WO2013083851A1 - Screening assays for compounds for use in the treatment of stress-related disorders - Google Patents

Screening assays for compounds for use in the treatment of stress-related disorders Download PDF

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WO2013083851A1
WO2013083851A1 PCT/EP2012/074995 EP2012074995W WO2013083851A1 WO 2013083851 A1 WO2013083851 A1 WO 2013083851A1 EP 2012074995 W EP2012074995 W EP 2012074995W WO 2013083851 A1 WO2013083851 A1 WO 2013083851A1
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Prior art keywords
stress
treatment
compound
protein
activity
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PCT/EP2012/074995
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French (fr)
Inventor
Nicole Zimmermann
Jürgen ZSCHOCKE
Theo Rein
Florian Holsboer
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority claimed from GBGB1121198.4A external-priority patent/GB201121198D0/en
Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Publication of WO2013083851A1 publication Critical patent/WO2013083851A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91011—Methyltransferases (general) (2.1.1.)
    • G01N2333/91017—Methyltransferases (general) (2.1.1.) with definite EC number (2.1.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/301—Anxiety or phobic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/304—Mood disorders, e.g. bipolar, depression

Abstract

The present invention relates to methods of identifying a compound having a therapeutic activity in the treatment of stress-related disorders. The invention also describes a method of providing a compound having a therapeutic activity in the treatment of stress-related disorders. In a further aspect, the invention relates to an antibody or a binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein for use as a medicament. The present invention also discloses an inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein and a vector comprising such an inhibitory polynucleotide molecule. The invention in a further aspect also relates to a pharmaceutical composition comprising an antibody or binding fragment thereof or an inhibitory polynucleotide molecule as described herein.

Description

Screening assays for compounds for use in the treatment of stress-related disorders
FIELD OF THE INVENTION
The present invention relates to methods of identifying a compound having an antidepressant activity and/or compounds having a therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event. The invention also describes a method of providing a compound having antidepressant activity and/or compounds having a therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event. In a further aspect, the invention relates to an antibody or a binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein for use as a medicament. The present invention also discloses an inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein and a vector comprising such an inhibitory polynucleotide molecule. The invention in a further aspect also relates to a pharmaceutical composition comprising an antibody or binding fragment thereof or an inhibitory
polynucleotide molecule as described herein.
BACKGROUND OF THE INVENTION Environmental stress represents a crucial factor that triggers and promotes the development of major depressive disorder (MDD) and bipolar depression (BPD) as well as anxiety disorders such as post-traumatic stress disorder (PTSD). Epigenetic processes are thought to link environmental cues to gene expression changes. Hence, they may play a fundamental role in the pathogenesis of these disorders and may constitute a target for novel treatment options (Tsankova et al, 2007).
DNA methylations at cytosines within CpG dinucleotides and histone modifications, including methylation or acetylation of histones, are intertwined epigenetic marks that work in concert to regulate gene expression by controlling chromatin structure and binding of transcription factors to DNA (Cheng and Blumenthal, 2010). In the nervous system, CpG methylation was recently recognized to change dynamically in response to various cues, such as psychosocial factors, diet and drug treatment (Weaver et al, 2004; Ptak and Petronis, 2008; Murgatroyd et al, 2009; Guidotti et al, 2011, Klengel at el, 2012).
Three DNA methyltransferase (DNMT) subtypes set DNA methylation marks in mammalian cells, DNMT1, DNMT3a and DNMT3b. They work cooperatively in perpetuating and establishing DNA methylation, with DNMT1 being the major DNMT that executes the maintenance of DNA methylation during the S-phase of replication, and DNMT3a /3b being mainly implicated in de novo methylation (Cheng and Blumenthal, 2008). Mounting evidence from human postmortem brain studies suggests that aberrant DNMT expression levels and promoter CpG methylation play a role in the pathogenesis of mood disorders (Poulter et al, 2008; McGowan et al, 2009; Guidotti et al, 2011). This concept is further substantiated by findings in rodent models of mood disorders (Tsankova et al, 2007). Interestingly, infusion of DNMT inhibitors into brain regions implicated in depression confers antidepressant- like effects on rodent behavior (LaPlant et al., 2010; Uchida et al., 2011; Sales et al, 2011). Conversely, exposure of rodent brain cells with psychoactive drugs, including imipramine (IMI), clozapine, sulpiride and VPA results in decreased promoter methylation and increased expression of candidate genes associated with mood disorders (Dong et al, 2007, 2008; Uchida et al, 2011). Also, electroconvulsive treatment leads to reduced methylation and increased expression of brain-derived neurotrophic factor BDNF (Krishnan and Nestler, 2008; Ma et al, 2009).
