimethylsilyl)ethanamine.hydrochloride, β-(dimethyl-2-phenylethylsilyl)ethanamine. hydrochloride, ethyl-4-fluorobenzylsilylmethanamine.hydrochloride, dimethyl-4-fluorobenzylsilylmethanamine. hydrochloride, dimethyl-3-fluoroenzylsilylmethanamine. hydrochloride, 3,4-difluorobenzyldimethylsilylmethanamine. hydrochloride, 2, 6-difluorobenzyldimethylsilylmethanamine. hydrochloride, 2,4-difluorobenzyldimethylsilylmethanamine. hydrochloride, dimethyl-2-fluoroenzylsilylmethanamine. hydrochloride, andcyclohexylmethyldimethylsilylmethanamine.hydrochloride, ethyl-4-fluorobenzylmethylsilylmethanamine. hydrochloride, dimethyl-4-fluorobenzylsilylmethanamine. hydrochloride, dimethyl-3-fluorobenzylsilylmethanamine. hydrochloride, 3,4-difluorobenzyldimethylsilylmethanamine. hydrochloride, 2,6-difluorobenzyldimethylsilylmethanamine, hydrochloride, 2,4-difluorobenzyl)dimethylsilylmethanamine. hydrochloride, dimethyl-2-fluorobenzylsilylmethanamine. hydrochloride, and eye Io hexy lmethy ldimethy lsily lmethanamine . hydrochloride .
Examples of phthalimido derivatives include, but are not limited to, 2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl]-acetamide, (S)-2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (S)-2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-iso indol-2-yl]-3-hydroxy- propionamide,
(R)-2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-iso indol-2-yl]-propionamide, 2-[5-(3-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl]-propionamide, (2-[5-(3-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-iso indol-2-yl]-acetamide, 2-[5-(3-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl]-3-hydroxy- propionamide,
N- (2-[5-(4-fluoro-benzyloxy)- 1 ,3-dioxo- 1 ,3-dihydro-isoindol-2-yl]-ethyl} -acetamide, 2-(2-amino-ethyl)-5-(4-fluoro-benzyloxy)-iso indole- 1 ,3-dione, 5-(4-fluoro-benzyloxy)-2-piperidin-4-yl-iso indole- 1 ,3-dione, 5-(4-fluoro-benzyloxy)-2-(2-hydroxy-ethyl)-iso indole- 1 ,3-dione, 5-(4-fluoro-benzyloxy)-2-(2-methoxy-ethyl)-iso indole- 1 ,3-dione, 5-(3-fluoro-benzyloxy)-2-(2-methoxy-ethyl)-iso indole- 1 ,3-dione, (S)-5-(4-fluoro-benzyloxy)-2-(2-methoxy- 1 -methyl-ethyl)-iso indole- 1 ,3-dione, (S)-5-(3-fluoro-benzyloxy)-2-(2-methoxy- 1 -methyl-ethyl)-iso indole- 1 ,3-dione, (S)-5-(2-fluoro-benzyloxy)-2-(2-methoxy- 1 -methyl-ethyl)-iso indole- 1 ,3-dione, (S)-2-(2-methoxy- 1 -methyl-ethyl)-5-(4-trifluoromethyl-benzyloxy)-iso indole- 1 ,3- dione,
(S)-5-(4-bromo-benzyloxy)-2-(2-methoxy- 1 -methyl-ethyl)-iso indole- 1 ,3-dione, (S)-5-(3,4-difluoro-benzyloxy)-2-(2-methoxy-l-methyl-ethyl)-isoindole-l,3-dione, 5-(3-fluoro-benzyloxy)-2-(2-hydroxy-ethyl)-iso indole- 1 ,3-dione, 5-(4-fluoro-benzyloxy)-2-(3,3,3-trifluoro-2-hydroxy-propyl)-isoindole-l,3-dione, 5-(3,5-bis-trifluoromethyl-benzyloxy)-2-(2-methoxy-l-methyl-ethyl)-iso indole- 1,3- dione, and pharmaceutically acceptable salts thereof.
Examples of isoquinolino derivatives include, but are not limited to,
2-[6-(3-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-acetamide,
2-[6-(3-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide
2-[6-(4-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-[6-(3,4-difluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-[6-(3-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-(R)-[6-(3-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-(R)-[6-(4-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-(S)-[6-(4-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-(S)-[6-(4-fluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-3-hydroxy- propionamide,
2-(R)-[6-(2,6-difluoro-benzyloxy)-l-oxo-3,4-dihydro-lH-isoquinolin-2-yl]- propionamide, 2-[6-(3-fluoro-benzyloxy)3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-[6-(4-fluoro-benzyloxy)3,4-dihydro-lH-isoquinolin-2-yl]-acetamide,
2-[6-(3-fluoro-benzyloxy)-3,4-dihydro-lH-isoquinolin-2-yl]-acetamide,
2-[6-(4-fluoro-benzyloxy)3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2-(R)-[6-(4-fluoro-benzylo xy)-l, 3-dioxo-3,4-dihydro-lH-isoquinolin-2-yl]- propionamide,
2-(S)-[6-(4-fluoro-benzyloxy)-l,3-dioxo-3,4-dihydro-lH-isoquinolin-2-yTJ- propionamide,
2-(S)-[6 (4-fluoro-benzyloxy)-3-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide,
2(R)-[6-(4-fluoro-benzyloxy)-3-oxo-3,4-dihydro-lH-isoquinolin-2-yl]-propionamide, and pharmaceutically acceptable salts thereof.
Examples of pyridine amidos include but are not limited to
5-(3-fluoro-benzyloxy)-pyridine-2-carboxylic acid carbamoylmethyl-amide,
5-(4-fluoro-benzyloxy)-pyridine-2-carboxylic acid carbamoylmethyl-amide,
5-(3,4-difluoro-benzyloxy)-pyridine-2-carboxylic acid carbamoylmethyl-amide,
(S)-5-(3-fluoro-benzyloxy)-pyridine-2-carboxylic acid (1 -carbamoyl-ethyl)-amide,
(S)-5-(4-fluoro-benzyloxy)-pyridine-2-carboxylic acid (1 -carbamoyl-ethyl)-amide,
(S)-5-(3,4-difluoro-benzyloxy)-pyridine-2-carboxylic acid (l-carbamoyl-ethyl)-amide,
6-Benzyloxy-N-carbamoylmethyl-nicotinamide,
N-Carbamoylmethyl-6-(3-fluoro-benzyloxy)-nicotinamide,
N-Carbamoylmethyl-6-(4-fluoro-benzyloxy)-nicotinamide,
(S)-6-Benzyloxy-N-( 1 -carbamoyl-ethyl)-nicotinamide,
(S)-N-(I -Carbamoyl-ethyl)-6-(3-fluoro-benzyloxy)-nicotinamide, and
(S)-N-( 1 -Carbamoyl-ethyl)-6-(4-fluoro-benzyloxy)-nicotinamide, and pharmaceutically acceptable salts thereof.
Examples of oxazolo[3,4-a]quinolin-l-ones, include, but are not limited to, 3-methoxymethyl-7-(4,4,4-trifluoro-3-hydroxybutoxy)-3,3a,4,5-tetrahydro-lH-oxazolo [3,4-a]quinolin-l-one, 3-methoxymethyl-7-(4,4,4-trifluorobutoxy)-3,3a,4,5-tetrahydro- lH-oxazolo[3,4-a]quinolin-l-one, 7-(4,4,4-trifluorobutoxy)-3,3a,4,5-tetrahydro-lH- oxazolo[3,4-a]quinolin-l-one, 7-(3-hydroxy-4,4,4-trifluorobutoxy)-3,3a,4,5-tetrahydro- 1 H-oxazolo [3 ,4-a]quino lin- 1 -one, 3 -methoxymethyl-7- [(2-( 1 -hydroxy cyclopentyl) ethoxy]-3,3a, 4,5-tetrahydro-lH-oxazolo[3,4-a]quinolin-l-one.
Examples of a 3,4-Dihydro-2H-pyrimido(2,l-b)benzothiazoles include, but are not limited to, N-(l-ethylpropyl)-3,4-dihydro-2H-pyrimido[2,l-b]benzothiazol-7-amine, the pharmaceutically acceptable acid addition salts thereof, and the pyrimido[2,l- b]benzothiazolium salts thereof.
Examples of thioxanthen-9-ones, include, but are not limited to, 7-isopropyl-3-(2- methyl)-2H-tetrazol-5-yl)thioxanthen-9-one 10,10-dioxide, 3-(2-methyl-lH-tetrazol-5- yl)thioxanthen-9-one 10,10-dioxide, and 3-(l-methyl-lH-tetrazol-5yl)thioxanthen-9- one 10,10-dioxide, and pharmaceutically acceptable salts thereof.