Further, there is an association of childhood abuse with DNA methylation levels in the promoters of ribosomal RNA genes (McGowan et al, 2008) and exon If of the glucocorticoid receptor gene promoter (McGowan et al., 2009). Exposure of infant rats to stressed caretakers produced persisting changes in DNA methylation of the brain derived nerve growth factor gene promoter in the adult prefrontal cortext (Roth et al, 2009). Early-life stress (Graf et al., 2007) in mice caused sustained DNA hypomehylation in the arginine vasopressin gene (Murgatroyd et al, 2009). These data support that changes in the DNA methylation pattern are involved in registering early-life adversity in the DNA.
Recent advances in the field of neuroscience showed that the antidepressant amitriptyline (AMI) reduces genome-wide DNA methylation at CCpGG sites and inhibits DNMT activity in primary cortical astrocytes (Perisic et al., 2010). Glial cells possibly significantly impact on the pathophysiology of mood disorders (Cotter et al, 2001).
However, there is still a need to identify novel compounds having a therapeutic effect in the treatment of stress-related disorders. In particular, there is a need to identify novel compounds with antidepressant effects and/or a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event.
OBJECTS AND SUMMARY OF THE INVENTION The present invention provides methods for identifying compounds having a therapeutic effect in the treatment of stress-related disorders. In particular, the present invention provides methods for identifying novel antidepressants. Further, the present invention provides methods for identifying compounds having a therapeutic activity in the treatment of anxiety disorders and/or disorders caused by the experience of a traumatic event.
Histone methyltransferase G9a represents a crucial cellular factor which regulates DNA methylation in neural cells by its impact on Dnmtl . DNA methylation plays a crucial role in the regulation of stress-related disorders such as depression and anxiety disorders (e.g. PTSD) on a molecular level. For example, traumatic events may induce a coordinated DNA methylation response in multiple promoters throughout the genome. In particular, DNA methylation is associated with increased behavioral stress-reactivity subsequent to early life stress. Hence, the pathogenesis of depression and other stress-related disorders seems to be accompanied by the establishment of aberrant methylation patterns which might contribute to consolidate the disease state. Targeting the cellular factor histone methyltransferase G9a which is involved in the regulation of DNA methylation by its impact on Dnmtl is thus a promising approach in the treatment of stress-related disorders such as depression or disorders caused by traumatic events. The present invention now demonstrates for the first time that the tricyclic antidepressants (TCAs) such as amitriptyline (AMI) and imipramine (IMI) as well as the selective serotonin reuptake inhibitor (SSRI) paroxetine (PAR) reduce DNMTl enzymatic activity in neural cells and regulate DNMTl via the decreased expression of histone methyltransferase G9a. Further, the present invention shows that interactions of G9a and DNMTl are decreased upon exposure to antidepressants. The present invention is thus based on the finding that antidepressants target the histone methyltransferase G9a.
Based on this finding the above objective is in particular accomplished by providing a method of identifying whether a compound has a therapeutic activity in the treatment of stress-related disorders, comprising the steps of contacting a compound with a neural cell under conditions suitable for cellular uptake of the compound, and determining a value indicative for the cellular level of histone methyltransferase G9a in the neural cell, wherein reduction of the cellular level compared to a control level is indicative of an therapeutic activity of the compound in the treatment of stress-related disorders.
The stress-related disorder may be selected from the group consisting of depression, disorders caused by experience of a traumatic event, and anxiety disorders such as PTSD.
In particular, the present invention provides a method of identifying whether a compound has an antidepressant activity, comprising the steps of contacting a compound with a neural cell under conditions suitable for cellular uptake of the compound, and determining a value indicative for the cellular level of histone methyltransferase G9a in the neural cell, wherein reduction of the cellular level compared to a control level is indicative of an antidepressant activity of the compound.
Further, the present invention provides a method of identifying whether a compound has a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event, comprising the steps of contacting a compound with a neural cell under conditions suitable for cellular uptake of the compound, and determining a value indicative for the cellular level of histone methyltransferase G9a in the neural cell, wherein reduction of the cellular level compared to a control level is indicative of a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event. Further, the present invention provides a method of identifying whether a compound has a therapeutic activity in the treatment of anxiety disorders, comprising the steps of contacting a compound with a neural cell under conditions suitable for cellular uptake of the compound, and determining a value indicative for the cellular level of histone methyltransferase G9a in the neural cell, wherein reduction of the cellular level compared to a control level is indicative of a therapeutic activity of the compound in the treatment of anxiety disorders.
Further, the present invention provides a method of identifying whether a compound has a therapeutic activity in the treatment of post-traumatic stress disorders, comprising the steps of contacting a compound with a neural cell under conditions suitable for cellular uptake of the compound, and determining a value indicative for the cellular level of histone
methyltransferase G9a in the neural cell, wherein reduction of the cellular level compared to a control level is indicative of a therapeutic activity of the compound in the treatment of posttraumatic stress disorders. In a preferred embodiment of the present invention determining the value indicative for the cellular level of histone methyltransferase G9a comprises measuring the expression level and/or the amount of histone methyltransferase G9a protein in the neural cell.