Examples of such ethylenediamine monoamides include, but are not limited to,
N-(2-aminoethyl)-4-methoxypyridine-2-carboxamide,
N-(2-aminoethyl)thiazole-2-carboxamide,
N-(2-aminoethyl)-4-bromopyridine-2-carboxamide,
N-(2-aminoethyl)-4-chloropyridine-2-carboxamide,
N-(2-aminoethyl)-2-chlorothiazole-4-carboxamide,
N-(2-aminoethyl)-5-methylisoxazole-3-carboxamide,
N-(2-aminoethyl)-6-bromopyridine-2-carboxamide,
N-(2-aminoethyl)-6-chloropyridine-2-carboxamide,
N-(2-aminoethyl)-5-bromothiazole-4-carboxamide,
N-(2-aminoethyl)-3-aminopyridine-2-carboxamide,
N-(2-aminoethyl)pyridine-2-carboxamide,
N-(2-aminoethyl)-5-chloropyridine-2-carboxamide, and pharmaceutically acceptable salts thereof.
Examples of l,2,3,4-tetrahydrocyclopent[b]indoles and l,2,3,3a,4,8a- hexahydrocyclopent[B]indoles include, but are not limited to, 4-methyl-3- phenylmethylamino-l,2,3,4-tetrahydrocyclopent[b]indol7-yl methylcarbamate, 3-(N- cyclopropyl)amino-4-methyl- 1 ,2,3 ,4-tetrahydrocyclopent[b]indol7-yl methylcarbamate, l,2,3,3a,4,8-hexahydro-4-methyl-3-(N-phenylmethyloxycarbonyl)amino-cyclopent[b] indol-7-yl methylcarbamate, 1 ,2,3,3a,4,8a-hexahydro-4-methyl-3-(N-phenylmethyl-N- methylaminocarbonyl)aminocyclopent[b]indol-7-yl phenylmethylcarbamate, 4-methyl- 3-(2-phenylethyl)amino-l,2,3,4-tetrahydrocyclopent[b]indol7-yl methylcarbamate, 4-methyl-3-(2-phenylethyl)amino-l,2,3,4-tetrahydrocyclopent[b]indol7-yl benzylcarbamate, and pharmaceutically acceptable salts thereof.
Examples of arylethynylphenylcyclopropylamines include, but are not limited to,
1 - [4-(biphenylylethynyl)phenyl] cyclopropylamine;
1 - [4-(p-tolylethynyl)phenyl] cyclopropylamine; l-[4-(2-methoxyphenylethynyl)phenyl]cyclopropyl-N-methylamine; l-[4-(3-fluorophenylethynyl)phenyl]cyclopropylmorpholine;
1 - [4-(phenylethynyl)phenyl] cyclopropyl-N,N-di-t-butylamine; l-[4-(3-iodophenylethynyl)phenyl]-cyclopropylamine;
1 - [4-(3 ,5 -dibromophenylethynyl)phenyl] cyclopropyl-N-propylamine; l-[4-(4-cyclohexylphenylethynyl)phenyl]cyclopropyl-N-cyclopropylamine;
1 -[4-(phenylethynyl)phenyl]cyclopropyl-N-hexylamine and pharmaceutically acceptable salts thereof
Examples of cyclopent[b]indoles include, but are not limited to, 4-Methyl-3- (2propynyl)amino-l,2,3,4-tetrahydrocyclopent[b]-indol-7-yl-methylcarbamate, l,2,3,3a,4,8b-Hexahydro-4-methyl-3-(N-phenylmethoxycarbonyl)aminocyclopent[b] indol-7-yl methylcarbamate, 1 ,2,3,3a,4,8b-Hexahydro-4-methyl-3-(N-phenylmethyl-N- methylaminocarbonyl)aminocyclopent[b] indo 1-7-yl-phenylmethylcarbamate, and pharmaceutically acceptable salts thereof.
Examples of benzamides include, but are not limited to, N-(2-Aminoethyl)-p- chlorobenzamide, N-(2-aminoethyl)-p-fluorobenzamide, N-(2-aminoethyl)-p- bromobenzamide, N-(2-aminoethyl)-3,4-dichlorobenzamide, N-(2-aminoethyl)-2,4- dichlorobenzamide and N-(2-aminoethyl)benzamide, and pharmaceutically acceptable salts thereof. Examples of 1,2,4-oxadiazoles include, but are not limited to, 3-[4-[3-(lH-imidazol- lyl)propoxy]phenyl]-5-ethyl-l,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-trichloromethyl-l,2,4-oxadiazo Ie, 3-[4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-propyl-l,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-cyclopropyl-l,2,4-oxadiazole, 3- [4-[3-(lH-imidazol-l- yl)propoxy]phenyl]-5-phenyl-l,2,4-oxadiazole, 3-[4- [3-(3-pyridyl)propoxy]phenyl]-5- ethyl-l,2,4-oxadiazole, 3-[4-[2-(lH-imidazol-l-yl)ethoxy]phenyl]-5-ethyl-l,2,4- oxadiazole, 3-(4-benzyloxy)phenyl-5-ethyl- 1 ,2,4-oxadiazole, 3-(4-benzyloxy)phenyl-5- trichloromethyl-l,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l-yl)propoxy]phenyl]-5- trifluoromethyl-l,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l-yl)propoxy]phenyl]-5- pentafluoroethyl-l,2,4oxadiazole, 3-[4-[3-(lH-imidazol-l-yl)propoxy]phenyl]-5- heptafluoropropyl-l,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l-yl) propoxy]phenyl]-5- methyl- 1 ,2,4-oxadiazole, 3-[4-(3pyridylmethyloxy)phenyl]-5-methyl- 1 ,2,4-oxadiazole, 3-[4-(4pyridylmethyloxy)phenyl]-5-methyl- 1 ,2,4-oxadiazole,
3-[4-(3phenylpropoxy)phenyl]-5-methyl- 1 ,2,4-oxadiazole, 3-(4-benzyloxy) phenyl]-5- methyl- 1 ,2,4-oxadiazole, 3-[4-(3-chlorobenzyloxy)phenyl]-5-methyl- 1 ,2,4-oxadiazole, 3-[4-[3-(lH-imidazol-l-yl)propoxy]phenyl]-5-methylamino-l,2,4-oxadiazole and 3-(4- benzyloxyphenyl)-5-methylamino-l,2,4-oxadiazole, and pharmaceutically acceptable salts thereof.
Examples of oxazolidones include, but are not limited to, 3-[2-(l-hydroxy-3- cyanopropyl)benzothiazo 1-6-yl] -5 -methoxymethyl-2-oxazo lidone; 3 - [2-( 1 (S)-hydroxy- 3 -cyanopropyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone; 3 - [2-( 1 (R)- hydroxy-3 -cyanopropyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone; 3 - [2- (3 -cyanopropyl)benzothiazo 1-6-yl] -5 -methoxymethyl-2-oxazo lidone; 3-[2-(3- cyanopropyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone; 3-[2-(3- cyanopropyl)benzothiazo 1-6-yl] -5 (S)-methoxymethyl-2-oxazo lidone; 3 - [2-( 1 -hydroxy- 4-cyanobutyl)benzothiazo 1-6-yl] -5 -methoxymethyl-2-oxazo lidone; 3-[2-(4- cyanobutyl)benzothiazo 1-6-yl] -5 -methoxymethyl-2-oxazo lidone; 3 - [2-(4- cyanobutyl)benzothiazo 1-6-yl] -5 (S)-methoxymethyl-2-oxazo lidone; 3-[2-(4- cyanobutyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone; 3 - [2-(2- cyanoethyl)benzothiazo 1-6-yl] -5 -methoxymethyl-2-oxazo lidone; 3 - [2-(3 - cyanopropyl)benzothiazo 1-6-yl] -5 (S)-hydroxymethyl-2-oxazo lidone; 3-[2-(3- cyanopropyl)benzothiazol-6-yl]-5(R)-hydroxymethyl-2-oxazolidone; 3-[2-(3-(cyano)- l-propenyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidone; 3-[2-(4- cyanobutyl)benzothiazol-6-yl]-5(S)-hydroxymethyl-2-oxazolidone; 3-[2-(3-(cyano)- 1 , 1 -dimethylpropyl)benzothiazol-6-yl ]-5(R)-methoxymethyl-2-oxazolidone; 3-[2-(3- (cyano)- 1 , 1 -dimethylpropyl)benzothiazo 1-6-yl] -5 (R)hydroxymethyl-2-oxazo lidone; 3 - [2-(3-(cyano)- 1 (R)-hydroxy, 1 (S)methyl-propy)benzothiazo 1-6-yl] -5 (R)- methoxymethyl-2-oxazo lidone; 3 - [2-(3 -(cyano)- 1 (S)-hydroxy , 1 (R)methyl- propyl)benzothiazo 1-6-yl] -5 (R)-methoxymethyl-2-oxazo lidone, and pharmaceutically acceptable salts thereof.
Examples of 3-(aminoalkylamino)-l,2-benzisoxazoles include, but are not limited to, 6-
Methoxy-N-methyl-N- [2-(4-morpholinyl)ethyl] - 1 ,2-benzisoxazo 1-3 -amine;
3-[[2-(4-Morpholinyl)-ethyl]methylaminol-l,2-benzisoxazol-6-ol;
3-[[2-(4-Morpholinyl)-ethyl]methylamino]- 1 ,2-benzisoxazo 1-6-yl methylcarbamate;
3 - [ [2-(4-Morpholin-yl)ethyl]methylamino] - 1 ,2-benzisoxazo 1-6-ylphenylmethyl carbamate;
3 - [ [2-(4-Morpholinyl)-ethyl]methylamino 1- 1 ,2-benzisoxazo 1-6-yl- 1 -methylethyl- carbamate;
N-methyl-N-[2-(4-morpholinyl)ethyl]- 1 ,2-benzisoxazo 1-3-amine;
N-[2-(4-Morpholinyl)ethyl]- 1 ,2-benzisoxazo 1-3-amine;
6-Methoxy-N-[2-(4-morpholinyl)ethyl]- 1 ,2-benzisoxazo 1-3 -amine;
3-[[2-(4-Morpholinyl)ethyl]amino]- 1 ,2-benzisoxazo l-6-ol;
3-[[2-(4-Morpholinyl)-ethyl]methylaminol-l,2-benzisoxazol-5-ol;
3-[[2-(4-Morpholinyl)-ethyl]amino]- 1 ,2-benzisoxazol-6-yl methylcarbamate;
3-[[2-(4-Morpholinyl)ethyl]amino]- 1 ,2-benzisoxazo 1-5 -yl methylcarbamate;
6-Chloro-N-[2-(4-morpholinyl)ethyl]- 1 ,2-benzisoxazo 1-3 -amine;
1 -Methyl-N-[2-(4-morpholinyl)ethyl]- 1 ,2-indazol-3-amine;
N-Methyl-N- [2-(4-morpho linyl)ethyl] - 1 ,2-benzisothiazo 1-3-amine;
5-Methoxy-N-[2-(4-morpholinyl)ethyl]-l,2-benzisoxazole-3-amine;
7-Bromo-6-methoxy-N-[2-(4-morpholinyl)ethyl]-l,2-benzisoxazo 1-3-amine;
5-Bromo-6-methoxy-N-[2-(4-morpholinyl)ethyl]-l,2-benzisoxazo 1-3-amine;
3-[[2-(4-Morpholinyl)ethyl]amino]- 1 ,2-benzisoxazo 1-6-yl dimethylcarbamate; 3-[[(Methylamino)carbonyl] [2-(4-morpholinyl)ethyl] amino]- 1 ,2-benzisoxazolo- 6-yl methylcarbamate; 3 - [ [(Methylamino)carbonyl] [2-(4-morpholinyl)ethyl] -amino] - 1,2- benzisoxazol- 5-yl methylcarbamate; 6-Methoxymethoxy-N-[2-(4- thiomorpholinyl)ethyl]- 1 ,2-benzisoxazol-3-amine; 3-[[2-(4-
Thiomorpholinyl)ethyl] amino]- 1 ,2-benzisoxazol-6-ol; 6-Methoxy-N-methyl-N-[2-[4- ( 1 -phenylmethyl)piperdinyl] - 1 ,2-benzisoxazo 1-3 -amine; 7-Bromo-3 - [N-methyl, N-2- (4-morpholinyl)ethyl]amino- 1 ,2-benzisoxazo l-6-ol; 7-Bromo-3-[N-methyl,N-2-(4- morpholinyl)ethyl]amino-l,2-benzisoxazol-6-yl-dimethylcarbamate, and pharmaceutically acceptable salts thereof.
Examples of the preparation of these compounds and pharmaceutical compositions comprising the compounds are disclosed in e.g., US patents 5,486,541 (issued January
23, 1996); 5,130,327 (issued July 14, 1992); 5,169,868 (issued December 8, 1992); patent 5,326,770 (issued July 5, 1994); 5,380,755 (issued January 10, 1995); 5,811,456 (issued September 22, 1998); 5,525,619 (issued June 11, 1996); 6,060,516 (issued May
9, 2000); 7,235,581 (issued June 26, 2007); 7,173,023 (issued February 6, 2007); 7,087,612 (issued August 8, 2006); 7,053,245 (issued May 30, 2006); 6,762,320 (issued July 13, 2004); 6,900,354 (issued May 31, 2005); 6,951,884 (issued October 4, 2005); 6,846,832 (issued January 25, 2005); 4,650,907 (issued March 17, 1987); 4,699,928 (issued October 13, 1987); 5,380,861 (issued January 10, 1995); 5,384,312 (January 24, 1995); 5,529,988 (June 25, 1996); 5,532,397 (July 2, 1996); 6,660,736 (issued December 9, 2003); 6,818,774 (issued November 16, 2004); 6,667,327 (issued December 23, 2003); 4,470,993 (issued September 11, 1984); 5,641,785 (issued June
24, 1997); 4,262,004 (issued April 14, 1981); 4,346,102 (issued August 24, 1982); 4,600,719 (issued July 15, 1986); 4,471,117 (issued September 11, 1984); 5,356,916 (issued October 18, 1994); 5,494,908 (issued February 27, 1996); 5,475,014 (issued December 12, 1995); 5,238,962 (issued August 24, 1993); 5,380,755 (issued January
10, 1995); 5,298,626 (issued March 29, 1994); 4,210,655 (issued July 1, 1980); 4,042,584 (issued August 16, 1977); 5,100,891 (issued March 31, 1992); 4,822,812 (issued April 18, 1989); 4,764,522 (issued August 16, 1988); 4,705,796 (issued November 10, 1987); and 4,616,032 (October 7, 1986), each of which is hereby incorporated by referenced in their entireties. In one embodiment of the invention, the compound useful for increasing UCHLl activity (e.g., mRNA, protein levels, and/or hydrolase activity) is a polyamine, or an analog or derivative thereof. Examples of such compounds include, but are not limited to, alkylpolyaminoguanidines, alkylpolyaminobiguanides, tetramines, and pentamines and pharmaceutically acceptable salts thereof. In specific aspects of this embodiment, the polyamine is a guanidine or biguanides analog or derivative.
In one embodiment of the invention, the polyamine compound (or analog or derivative thereof) useful for increasing UCHLl activity (e.g., mRNA, protein levels, and/or hydrolase activity) is of the formula E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH-B-A-B- NH-E. According to this embodiment, A is independently chosen from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl; B is independently chosen from the group consistingof a single bond, C1-C6 alkyl, and C2-C6 alkenyl; E is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, C3-C6 cycloalkenyl; with the proviso that either at least one A moiety is chosen from the group consisting of C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl, or at least one B moiety is chosen from the group consisting of C2-C6 alkenyl; and any salt or stereoisomer thereof.
In one embodiment of the invention, the polyamine compound (or analog or derivative thereof) useful for increasing UCHLl activity (e.g., mRNA, protein levels, and/or hydrolase activity) is of the formula E-NH-B-A-B-NH-B-A-B-NH-B-A-B-NH(-B-A- B-NH)x-E. According to this embodiment, A is independently chosen from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl; B is independently chosen from the group consisting of a single bond, C1-C6 alkyl, and C2-C6 alkenyl; E is independently chosen from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl; and x is an integer from 2 to 16; with the proviso that either at least one A moiety is chosen from the group consisting of C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 aryl, and C3-C6 cycloalkenyl, or at least one B moiety is selected from the group consisting of C2-C6 alkenyl; and any salt or stereoisomer thereof. Specific polyamine compounds include l,l l-bis{Λ/2,Λ/3-dimethyl-N1-guanidino}-4,8- diazaundecane; l,15-bis{Λ/5-[3,3-(diphenyl)propyl]-Λf1-biguanido}-4,12- diazapentadecane; BENSpm; N1,N11-bis(ethyl)norspermine; CPENSpm, N'-ethyk/V11- [(cyclopropyl)methyl]-4,8,-diazaundecane; CHENSpm; N1StKyI-N11-
[(cyclohepthyl)methyl]-4,8,-diazaundecane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide.