In a further preferred embodiment of the present invention the neural cell is an astrocyte.
In another preferred embodiment of the present invention the neural cell is derived from a mammal.
In a yet another preferred embodiment of the present invention the neural cell is a human neural cell.
In a preferred embodiment of the present invention the value indicative for the cellular level of histone methyltransferase G9a is determined by using a total or nuclear extract of said neural cell. In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 80% is indicative of a therapeutic activity of the compound in the treatment of stress-related disorders.
In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 80% is indicative of an antidepressant activity of the compound. In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 80% is indicative of a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event.
In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 80% is indicative of a therapeutic activity of the compound in the treatment of anxiety disorders.
In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 80% is indicative of a therapeutic activity of the compound in the treatment of post-traumatic stress disorders.
In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 70% is indicative of a therapeutic activity of the compound in the treatment of stress-related disorders.
In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 70% is indicative of an antidepressant activity of the compound. In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 70% is indicative of a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event. In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 70% is indicative of a therapeutic activity of the compound in the treatment of anxiety disorders. In a further preferred embodiment of the present invention reduction of the cellular level of histone methyltransferase G9a to less than 70% is indicative of a therapeutic activity of the compound in the treatment of post-traumatic stress disorders.
In another preferred embodiment of the invention the method of identifying a compound having a therapeutic activity in the treatment of stress-related disorders, e.g. having an antidepressant activity, further comprises a step of determining whether the compound is capable of decreasing the activity of a DNA methyltransferase.
In yet another preferred embodiment of the method of identifying a compound having a therapeutic activity in the treatment of stress-related disorders, e.g. having an antidepressant activity, comprises a step of determining whether the compound is capable of decreasing the activity of DNA methyltransferase DNMT1.
In a preferred embodiment of the present invention the activity of the DNA methyltransferase is determined by i) contacting a DNA substrate with a methyl group donor and a total or nuclear extract of said neural cell and ii) detecting DNA methylation at cytosine residues.
In a preferred embodiment of the present invention the DNA substrate is poly(dI-dC)-Poly(dI- dC) or hemi/unmethylated DNA substrate.
In a further preferred embodiment of the present invention the decrease of DNA
methyltransferase activity is at least about 30% is indicative of a therapeutic activity of the compound in the treatment of stress-related disorders, e.g. indicative of an anti-depressant activity, indicative of a therapeutic activity of the compound in the treatment of disorders caused by experience of a traumatic event, and/or indicative of a therapeutic activity of the compound in the treatment of anxiety disorders such as PTSD.
In a further preferred embodiment of the present invention the decrease of DNA
methyltransferase activity is at least about 40% is indicative of a therapeutic activity of the compound in the treatment of stress-related disorders, e.g. indicative of an anti-depressant activity, indicative of a therapeutic activity of the compound in the treatment of disorders caused by experience of a traumatic event, and/or indicative of a therapeutic activity of the compound in the treatment of anxiety disorders such as PTSD.
In another preferred embodiment of the invention the method of identifying a compound having a therapeutic activity of the compound in the treatment of stress-related disorders, e.g. an antidepressant activity and/or a therapeutic activity of the compound in the treatment of disorders caused by experience of a traumatic event, further comprises a step of determining whether the decrease of the activity of the DNA methyltransferase can be restored by the addition of purified histone methyltransferase G9a protein.
In a further preferred embodiment of the invention reduction of the cellular level comprises inhibiting histone methyltransferase G9a by directly binding to histone methyltransferase G9a protein. In this embodiment, reduction of the cellular level of histone methyltransferase G9a is indicative of a compound capable of inhibiting histone methyltransferase G9a by directly binding to histone methyltransferase G9a.
In a further preferred embodiment of the invention reduction of the cellular level comprises decrease or inhibition of histone methyltransferase G9a protein expression. In this embodiment, reduction of the cellular level of histone methyltransferase G9a is indicative of compound capable of decreasing or inhibiting histone methyltransferase G9a protein expression.
In a yet further preferred embodiment of the invention reduction of the cellular level comprises enhancing degradation of histone methyltransferase G9a protein in the neural cell. In this embodiment, reduction of the cellular level of histone methyltransferase G9a is indicative of a compound capable of enhancing degradation of histone methyltransferase G9a in the neural cell.