Examples of the synthesis of such polyamine compounds are known in the art and given in e.g., EP 1 177 197 Bl (WO 2000/066587), Bacchi et al. Antimicrobial Agents and Chemotherapy, January 2002, p. 55-61, Vol. 46, No. 1, Huang et al. Clinical Cancer Research Vol. 9, 2769-2777, July 2003, each of which is hereby incorporated by reference in its entirety.
Inhibitors of the function of AOF2 include Spermine; N-Acetyl-D-Glucosamine; Mdl72527 (N,N'-bis(2,3-butadienyl)-l,4-butane-diamine); alpha-D-mannose; alpha-D- fucose; Flavin- Adenine Dinucleotide; octane 1,8-diamine; L-deprenyl and tranylcypromine .
Demethylation agents such as 5-azacytidine and 5-aza-2'-deoxycytidine may also be useful in the method of the invention.
Any of these small molecule chemical agents would be useful in the treatment or prevention of neurodegenerative diseases, and particularly in the treatment of Lewy body disorders.
Further, a cell based screen for molecules that inhibit the UCHLl inhibitory complex may be done by using a cell line expressing REST with low/no levels of UCHLl in which a reporter cassette (e.g. GFP or luciferin) was inserted in its original genomic environment or in which a reporter construct fusing the UCHLl regulatory domains with a reporter cassette was inserted randomly into the genome; and the screening for reactivation and expression of the reporter gene. In this way; inhibitors directed against additional components of the repressor complex can be discovered. We would expect such inhibitors to be useful in the treatment or prevention of neurodegenerative diseases, and particularly in the treatment of Lewy body disorders. Such inhibitors may include compounds, extracts or biological molecules, such as siRNA, miRNA, antibodies, and proteins.
In one embodiment, the invention provides a method of treating a patient by administering a combination of two or more inhibitors; for example the first may inhibit one protein belonging to the transcriptional repressor complex from the UCHLl gene such as REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl, and the second may inhibit a different member of the aforementioned complex. Alternatively or additionally the inhibitor may indirectly inhibit the complex by altering the transcription, translation, subcellular localisation or activity of one or several components of the complex. Any combination of inhibitors may be used and is not restricted to the example above.
In one embodiment, the invention uses an RNAi approach to directly or indirectly inhibit proteins in the transcriptional repressor complex, as would be apparent to the person skilled in the art.
An alternative approach uses microRNAs (miRNAs; miRs). miRs are small double stranded RNA molecules that are encoded in miRNA precursor genes. miRNA precursors mRNAs are transcribed; fold and are processed by the proteins Drosha and DICER to 20-25 base pair double-stranded RNA molecules. miRs negatively regulate expression of their target genes at the posttranscriptional level. In recent publications; computer algorithms were used to identify REST/NRSF target sequences (UCHLl was not among the targets identified). Wu and Xi (2006) have used these algorithms to identify a set of miRNAs (hsa-miR-124a, hsa-miR-132, hsa-miR-135a , hsa-miR-153, hsa-miR-218, hsa-miR-29b, hsa-miR-9, hsa-miR-9*) whose expression may be regulated by the REST complex. The targets of the identified miRNAs, on the other hand, are the different components of the REST complex (a regulation feedback loop). Given that according to this invention REST was shown to regulate the UCHLl promoter, the exogenous application of miRs that target the components of the REST complex could, (alone or in a mix containing at least one but possibly preferably several of these miRNAs), be an efficient way to release UCHLl expression which would not be subject to the negative feedback loop existing in the human body. Any of the miRNAs listed above (available from Ambion) are suitable for use according to the method of the invention.
Yet another alternative approach uses small double stranded interference RNAs (siRNAs) directed against the members of the complex regulating the UCHLl promoter. These can directly or indirectly inhibit the repression complex and release the control over the expression of UCHLl. A similar effect can be achieved with or short hairpin RNAs (shRNAs).
Examples of suitable siRNAs are listed in the table below:
Figure imgf000045_0001
A further approach is to use antisense technology to inhibit proteins in the transcriptional repressor complex, as would be apparent to the person skilled in the art.
Another approach is the use of monoclonal antibodies directed against proteins in the transcriptional repressor complex. As the complex exerts its activity in the nucleus, the antibodies employed are preferably designed to be able to target and act in the cell nucleus (e.g. by fusion to the monoclonal antibody 3E10 Fv fragment (Hansen JE et al. Antibody-mediated p53 protein therapy prevents liver metastasis in vivo. Cancer Res. 2007 Feb 15;67(4): 1769-74). Alternatively the antibodies may be designed to sequester proteins of the complex in the cytosol. Monoclonal antibodies have been described in the literature that would be suitable for use according to the present invention (e.g. Battaglioni et al. REST repression of neuronal genes requires components of the hSWI.SNF complex. J Biol Chem. 2002 Oct 25;277(43):41038-45). Antibodies directed against the members of the complex regulating the UCHLl promoter can be generated by standard methods involving the fusion of antibody secreting B cells with cell lines selected for their ability to confer in vitro immortality on the antibody secreting cells. Alternatively, DNA encoding monoclonal antibodies, antigen binding chains or domains can be cloned and expressed using standard methods of recombinant DNA technology. Recombinant antigen binding molecules can be manipulated to improve therapeutic properties such as specificity, affinity, half-life and lack of immunogenicity.
Rodent (e.g. rat or mouse) or other non-human animal (e.g. horse) antibodies can be used according to the invention. However, for use in man it is preferred that the antibody has been engineered to limit the anti-globulin response. Examples of antibodies engineered in this way are chimeric antibodies (where the constant regions of a non-human antibody are replaced by human constant regions) and humanised antibodies where the antibody is engineered to appear human to the immune system of the recipient. Examples of humanised antibodies are CDR-grafted antibodies where as well as replacing the constant regions of a non-human antibody with a human constant region, the framework regions of the variable regions are also replaced by human variable regions. The agent that represses the transcriptional complex that represses the promoter of the UCHLl gene may be administered to the patient in a number of ways. For example use could be made of liposomes, nanoparticles, viral vectors, and the like. In particular it is preferred to use a delivery method that will allow the agent to cross the blood-brain barrier.
The products and methods of the invention will now be illustrated by the following examples which are not intended to limiting. The scope of the invention is defined by the appended claims.
Example 1: Weak potential NRSF/REST binding sites are present in the UCHLl regulatory region
As a result of the previous findings showing that methylation of the UCHLl gene promoter was not responsible for reduced UCHLl levels in LBDs, an in silico analysis of UCHLl gene promoter sequence was performed using the Matlnspector software (Cartharius et al, 2005, Bioinformatics 21, 2933-2942). The analysis predicted three potential functional neuron-restrictive silencer elements (NRSE) which are the binding site for the neuronal restrictive silencer factor (NRSF/REST). NRSEl (0.69) was located in the complementary DNA chain, upstream from the transcription start site between positions -121 and -101 bp (Figure IA). NRSE2 (0.71) and NRSE3 (0.67) were located in the intron 1, in the coding and complementary chains, respectively. Both NRSE elements were located very close together, being separated only by 11 bp. The sequence of all NRSE elements and the consensus sequence are shown in Figure IB.
Example 2: NRSF/REST is a putative regulator of UCHLl, and its expression level is inversely related to UCHLl in the frontal cortex in PD and DLB.
We recently showed that UCHLl protein levels are reduced in the cerebral cortex in DLB but remain unchanged in the cerebral cortex of PD samples (Barrachina et al., 2006, Neurobiol. Dis. 22, 265-273). As NRSF is a repressor transcription factor, we tested whether there was an inverse relationship between UCHLl and NRSF protein levels, as determined by Western blotting, in the frontal cortex in PD, DLBp, DLBc and agematched controls. NRSF levels were not detected in control and PD samples but were increased in DLBp and DLBc samples (Figure 2). Moreover, increased NRSF protein levels occurred, in the cerebral cortex, in parallel with reduced UCHLl protein levels (Barrachina et al., 2006).
Example 3: NRSF and UCHLl expression levels are inversely related in cell lines.
To verify the functional relationship between NRSF and UCHLl, we used a human lung carcinoma cell line (DMS53, small cell lung cancer), human glioblastoma cell line (U87-MG) and human cervical cancer cell line (HeLa). NRSF mRNA levels were absent in DMS53 cells but higher in U87-MG and, particularly, in HeLa cells (Figure 3A). In contrast, UCHLl mRNA levels were very high in NRSF-negative DMS53 cells, lower in U87-MG and undetectable in NRSF-positive HeLa cells. The same situation was found in relation to NRSF and UCHLl protein (Figure 3B).
Example 4. Exogenous expression of REST/NRSF reduces UCHLl expression
We then tested whether exogenous expression of NRSF attenuated endogenous UCHLl mRNA levels in cells with very low levels of NRSF. The REEXl vector, coding for human full-length NRSF cDNA, was transiently transfected in DMS53 cells, thereby increasing NRSF mRNA and protein levels (Figure 4A). This over-expression reduced by 27% the expression levels of UCHLl mRNA in the same cells (p<0.01, ANOVA with post-hoc LSD test) (Figure 4B). As a positive control, we detected that NRSF over-expression reduced by 34% the expression levels of synaptophysin mRNA (p<0.01, ANOVA with post-hoc LSD test) (Figure 4C), as previously described (Lietz et al., 2003).
Example 5: Inhibition of NRSF/REST releases UCHLl repression
We also tested the effect of NRSF siRNA transfection in U87-MG cells. As shown in figure 5 A, endogenous NRSF protein levels were reduced after transfection with NRSF siRNA#l and siRNA#2, but remained unchanged with scramble siRNA. Reduction of NRSF expression was accompanied by increased expression of endogenous UCHLl mRNA levels (p<0.01, ANOVA with post-hoc LSD test). No changes were found after transfection of scramble siRNA (Figure 5B). Transfection of siRNA#l upregulated endogenous expression of synaptophysin mRNA levels (p<0.001, ANOVA with post- hoc LSD test) (Figure 5C). Example 6: NRSF interacts directly with the UCHLl promoter.
Having identified a functional relationship between NRSF and UCHLl, we proceeded to examine the interaction of NRSF with the UCHLl gene promoter to further support the concept that NRSF regulates endogenous UCHLl transcription by binding to its promoter. For this purpose, we carried out ChIP assays in U87-MG, HeLa and DMS53 cells. After the cross-linking of proteins and DNA with formaldehyde, sonicated cell lysates from each cell line were subjected to immunoprecipitation with the goat polyclonal anti-NRSF antibody. The precipitated DNA fragments were amplified with two sets of primers: set 1 spanned a 247 bp region covering the NRSEl and set 2 spanned a 214 bp region covering the NRSE2 and NRSE3 of the UCHLl gene promoter (Figure 6A). In U87-MG and HeLa cells, ChIP PCR products were detected with the NRSF and acetyl-histone 3 antibodies but not with a goat serum used as a negative control (Figure 6B). These results demonstrate that NRSF binds to the NRSE regions of UCHLl gene promoter in both cell lines. By contrast, the same analysis performed in DMS53 cells revealed the absence of binding of NRSF to NRSEl and the region covering NRSE2 and NRSE3 of UCHLl gene promoter as no PCR amplification is obtained using both set of primers in the anti-NRSF DNA immunoprecipitated (Figure 6C).
Example 7: Identification of NRSF/REST repressor complex components required for negative regulation of UCHLl.
To identify other components of the NRSF/REST repressor complex that regulates UCHLl; we trans fected the NRSF/REST expressing human glioblastoma cell line (U87-MG) and evaluated the effect of the transfection of siRNAs directed to candidate components of the NRSF/REST repressor complex binding to the UCHLl promoter on UCHLl mRNA expression by Taqman PCR.
No increase of UCHLl mRNA expression was observed in non transfected cells or in cells transfected at 10OnM concentration with siRNAs scrambled siRNA. A clear induction of expression was observed using siRNAs directed against MeCP2, RCORl, sin3A, HDACl, HDAC2, and AOF2 (direct inhibition of components the repression complex). In this experiment, lower inductions were observed with siRNAs directed against REST and JARIDlC (Figure 7).
Example 8: Identification of factors NRSF/REST repressor complex components required for negative regulation of UCHLl.
We also transfected the NRSF/REST expressing human glioblastoma cell line (U87- MG) with an siRNA directed against HDAC6. HDAC6 is not thought to participate directly in the regulatory complex that regulates the expression of the UCHLl promoter. Nevertheless, a strong induction of UCHLl mRNA expression was observed using an siRNA directed against HDAC6 at 10OnM concentration. (Figure 7). Analysis ofthe role ofHDACβ in
Example 9: Inhibition of the REST/NRSF complex and expression of UCHLl by application of miRs targeting components of the REST/NRSF complex.
We transfected the NRSF/REST expressing human glioblastoma cell line (U87-MG) and evaluated the effect of the transfection of miRs targeting the NRSF/REST repressor complex on UCHLl mRNA expression by Taqman PCR.
No increase of UCHLl mRNA expression was observed in non transfected cells or in cells transfected at 10OnM concentration with scrambled siRNA. On the contrary, transfection with a miR mix composed of hsa-miR-124a, hsa-miR-132, hsa-miR-135a , hsa-miR-153, hsa-miR-218, hsa-miR-29b, hsa-miR-9, and hsa-miR-9* (10OnM total miRNA concentration) caused a small increase in the UCHLl mRNA expression levels (Figure 7).
Example 10: Application of HDAC inhibitors increases UCHLl expression levels
NRSF has been known to recruit histone deacetylases (HDAC) to act as repressors through chromatin remodelling (Naruse et al., 1999; Huang et al., 1999). To assess whether the repression of UCHLl promoter activity is HDAC-dependent, we used TSA, a specific inhibitor of HDAC in U87-MG, HeLa and DMS53 cells. As shown in Figure 8 A, the inhibition of histone deacetylase activity by TSA 100 nM for 24 h was sufficient to increase UCHLl mRNA levels in U87-MG cells (p<0.01, ANOVA with post-hoc LSD test) and to induce its expression in HeLa cells Figure 8B with respect to non-treated cells (p<0.001, ANOVA with post-hoc LSD test). By contrast, the mRNA levels of endogenous UCHLl in DMS53 cells remained unchanged after TSA treatment, a phenomenon compatible with the low NRSF protein levels detected in these cells. TSA treatment did not affect the endogenous NRSF mRNA levels in U87- MG and HeLa cells.
We treated the NRSF/REST expressing human glioblastoma cell line (U87-MG) during 72h with 5μM HDAC inhibitors M344 (selective for HDAC6 over HDACl), MOCPAC (selective for HDAcI over HDAC6) and BATCP (selective for HDAC6 over HDACl), and evaluated the effect on UCHLl mRNA expression by Taqman PCR.
No increase of UCHLl mRNA expression was observed in non treated cells or cells treated with the vehicle. On the contrary, the treatment with HDAC inhibitors increased the UCHLl mRNA expression levels.
Example 11: UCHLl promoter methylation is not consistently increased in Lewy body diseases.
We analysed the methylation status of the minimal promoter region of the UCHLl gene, spanning the transcription start site and exons 1 and 2 (GenBank accession n0 Xl 7377). For this purpose, genomic DNA from post-mortem cortical brain samples of patients with Dementia with Lewy Bodies pure form (DLBp) and common form (DLBc) and age-matched controls was purified and treated with bisulfite. This treatment allows the analysis of the methylation status at each cytosine in a CpG island. 5-methylcytosine remains non-reactive to bisulfite whereas non-methylated cytosines are replaced by thymine. All remaining cytosines represent, after sequence analysis, methylated cytosines (compare Figure 9A and B). We found that the vast majority of the 35 CpG islands described in the UCHLl gene promoter were non-methylated in age-matched controls and in DLB samples (Figure 9C). For example, the sample number 3 presented only three methylated positions (CpG islands 3, 24 and 29) in two of three clones analysed. Sample 4 presented three methylated positions in one of the four clones examined (CpG islands 1, 2 and 8). Sample 9 presented three methylated positions in one of the four clones analysed (positions 10-12). Only position 1 was methylated in one of the two analysed clones of sample 11. A slight increase in the number of methylated positions was found in the clone sequence from DLBc samples, although in no case did methylation sites exceed 25% of the 35 CpG islands analysed in the minimal UCHLl gene promoter. Although we did not observe consistent methylation of the UCHLl promoter in the frontal cortex of the analyzed DLB cases, it is known that transcriptional repression by REST can prime the DNA sequence for more stable repression by DNA methylation (Lunyak et ah. Corepressor-dependent silencing of chromosomal regions encoding neuronal genes. Science. 2002 Nov 29;298(5599): 1747-52. Epub 24Oct 2002)
Example 12: Application of demethylating agents increases UCHLl expression in U87-MG and HeLa cells
The proclivity for UCHLl gene de-repression by a demethylating agent was also tested in U87-MG and HeLa cells. The treatment with 5-azacitidine 5 μM for 72 h up- regulated the expression of UCHLl in U87-MG cells and induced its expression in HeLa cells (p<0.05, ANOVA with post-hoc LSD test) (Figure 10A) without affecting the NRSF mRNA levels in either cell line (Figure 10B). The induction was especially clear in HeLa cells; in which the UCHLl promoter is known to be methylated.