In a second aspect, the present invention describes a method of identifying whether a compound has a therapeutic activity in the treatment of stress-related disorders, comprising the steps of contacting said compound with a neural cell under conditions suitable for cellular uptake of the chemical compound, and determining a value indicative for a loss of interaction between histone methyltransferase G9a and DNA methyltransferase DNMT1, wherein said loss of interaction is indicative of a therapeutic activity of the compound in the treatment of a stress related disorder. Also in this aspect of the invention, the stress-related disorder may be selected from the group consisting of depression, disorders caused by experience of a traumatic event, and anxiety disorders such as PTSD.
In one embodiment, the method is thus a method of identifying whether a compound has an anti-depressant activity, comprising the steps of contacting said compound with a neural cell under conditions suitable for cellular uptake of the chemical compound, and determining a value indicative for a loss of interaction between histone methyltransferase G9a and DNA methyltransferase DNMT1, wherein said loss of interaction is indicative of an antidepressant activity of the compound.
In another embodiment, the method is a method of identifying whether a compound has a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event, comprising the steps of contacting said compound with a neural cell under conditions suitable for cellular uptake of the chemical compound, and determining a value indicative for a loss of interaction between histone methyltransferase G9a and DNA methyltransferase DNMT1, wherein said loss of interaction is indicative of a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event.
In another embodiment, the method is a method of identifying whether a compound has a therapeutic activity in the treatment of anxiety disorders, comprising the steps of contacting said compound with a neural cell under conditions suitable for cellular uptake of the chemical compound, and determining a value indicative for a loss of interaction between histone methyltransferase G9a and DNA methyltransferase DNMT1, wherein said loss of interaction is indicative of a therapeutic activity of the compound in the treatment of anxiety disorders.
In another embodiment, the method is a method of identifying whether a compound has a therapeutic activity in the treatment of PTSD, comprising the steps of contacting said compound with a neural cell under conditions suitable for cellular uptake of the chemical compound, and determining a value indicative for a loss of interaction between histone methyltransferase G9a and DNA methyltransferase DNMTl, wherein said loss of interaction is indicative of a therapeutic activity of the compound in the treatment of PTSD.
In a preferred embodiment of the invention the loss of interaction is detected by determining the loss of binding of histone methyltransferase G9a to DNA methyltransferase DNMTl .
In a further preferred embodiment of the invention the loss of interaction is determined by using a method selected from the group consisting of co-immunoprecipitation, pulldown- assay, resonance energy transfer (FRET), protein complementation assay, enzyme
complementation assay, yeast two hybrid, mammalian two hybrid, fluorescent polarization, and surface plasmon resonance.
In a third aspect, the present invention relates to a method of providing a compound having a therapeutic activity of the compound in the treatment of stress-related disorders comprising the steps of conducting the method of identifying a compound having a therapeutic activity of the compound in the treatment of stress-related disorders according to the present invention as described above, and synthesizing the identified compound.
The stress-related disorders may be selected from the group consisting of depression, disorders caused by experience of a traumatic event, and anxiety disorders such as PTSD.
Hence, in one embodiment, the method is a method of providing a compound having antidepressant activity comprising the steps of conducting the method of identifying a compound having antidepressant activity according to the present invention as described above, and synthesizing the identified compound.
In another embodiment, the method is a method of providing a compound having a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event comprising the steps of conducting the method of identifying a compound having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event according to the present invention as described above, and synthesizing the identified compound. In another embodiment, the method is a method of providing a compound having a therapeutic activity of the compound in the treatment of anxiety disorders comprising the steps of conducting the method of identifying a compound having a therapeutic activity in the treatment of anxiety disorders according to the present invention as described above, and synthesizing the identified compound.
In another embodiment, the method is a method of providing a compound having a therapeutic activity of the compound in the treatment of PTSD comprising the steps of conducting the method of identifying a compound having a therapeutic activity in the treatment of PTSD according to the present invention as described above, and synthesizing the identified compound.
In a further aspect, the present invention describes an antibody or a binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein for use as a medicament.
In one embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of stress- related disorders.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of depression.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of disorders caused by the experience of a traumatic event.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of anxiety disorders.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of PTSD. In a further preferred embodiment of the invention the antibody comprises a human, humanized, murine, xenogeneic or chimeric human-murine antibody or binding fragment thereof. In a further preferred embodiment of the invention the antibody is a monoclonal antibody.
In a further aspect, the present invention describes an inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein comprising
a) an antisense nucleic acid specific for histone methyltransferase G9a,
b) a small interfering RNA specific for histone methyltransferase G9a , or
c) a microRNA specific for histone methyltransferase G9a,
for use as a medicament. In one embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of stress- related disorders.
In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of depression.
In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of disorders caused by the experience of a traumatic event.
In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of anxiety disorders.
In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of PTSD. In another aspect, the present invention relates to a vector comprising the inhibitory polynucleotide molecule as described herein.
In yet another aspect, the present invention relates to a pharmaceutical composition comprising the antibody or binding fragment thereof as described herein and/or the inhibitory polynucleotide as described herein and at least one pharmaceutically acceptable excipient.
In one embodiment, the pharmaceutical composition is for use in the treatment of stress- related disorders.
In another embodiment, the pharmaceutical composition is for use in the treatment of depression.
In another embodiment, the pharmaceutical composition is for use in the treatment of disorders caused by the experience of a traumatic event.
In another embodiment, the pharmaceutical composition is for use in the treatment of anxiety disorders.
In another embodiment, the pharmaceutical composition is for use in the treatment of PTSD.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the characterization of anti-depressant's (AD's) inhibitory effect on DNMT activity. DNMT activity was measured in nuclear extracts from cortical astrocytes (A-D), neurons and NSCs (F) using Poly(dI-dC)-Poly(dI-dC) and S-adenosyl-L[methyl-3H]- methionine. (A) Cortical astrocytes were treated with the respective drugs for 72 h and DNMT activity was measured. (B) Nuclear extracts from untreated cortical astrocytes were supplemented with 10 μΜ AMI and relative DNMT activity was determined. (C, D) Cortical astrocytes were treated with AMI for different times (C) or at different concentrations (D). (F) Cortical neurons and subventricular neural stem cells (NSCs) were treated with AMI or PAR for 72 h. DNMT activity is presented relative to the mean ± SEM of the signal from control extracts of n > 3 independent experiments, (n > 3; *p<0.05, *** p<0.0005 vs. control). Figure 2 shows that anti-depressant (AD) treatment reduces enzymatic activity but not protein expression of the DNMT1 subtype. (A) The relative expression levels of DNMT1, DNMT3a and DNMT3b mR A from cortical neurons, subventricular NSCs and cortical astrocytes were assessed by Real-Time PCR using HPRT mRNA levels for normalization. Bar graphs represent means ± SEM (n > 3). (B) Proteins from astrocytic nuclei were successively extracted with buffer containing increasing NaCl concentrations (100- 400 mM). The fraction > 400 mM NaCl contains all proteins past the last extraction step. Fractions were probed for DNMT1 and 3 a immunoreactivity. (C) Nuclear extracts of untreated astrocytes obtained by extraction with 250 mM NaCl show major DNMT activity on hemimethylated and only minor activity on unmethylated oligonucleotides. (D, E) Cortical astrocytes were treated with 10 μΜ AMI for 72 h. DNMT activity was measured and is presented relative to the mean of the signal from control extracts. Nuclear extracts were assayed on hemi- and unmethylated DNA (D). Total extracts were assayed on Poly(dI-dC)-Poly(dI-dC) and unmethylated DNA (E) was used as substrate. Bar graphs represent means ± SEM (n>3;
**p<0.005, *** p<0.0005 vs. control). (F) Cell cycle profiles of cortical astrocytes treated with AMI or PAR (each at 10 μΜ, 72 h) were obtained by PI/FACS analysis. (G) Astrocytes were treated with the respective drugs at 10 μΜ except for CBZ (100 μΜ) and VPA (10 mM). Total (n>3) or nuclear extracts (=8) were probed for DNMT1 and HSC70/actin
immunoreactivity. Bar graphs represent mean ± SEM of relative protein levels.
Figure 3 illustrates that the DNMT1 regulator G9a is affected by ADs. Cortical astrocytes were treated with 10 μΜ AMI /PAR or 100 μΜ CBZ for 72 h (A) or 10 μΜ AMI for 24, 48, or 72 h (B). Relative G9a or NAAIO protein levels in total (A) or nuclear extracts (A, B) were determined using Western blot analysis. HSC70 or actin served as housekeeper proteins. Bar graphs represent the mean ± SEM (n > 3, *p<0.05). (C) Nuclear extracts (250 mM NaCl) from astrocytes treated with 10 μΜ AMI for 72 h were supplemented with 500 pg of recombinant G9a or vehicle and DNMT activity was measured. G9a immunoreactive signals of a concentration series of recombinant G9a and nuclear extracts from control/ AMI treated astrocytes are depicted. Actin served as a housekeeper.
Figure 4 shows the results of astrocytes treated with 10 μιηΑΜΙ for 72 hours. Interaction between G9a and DNMT1 was assessed by Co-immunoprecipitation (CoIP) using an anti- G9a antibody. Figure 5A depicts the relative G9a protein levels in BL6 mice which were either untreated (CO) or treated with paroxetine (PAR). Figures 5B and 5C show the results obtained in BL6 mice treated with paroxetine (~20 mg/kg) for three weeks. Relative G9a protein levels in brain extracts were determined using Western blot analysis. Hsc70 served as a housekeeper.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention generally relates to methods for identifying a compound having a therapeutic activity in the treatment of stress-related disorders such as depression, disorders caused by the experience of a traumatic event, and anxiety disorders including PTSD, and further provides suitable compounds for use in the treatment of such stress-related disorders.