Example 13: Cell-based screening method for molecules that inhibit the UCHLl inhibitory complex
Human reporter lines, for example, can be produced through plasmid or recombinant adeno associated viral vectors (rAAV) delivery of knock in constructs and homologous recombination with the endogenous UCHLl gene; or by transient or stable transfection with promoter reporter fusion constructs.
Targeting constructs
Regions of homology at the UCHLl locus can be amplified from genomic DNA obtained from U87-MG; HeLa cells or other cells in which UCHLl expression is downregulated, using a High Fidelity DNA Polymerase (e.g. Pfu DNA Polymerase). Typically, 5 to 7kb fragments are amplified from the upstream homology arm. For example, a DNA fragment covering 1.5kb of the promoter of UCHLl, exon 1, intron 1, exon 2, intron 2, exon 3, intron 3 and exon 4 (a total of around 5kb) can be amplified for the upstream homology region; which includes all the known regulatory elements for expression of the UCHLl gene. While shorter fragments of 2 to 3 kb are sufficient for the downstream homology arm; for example the DNA fragment covering exon 5, intron 5, exon 6, intron 6 and exon 7 of the UCHLl gene.
Unique restriction sites are embedded in the oligonucleotide primers used to amplify all fragments to facilitate cloning. Targeting plasmids are constructed by ligating the homology arms, and targeting/reporter cassette in the MCS of pBR322.
Construction of the targeting/reporting cassette
As an example, an adequate targeting/reporting cassette contains a hybrid 5 '-regulatory element containing a short length of intron sequence followed by a splice acceptor site, an IRES, which permits the translation of the open reading frame (ORF) of the reporter gene (preferentially Luciferase; the synthetic firefly Iuc2 (Photinus pyralis) and Renilla hRluc {Renilla reniformis) included in the pGL4 vectors from Promega) from RNA transcripts initiating from upstream exons (Topaloglu et al. Nucleic Acids Res. 2005; 33(18): el58), followed by a polyadenylation site. Preferentially, the components of the targeting/reporter cassette have been codon optimized and engineered to reduce the number of consensus transcription factor binding sites to reduce the risk of anomalous transcription.
Integration of this cassette in the UCHLl gene is useful to assess the effect of treatments on the transcriptional control of the UCHLl gene. Alternative; the reporter gene may be translationally fused to part of the UCHLl protein coding sequence. Integration of this cassette in the UCHLl gene is useful to assess the effect of treatments on the transcriptional+translational control of the UCHLl gene. Finally, the targeting/reporting cassette contains resistance gene (e.g. neo) expressed from its own promoter and provided with its own polyadenylation site for selection of stably transformed cells.
rAAV targeting constructs are assembled by ligation of homology arms and selectable marker cassettes, amplified using a high fidelity DNA Polymerase (e.g. DNA polymerase) from the targeting plasmid vector using oligonucleotide primers with embedded unique restriction sites allow inserted between the two Notl sites of p AAV- MCS, an AAV shuttle vector that carries the two inverted terminal repeat (ITR) sequences necessary for viral packaging (Stratagene). Typically, homology arms in rAAV targeting constructs need not be as long those for plasmid targeting vectors, lkb being sufficient.
Packaging of rAAV targeting/reporting constructs
Infectious rAAV stocks can be produced with the AAV Helper-Free System (Stratagene) according to the manufacturer's protocols. Briefly, ITR-containing targeting constructs are co-transfected with the plasmids pAAV-RC and pHELPER. Approximately 5 x 106 AAV-293 cells are transfected with a mixture of 2.5 μg of each of the above three plasmids, using 54 μl of Lipofectamine (Invitrogen) as described by the manufacturer. Two days after transfection, cells are scraped into 1 ml of phosphate- buffered saline and frozen and thawed three times. The crude lysate is clarified by centrifugation.
Gene targeting and isolation of recombinant cell lines using rAAVs Cells are grown in 25 cm2 flasks and infected with rAAV when -75% confluent. At the time of infection, medium was aspirated and 4 ml of medium containing rAAV lysate (0.5-2.5 x 105 viral particles) is added to each flask. Cells are washed with Hanks buffered saline solution and detached with trypsin (Invitrogen), 24 h after infection. Cells are replated in eight 96-well plates in medium containing geneticin (Invitrogen) at a final concentration of 0.4 mg/ml. Drug resistant colonies are grown for 3-4 weeks.
Gene targeting and isolation of recombinant cell lines using targeting/reporter plasmids
Approximately 5 x 106 cells are transfected with a mixture of 2.5 μg of the targeting/reporter plasmid, using 54 μl of Lipofectamine (Invitrogen) as described by the manufacturer. Cells are replated in eight 96-well plates in medium containing geneticin (Invitrogen) at a final concentration of 0.4 mg/ml. Drug resistant colonies are grown for 3-4 weeks.
Analysis of drug resistant colonies
Locus-specific integration of the targeting/reporting constructs (either plasmid or rAAV based) is assessed by PCR using primers outside the homology arms in combination with targeting cassette specific primers. In all cases, DNA polymerases fit for long PCR reactions (e.g. Pfu DNA polymerase) are employed.
Promoter fusions
Alternatively, the UCHLl regulatory sequences, including the promoter sequences and the first intron of the UCHLl gene, inserted in a pGL4 vector (Promega), transfected in transient or stable manner.
Analysis of reporter gene expression levels
After generation of the reporter cell lines, the low expression level of the reporter gene in the cell lines and its induction by the application of the agents previously identified to effectively induce UCHLl expression are verified, and cell lines with the correct response are selected.
Luciferase activity is assayed as described by the manufacturer (Promega). Sufficient
GIo Lysis Buffer, equilibrated at 220C, is added to the cells, equilibrated to room temperature; and incubated for 5 minutes at room temperature to allow lysis to occur.
The lysates are transferred to lumino meter tubes or plates and a volume of Bright
Glo™ Assay Reagent equal of GIo Lysis Buffer is added and luminescence is measured with a luminometer.
Screening for agents that induce UCHLl expression
A cell line with correct expression response selected above is then employed to perform high throughput evaluation of compounds, extracts or biologicals (siRNA, miRNA, antibodies, and proteins) to assess their effectiveness in inducing UCHLl expression.
Assays can be performed either in plate format or using reverse transfected/treated cell arrays.
Experimental procedures Brain samples
PD and DLB are considered α-synucleinopathies because abnormal α-synuclein is aggregated into Lewy bodies (LBs) and Lewy neurites in selected nuclei of the brain stem, spinal cord and autonomic ganglia. In addition, DLB is characterized by the widespread distribution of LBs and Lewy neurites in the cerebral cortex (Forno, 1996; Ince et al, 1998; Spillantini et al, 1998; Ince and McKeith, 2003; Jellinger and Mizuno, 2003). DLB is often accompanied by Alzheimer's disease (AD); this is considered the common form (DLBc). The pure form of DLB (DLBp) is characterized by minimal αA4-amyloid deposits and no tau pathology (Kosaka, 1993). The brains of six patients with PD, six DLBp, seven DLBc, and five aged-matched controls were obtained at autopsy, following informed consent of the patients or their relatives and the approval of the local ethics committees. Cases with prolonged agonal state, pyrexia, hypoxia, seizures or coma were excluded from the present study. Age range was between 57 and 91 years (mean age 75 years), and the average time between death and tissue processing was 6 h (between 2 and 13 h). pH range was between 6 and 7. Half of the brain was immediately cut into coronal sections, 1 cm thick, frozen on dry ice and stored at -8O0C until use. For morphological examinations, the brains were fixed by immersion in 10% buffered formalin for 2 or 3 weeks. Neuropatho logical characterization of PD was according to well-established neuropathological criteria (Jellinger and Mizuno, 2003).
Neuropathological characterization of DLB was according to consensus guidelines of the consortium on DLB international workshop (McKeith et al., 1996, 2000). Associated AD stages were further established depending on the amyloid deposition burden and neurofibrillary pathology, following the nomenclature of Braak and Braak (Braak and Braak, 1999). Stages of amyloid deposition refer to initial deposits in the basal neocortex (stage A), deposits extended to the association areas of the neocortex (stage B), and heavy deposition throughout the entire cortex (stage C). Stages of neurofibrillary pathology correspond to transentorhinal (I-II), limbic (III-IV) and neo cortical (V and VI).