In particular, the present invention relates to methods for identifying a compound having an antidepressive activity and further provides suitable compounds for use in the treatment of depression.
Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.
Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given.
As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.
In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, optionally ±15 %, specifically ±10 %, and more specifically ±5 %.
It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group, which optionally consists of these embodiments only.
Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" "i", "ii" etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.
A "peptide" consists of amino acids chains. A peptide as used within the context of a binding molecule may comprise or alternatively consist of a stretch of 2 to 35 amino acids, amino acid derivatives or a mixture thereof. The peptide may be linear, branched, circular or mixture thereof. A peptide affinity molecule may also be attached to a scaffold structure as defined herein above.
A "protein" is a polymer of amino acids linked by peptide bonds, which may comprise one polypeptides chain or more than one polypeptide chain put together typically in a biologically functional way. A protein as used within the context of binding molecule may comprise or alternatively consist of a stretch more than about 35 amino acids, amino acid derivatives or a mixture thereof. The protein may have a linear, branched, circular form or be comprised of a mixture of these forms. An "oligonucleotide" as used within the context of present invention may comprise or alternatively consist of a stretch of about 5 to 120 nucleotides, e.g. a stretch of 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, optionally of about 15 to 60 nucleotides. An
oligonucleotide used within the context of a binding molecule may be an RNA, DNA or PNA molecule, or a mixture thereof. In one embodiment, the oligonucleotide may be a complementary nucleic acid molecule.
The term "complementary nucleic acid molecule" refers to a molecule of a defined sequence, where the single strands are complementary to each other. It is known in the art that complementary strands of a double stranded nucleic acid molecule have a strong affinity to each other due to the formation of base pairing. The single stranded stretch is capable of recognizing and hybridizing to the complementary nucleotide sequence of interest with high affinity.
The determination of percent identity between two sequences may be accomplished using the mathematical algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873- 5877. Such an algorithm is e.g. incorporated into the BLASTn and BLASTp programs of Altschul et al. (1990) J. MoT Biol. 215: 403-410 available at NCBI
(http://www.ncbi.nlm.nih.gov/blast/Blast.cge).
The determination of percent identity may be performed with the standard parameters of the BLASTn and BLASTp programs. BLAST polynucleotide searches may be performed with the BLASTn program.
For the general parameters, the "Max Target Sequences" box may be set to 100, the "Short queries" box may be ticked, the "Expect threshold" box may be set to 10 and the "Word Size" box may be set to 28. For the scoring parameters the "Match/mismatch Scores" may be set to 1,-2 and the "Gap Costs" box may be set to linear. For the Filters and Masking parameters, the "Low complexity regions" box may not be ticked, the "Species-specific repeats" box may not be ticked, the "Mask for lookup table only" box may be ticked, the "Mask lower case letters" box may not be ticked.
BLAST protein searches may be performed with the BLASTp program.
For the general parameters, the "Max Target Sequences" box may be set to 100, the "Short queries" box may be ticked, the "Expect threshold" box may be set to 10 and the "Word Size" box may be set to "3". For the scoring parameters the "Matrix" box may be set to
"BLOSUM62", the "Gap Costs" Box may be set to "Existence: 11 Extension: 1", the
"Compositional adjustments" box may be set to "Conditional compositional score matrix adjustment". For the Filters and Masking parameters the "Low complexity regions" box may not be ticked, the "Mask for lookup table only" box may not be ticked and the "Mask lower case letters" box may not be ticked. One letter amino acid abbreviations used herein correspond to IUPAC nomenclature (see e.g. European Journal of Biochemistry, 138:9-37, 1984).
It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. As has been set out above, the present invention concerns in one aspect a method of identifying a compound having having a therapeutic activity in the treatment of stress-related disorders, e.g. having an antidepressant activity and/or a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event, comprising the steps of contacting a compound with a neural cell under conditions suitable for cellular uptake of the compound, and determining a value indicative for the cellular level of histone methyltransferase G9a in the neural cell, wherein reduction of the cellular level compared to a control level is indicative of a therapeutic activity in the treatment of stress-related disorders, e.g. an antidepressant activity of the compound and/or a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event. For example, the step of determining a value indicative for the cellular level of histone methyltransferase G9a may include determining a second value indicative for the cellular level of histone
methyltransferase G9a in the neural cell and comparing the second value with a first value of the cellular level determined from a control neural cell, wherein reduction of the cellular level, i.e. a second value being smaller than the first value is indicative of a therapeutic activity in the treatment of stress-related disorders, e.g. an antidepressant activity and/or a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event of the compound. A "control neural cell" may be a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of stress-related disorders, e.g. having an antidepressant activity, or a neural cell treated with a control compound not having a therapeutic activity in the treatment of stress-related disorders, e.g. not having an anti-depressant effect (negative control). A "control neural cell" may also be a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event, or a neural cell treated with a control compound not having a therapeutic activity in the treatment of other stress- related disorders, e.g. disorders caused by the experience of a traumatic event (negative control).
The term "antidepressant activity" or "antidepressant-like activity" as used herein refers to the activity a compound, e.g. a psychoactive drug, that typically alleviates mood disorders such as major depression, dysthymia and anxiety disorders such as social anxiety disorders. Typically, antidepressant activity treats or prevents depression or alleviates the symptoms of depression. It is to be understood that antidepressants generally exhibit an antidepressant activity or antidepressant-like activity when administered to an individual. The term„a compound having antidepressant activity" as used herein thus refers to a compound that exhibits the same or similar effect as an antidepressant, i.e. alleviation or treatment of depression. Typical antidepressant drugs, for instance, include the selective serotonin reuptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCA), and tetracyclic antidepressants.
Antidepressants are the most prescribed therapy for depression although the exact mechanism of action of antidepressants is often not understood. The prevailing theory is that
antidepressants increase the concentration of one or more brain chemicals (neurotransmitters) that nerves in the brain use to communicate with one another. Typical neurotransmitters affected by antidepressants are, for instance, norepinephrine, serotonin, and dopamine.
Generally, the different classes of antidepressants affect a different and individual set of neurotransmitters.
Human postmortem brain studies suggest that aberrant DNMT expression levels and promoter CpG methylation play a role in the pathogenesis of mood disorders (Poulter et al, 2008;
McGowan et al., 2009; Guidotti et al, 2011). Moreover, infusion of DNMT inhibitors into brain regions implicated in depression confers antidepressant- like effects on rodent behavior (LaPlant et al, 2010; Uchida et al, 2011; Sales et al., 2011.). It was reported that the antidepressant amitriptyline (AMI) is capable of reducing genome-wide DNA methylation at CCpGG sites in neural cells.
Amitriptyline as used herein refers to a tricyclic antidepressant (TCA) having antidepressant activity. Amitriptyline primarily acts as a serotonin-norepinephrine reuptake inhibitor, with strong actions on the serotonin transporter, and moderate effects on the norepinephrine transporter. Amitriptyline is commonly used for a number of medical conditions such as depressive disorders, anxiety disorders, attention deficit hyperactivity disorder, migraine prophylaxis, eating disorders, bipolar disorder, post herpetic neuralgia, and insomnia.
The Examples in the present invention convincingly demonstrate for the first time that also other antidepressants such as the TCA imipramine (IMI) and the selective serotonin reuptake inhibitor paroxetine (PAR) confer the anti-depressant activity as described herein. As can be seen in Figure 1 A the inventors have found that the psychoactive drugs AMI, IMI, and PAR significantly reduce the activity of DNA methyltranf erase DNMT1. Citalopram showed a trend in the same direction suggesting the presence of antidepressant activity of this drug.
The skilled person knows means and methods for measuring antidepressant and antidepressant activity. Without being limited thereto, one way of determining antidepressant activity envisaged by the present invention is the measurement of DNMT activity as described herein. In some embodiments the method of identifying a compound having an antidepressant activity and/or identifying compounds having a therapeutic activity in the treatment of other stress-related diseases , e.g. diseases caused by the experience of a traumatic event further comprises the step of determining whether said compound is capable of decreasing the activity of a DNA methyltransferase.
It has been previously reported that environmental stress represents a crucial factor that triggers and promotes the development of major depressive disorder (MDD) and bipolar depression. Envisaged by the present invention is the treatment of such mood disorders. In specific embodiments the present contemplates the treatment of major depressive disorder (MDD) or bipolar depression (BPD). „Major depressive disorder" also referred to as "recurrent depressive disorder", "clinical depression", "major depression", "unipolar depression", or "unipolar disorder" means a mental disorder characterized by an all-encompassing low mood accompanied by low self- esteem and by loss of interest or pleasure in normally enjoyable activities.
"Bipolar disorder" or "bipolar affective disorder" also known as manic-depressive disorder, refers to a psychiatric diagnosis describing a category of mood disorder defined by the presence of one or more episodes of abnormally elevated energy levels, cognition and mood with or without one or more depressive episodes. The elevated moods are clinically referred to as mania or hypomania. Individuals with manic episodes commonly experience depressive episodes, or symptoms, or a mixture thereof with features of both mania and depression present at the same time.
Another mood disorder envisaged by the present invention is termed "dysthymia", which is a mood disorder consisting of chronic depression with less severe symptoms than major depressive disorder.
Also envisaged is„anxiety disorder", which refers to several different forms of abnormal and pathological fear and anxiety. In particularly preferred embodiments, post-traumatic stress disorder is envisaged.