To further refine Alpha-synuclein pathology, staging of brain pathology related to sporadic PD proposed by Braak et al. (Braak et al., 2003) was used in the present study. Basically, stages 1 and 2 affect the medulla oblongata plus the pontine tegmentum; stage 3, the midbrain; stage 4, the basal prosencephalon and mesocortex; and stages 5 and 6, the neocortex. Clinically, all cases of PD had suffered from classical PD lasting from 8 to 15 years, and none of them had cognitive impairment. Cases with DLB fulfilled the clinical criteria proposed by the consortium on DLB international workshop (McKeith et al, 1996, 2000). Control cases were considered in the absence of neurological symptoms and signs, and no abnormalities in the neuropathological study. The main neuropathological data in the present series are summarized in Table I. Biochemical studies were carried out in frozen samples of the frontal cortex (area 8). Control and diseased brains were processed in parallel.
Brain samples were obtained from the Institute of Neuropathology and University of Barcelona Brain Banks following the guidelines and approval of the local ethics committees.
Bisulfite treatment and PCR amplification of bisulfite-treated DNA
1.5 μg of genomic DNA isolated from human frozen brain homogenate was re- suspended in 50 μl of water and denatured, adding 5.7 μl of 3M NaOH for 10 min at 370C. Then 33 μl of 20 mM hydroquinone (Sigma) and 530 μl of 4.3 M sodium bisulfite (Sigma) at pH 5.0 were added. The DNA solution was incubated for 16 h at 5O0C. After that, DNA samples were desalted through a column (Wizard DNA Clean- Up System, Promega) and eluted with 50 μl of water. Then, the eluted DNA was treated with 5.7 μl of 3M NaOH for 20 min at 370C. Finally, DNA was precipitated, adding 1 μl of 10 mg/ml glycogen, 17 μl of 1OM ammonium acetate and 450 μl of ethanol overnight at -8O0C. The bisulfite-modified genomic DNA was re-suspended in 50 μl of water.
The conditions used in PCR amplification of bisulfite-modified genomic DNA have been previously described (Bittencourt-Rosas et al., 2001). The primers used were the CPGP9.5-Fow: 5 ' -TT AAAAgg ATTgTTTT AT ATATTT AAggAAT-3' and CPGP9.5- Rev: 5 '-CACTCACTTTATTCAACATCTAAAAAAC-S ' . The PCR product (473 bp) was cloned in TA pCRII vector (Invitrogen) and transformed in OneShot TOPlO chemically competent bacteria (Invitrogen). Several clones from each bisulfite- modified genomic DNA sample were sequenced using SP6 (5'- ATTTAggTgACACTATAg-3') and T7 (5' -T AATACgACTC ACTATAggg-3') primers and the Big Dye Terminator v3.1 sequencing kit on an Abi Prism 3730 sequence detector (Applied Biosystems). Cell culture
HeLa cells were maintained in Dulbecco's minimal essential medium (DMEM, Gibco, Invitrogen) supplemented with 10% foetal bovine serum. U87-MG cells (ATCC® number: HTB- 14) were maintained in minimal essential medium (Eagle) with 2 mM L- glutamine and supplemented with 10% foetal bovine serum and 1 mM sodium pyruvate. DMS53 cells (ATCC® number: CRL-2062) were maintained in Waymouth's MB 752/1 medium (GIBCO) supplemented with 10% foetal bovine serum.
All cell lines were grown at 370C in a humidified atmosphere of 5% CO2. TSA was dissolved in ethanol and 5-azacytidine in water:acetic acid (1 :1). For TSA treatment, cells were plated in 6-well dishes at a concentration of 105 cell/well and cultured overnight before activation. Cells were plated at a concentration of 50,000 cell/well for 5-azacytidine and also cultured overnight before treatment.
Cell transfection
DMS53 cells were plated in 6-well dishes at a concentration of 105 cells/well and cultured overnight before transfection. 1 μg of REEXl vector (kindly provided by Dr. Gail Mandel) was trans fected using lipofectamineTM 2000 (Invitrogen) following the instructions of the manufacturer. After 5 hours of post-transfection the medium was replaced by fresh medium. The efficiency of transfection was around 40% using the pEGFP-Cl vector (BD Biosciences Clontech).
siRNA and miR transfection
U87-MG cells were plated in 6-well dishes at a concentration of 50,000 cells/well and cultured overnight before transfection. 100 nM of siRNA, a mix of miRs at 1OnM each or scramble siRNA (Ambion, Cat. N°4611) were transfected using lipofectamine™ 2000 (Invitrogen) following the instructions of the manufacturer. After 5 hours of post- transfection the medium was replaced by fresh medium. The analysis of the siRNA or miR transfection was performed 48 hours later. All siRNAs and miRs used were from Ambion (Applied Biosystems): NRSF/REST; 5'-GCUUAUUAUGCUGGCAAAUTT-S'; Ambion, Cat. N°16810, NRSF/REST; 5'-GCCUUCUAAUAAUGUGUCATT-S'; Ambion, Cat. N016708, HDACl; ID # 120418 (NM_004964); AM51320 HDAC2; ID # 120210 (NMJ)01527); AM51320
HDAC6; ID # 120452 (NM_006044); AM51320
AOF2; ID # 118783 (NM-OOlOOQQQQ5 NM-OlSOn); AM51320
REST; ID # 115696 (NM 005612); AM16708
MECP2; ID # 143Q38 (NM_004QQ2); AM 16708
SIN3A; ID # 108733 (NM_015477); AM 16708
RCORl; ID #1367Q6; (NM 015156); AM16708
JARIDlC; ID #115486; (NM_004187); AM 1670
Silencer® Negative control #1 siRNA; AM4611 hsa-miR-124a; PM 10245; AMI 7100 hsa-miR-132; PM10166; AM17100 hsa-miR-135a; PMl 1126; AM17100 hsa-miR-153; PMl 1007; AM17100 hsa-miR-218; PM10328; AM17100 hsa-miR-2Qb; PMl 0103; AMI 7100 hsa-miR-Q; PM10022; AM17100 hsa-miR-Q*; PM10156; AM17100
Western Blot
Frozen frontal cortex (area 8; 100 mg) was directly homogenized in 1 ml lysis buffer (20 mM Hepes, 10 mM KCl, 1.5 mM MgC12, 1 mM EDTA, 1 mM EGTA, 1 mM DDT, 2 mM PMSF, 1 μg/ml aprotinin, leupeptin and pepstatin) and then sonicated. Cell lines grown in 10 ml-plates were homogenized in the same way without sonication. Lysates were centrifuged at 2650 g for 10 minutes at 40C, and protein concentration was determined with BCA (Pierce) method. 30 μg of total protein was boiled at Q5°C for 3 min and loaded in SDS-polyacrylamide gels with Tris-glycine running buffer.
Proteins were electrophoresed using a mini-protean system (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad) with a Mini Trans-Blot electrophoresis transfer cell (Bio-Rad) for 1 h at 100 V. Nitrocellulose membranes were blocked with Tween 20 TBS (TBST), containing 5% skimmed milk, for 30 min. Subsequently, the membranes were incubated at 4°C overnight with one of the primary antibodies in TBST containing 3% BSA. The following antibodies were used: anti-REST (Abeam) used at a dilution of 1 :250, anti- UCHLl (AB5937, Chemicon) used at a dilution of 1 :500, and anti-S- actin (clone AC- 74, Sigma) diluted 1 :10,000. After incubation with the primary antibody, the membranes were washed three times with TBST for 5 min at room temperature, and then incubated with the corresponding anti-rabbit, anti-goat or anti-mouse IgG antibody labelled with horseradish peroxidase (Dako) at a dilution of 1 :1,000 (1 :10,000 for S- actin) for 1 h at room temperature.
Subsequently, the membranes were washed five times, 5 min each, with TBST at room temperature, and developed with the chemiluminescence ECL Western blotting system (Amersham/Pharmacia), followed by apposition of the membranes to autoradiographic films (Hyperfϊlm ECL, Amersham).
mRNA isolation
The RNA from cell lines was purified with RNeasy Midi kit (Qiagen) following the protocol provided by the manufacturer. The concentration of each sample was obtained from A260 measurements. RNA integrity was tested using the Agilent 2100 Bio Analyzer (Agilent).
cDNA synthesis
The retrotranscriptase reaction (100 ng RNA/μl) was performed using the High capacity cDNA Archive kit (Applied Biosystems) following the protocol provided by the supplier. Parallel reactions for each RNA sample were run in the absence of MultiScribe Reverse Transcriptase to assess the degree of contaminating genomic DNA. TaqMan PCR
The NRSF/REST TaqMan assay (HsOO 194498 ml, TaqMan probe 5 '-AGGAAGGCCGAATACAGTTATGGCC-S ') (Applied Biosystems) generates an amplicon of 79 bp and is located at position 341 between 1 and 2 exon boundary of NM_005612.3 transcript sequence.
The TaqMan assay for UCHLl (HsOOl 88233_ml, TaqMan probe 5'- CCTGCTGAAGGACGCTGCCAAGGTC-3 ) (Applied Biosystems) is located at position 648 between 8 and 9 exon boundary of NM 004181.3 transcript sequence. It generates an amplicon of 100 bp. The TaqMan assay for Synaptophysin (Hs00300531_ml, TaqMan probe 5 - CGAGTACCCCTTCAGGCTGCACCAA-3 ') (Applied Biosystems), generates an amplicon of 63 bp and is located at position 241 of NM_003179.2 transcript sequence.
TaqMan PCR assays for NRSF/REST, UCHLl and synaptophysin were performed in duplicate on cDNA samples in 96-well optical plates using an ABI Prism 7700 Sequence Detection system (Applied Biosystems). The plates were capped using optical caps (Applied Biosystems). For each 20 μl TaqMan reaction, 9 μl cDNA (diluted 1/50) was mixed with 1 μl 2Ox TaqMan® Gene Expression Assays and 10 μl of 2x TaqMan Universal PCR Master Mix (Applied Biosystems). Parallel assays for each sample were carried out with S-glucuronidase (GUSB) (Hs99999908_ml, TaqMan probe 5 '-GACTGAACAGTCACCGACGAGAGTG-S '), for normalization. The reactions were carried out using the following parameters: 5O0C for 2 min, 950C for 10 min, and 40 cycles of 950C for 15 sec and 6O0C for 1 min. Standard curves were prepared for NRSF/REST, UCHLl, synaptohysin and GUSB using serial dilutions of cDNA from U87-MG cell line. Finally, all TaqMan PCR data were captured using the Sequence Detector Software (SDS version 1.9, Applied Biosystems).
The amount of targets (NRSF/REST, UCHLl and synaptophysin) and endogenous reference (GUSB) was determined for each experimental sample from the appropriate standard curve, which was plotted showing the cycle threshold, Ct (y), versus log ng total control RNA (x). The amount of each target was divided by the endogenous reference amount to obtain a normalized target value (arbitrary units). Chromatin immunoprecipitation (ChIP assay)
ChIP assay was performed according to the manufacturer's protocol (Upstate) using 106 U87-MG, HeLa and DMS53 cells. 10 μg Anti-NRSF/REST (P-18X, SC-15118X Santa Cruz) and 10 μg antiacetylated H3 (residue Lys9, Cell Signalling) were used for immunoprecipitation. Purified DNA was resuspended in 20 μl of DNAse-free water and 1 μl was used as template in 25 μl of PCR reaction using GoTaq Flexi DNA Polymerase (Promega). Primer concentration was 200 nM. PCR primers were 5'- ACAAATCCCgTCTCCACAAC-3' and 5'-gCCTAgggAAgACgAAAAACA-3' for the amplification of NRSEl sequence of UCHLl gene promoter. The NRS E2 and NRSE3 sequences were amplified with 5'-gCTCCgTAgCTgTTTTTCgT-3' and 5 '-gCC ACTC ACTTTgTTC AgC A-3'. The reaction was carried out using the following parameters: 950C for 2 min and 35 cycles of 950C for 30 sec, 650C for 30 sec and 720C for 30 sec. Finally, a last hold of 720C for 5 min was performed.

Claims

Claims
1. A method of treating or preventing a neurodegenerative disease in a patient suffering from such a condition which comprises administering to such a patient a therapeutically effective amount of an agent that represses the transcriptional complex that represses the promoter of the UCHLl gene.
2. The method according to claim 1, where the agent affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
3. The method according to claim 1 or 2, wherein the disease is a Lewy Body Disorder.
4. The method according to claim 1 or 2, wherein the disease is Huntington's Disease.
5. The method according to any of claims 1 to 4, wherein the agent is a HDAC inhibitor.
6. The method according to claim 5, wherein the HDAC inhibitor is selected from Trichostatin A (TSA), Suberoylanilide hydroxamic acid (SAHA), N-Hydroxy-4-(Methyl{[5-(2-Pyridinyl)-2-Thienyl]Sulfonyl}Amino)Benzamide, 4-Dimethylamino-N-(6-Hydroxycarbamoyethyl)Benzamide-N-Hydroxy-7-(4- Dimethylaminobenzoyl)Aminoheptanamide,
7-[4-(Dimethylamino)Phenyl]-N-Hydroxy-4,6-Dimethyl-7-Oxo-2,4-Heptadienamide, and Docosanol.
7. The method according to claim 6, wherein the inhibitor is SAHA.
8. The method according to claim 5, wherein the HDAC inhibitor is a short-chain to medium-chain fatty acid or a derivative or analog thereof.
9. The method according to any of claims 1 to 4wherein the agent is a small molecule that inhibits the function of REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl in the transcriptional complex that represses the promoter of the UCHLl gene.
10. The method according to claim 9 wherein the agent is a small molecule that inhibits the function of AOF2, such as Spermine; N-Acetyl-D-Glucosamine; Mdl72527 (N,N'-bis(2,3-butadienyl)- 1 ,4-butane-diamine); alpha-D-mannose; alpha-D-fucose; Flavin- Adenine Dinucleotide; octane 1,8-diamine; L-deprenyl or tranylcypromine.
11. The method according to any of claims 1 to 4wherein the agent is selected from: Benzyl ((S)-[ 1 -(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5- propionylaminopentyl] carbamate; Molecular Formula: C27H31N3O6; (iS)-[5-Acetylamino- 1 -(2-oxo-4-trifluoromethyl-2H-chromen-7- ylcarbamoyl)pentyl]carbamic acid tert-butyi ester; Molecular Formula: C23Η28F3N3O6; 4-(Dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-benzamide; and N-Hydroxy-7- (4-dimethylaminobenzoyl)-aminoheptanamide.
12. The method according to any of claims 1 to 4wherein the agent is a small molecule that inhibits HDAC6.
13. The method according to any of claims 1 to 4 wherein the agent is a demethylation agent.
14. The method according to claim 13 wherein the demethylation agent is selected from 5-azacytidine and 5-aza-2'-deoxycytidine.
15. The method according to any of claims 1 to 4 wherein the inhibition is provided by administering a short hairpin RNA (shRNA) for at least one of the genes that codes for a protein belonging to the transcriptional repressor complex from the UCHLl gene, or for a gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
16. The method according to any of claims 1 to 4 wherein the inhibition is provided by administering a microRNA for at least one of the genes that codes for a protein belonging to the transcriptional repressor complex from the UCHLl gene, or for a gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
17. The method according to any of claims 1 to 4 wherein the inhibition is provided by administering an antisense oligonucleotide for at least one of the genes that codes for a protein belonging to the transcriptional repressor complex from the UCHLl gene, or for a gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
18. The method according to any of claims 1 to 4 wherein the inhibition is provided by administering a small double stranded interference RNA (siRNA) directed against at least one of the genes that codes for a protein belonging to the transcriptional repressor complex from the UCHLl gene, or for a gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
19. The method according to any of claims 1 to 4 wherein the inhibition is provided by administering monoclonal antibodies directed against at least one of the genes that codes for a protein belonging to the transcriptional repressor complex from the UCHLl gene, or for a gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene.
20. The method according to any of claims 15 to 19 wherein the gene that codes for a protein belonging to the transcriptional repressor complex from the UCHLl gene is selected from REST, sin3a, HDACl, HDAC2, MeCP2, AOF2, RCORl, JARIDlC, BAF57, BAF170 and BRGl.
21. The method according to any of claims 15 to 19 wherein the gene that codes for a protein that affects the transcription, translation, subcellular localization or activity of one or several of the components of the transcriptional complex that represses the promoter of the UCHLl gene is HDAC6.
22. A method of screening for molecules that inhibit the transcriptional complex that represses the promoter of the UCHLl gene comprising providing a cell line containing a reporter gene fused with the UCHLl regulatory domains which expresses no or low levels of UCHLl, incubating the cell line with a molecule of interest and screening for expression of the reporter gene, wherein expression of the reporter gene indicates inhibition of the transcriptional complex that represses the promoter of the UCHLl gene by the molecule of interest.
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