The term "disorder(s) caused by the experience of a traumatic event" includes any disorder and/or symptoms or combinations thereof caused by a traumatic event which negatively affects the psychologic and/or physiologic health of a patient. A traumatic event is one or more event(s) that is/are powerful and upsetting experiences, e.g. a threat to live and/or safety, but also any situation that leaves the person overwhelmed. Hence, traumatic events do not necessarily involve physical violence and/or harm. Furthermore, the disorder and/or symptoms not need occur immediately at the traumatic event but may be delayed for e.g. days, weeks or years after the event.
Exemplary disorders caused by the experience of a traumatic event are selected from the group consisting of psychological trauma, excessive alertness, exhaustion, sleeping disorders, poor concentration and/or memory, social withdrawal, loss of interest in normal activities, acute stress reaction, fear, depression, anxiety, panic and/or posttraumatic stress disorder (PTSD).
In particular, the disorder caused by the one or more traumatic event may be a psychological trauma. Psychological trauma as used herein relates to damage to the psyche caused by the experience of a traumatic event. Further disorders and/or symptoms which may be caused by the experience of a traumatic event include excessive alertness, exhaustion, sleeping disorders (e.g. disturbed sleep, nightmares), poor concentration and/or memory, social withdrawal, loss of interest in normal activities, acute stress reaction ("shock"), fear, depression, anxiety and/or panic as well as posttraumatic stress disorder (PTSD). It is understood, that one or more of the aforementioned disorders or symptoms may occur after the traumatic event.
As used herein "PTSD" relates to a severe anxiety disorder which develops as a result of exposure to a traumatic event. According to the National Institute of Mental Health (NIMH; http://www.nimh.nih.gov/health/publications/post-traumatic-stress-disorder-ptsd/what-are- the-symptoms-of-ptsd.shtml) symptoms of PTSD include re-experiencing symptoms such as flashbacks and frightening thoughts, avoidance symptoms such as avoidance of places that are reminders of the experience and/or hyperarousal symptoms such as anger and aggression. Diagnostic criteria for PTSD have been proposed in the American Psychiatric Association's diagnostic manual of mental disorders (fifth edition). In one embodiment, the disorder caused by the experience of a traumatic event is PTSD.
The term "neural cell" as used herein refers to any cell type of the animal central nervous system (CNS). Neural cells may form networks and establish the higher order structures of the nervous system such as the brain, spinal cord, and peripheral ganglia. Neural cells exist in a number of different shapes and sizes and can be classified by their morphology and function. The present invention contemplates the use of any neural cell type including, but without being limited thereto, neural progenitors, neurons, astrocytes, mircroglia, and oligodendrocytes. The present invention also contemplates the use of neuronal stem cells (NSCs) and progenitor cells, which may undergo differentiation to the neural cell type of interest.
A "neuron" as used herein generally refers to an electrically excitable cell capable of processing and transmitting information by electrical and chemical signaling. Typically, chemical signaling occurs via synapses, which form the connections to other neuronal cells. A typical neuron is built of a cell body (soma), dendrites, and an axon.
An "astrocyte" refers to a sub-type of glial cell in the central nervous system also known as astrocytic glial cell. Typically, astrocytes have a characteristic star-shaped appearance and represent the most abundant glial cells in the brain that are closely associated with neuronal synapses. Astrocytes have many structural and biochemical functions such as structuring of the brain, metabolic support of neurons with nutrients, maintenance of the blood brain barrier, transmitter uptake and release, regulation of ion concentration in the extracellular space, modulation of synaptic transmission, vasomodulation, promotion of the myelinating activity of oligodendrocytes, nervous system repair, and long-term potentiation. In one embodiment the neural cell is an astrocyte.
"Glial cells" as used herein also referred to as" neuroglia" or "glia" mean non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the brain, and for neurons in other parts of the nervous system such as in the autonomous nervous system. "Microglia" refers to a specific type of glial cell that are considered as the resident macrophages of the brain and spinal cord. Microglia thus act as the immune defense in the central nervous system (CNS). Microglia constitute 20% of the total glial cell population within the brain. Microglia and astrocytes are distributed in large non-overlapping regions throughout the brain and spinal cord.
"Neural stem cells (NSCs)" as used herein refers to self-renewing, multipotent cells that generate the main phenotypes of the nervous system. NSCs have an important role during development producing the enormous diversity of neurons, astrocytes and oligodendrocytes in the developing CNS. They also have important role in adult animals, for instance in learning and hippocampal plasticity in the adult mice in addition to supplying neurons to the olfactory bulb in mice. The skilled person knows various suitable means and methods for the isolation, separation, and cultivation of primary neural cells. One example of how cells can be isolated is described in the Examples section of the present application. It will be appreciated immediately by the person skilled in the art that neural cells can be advantageously and specifically separated from a pool of brain cells using automated cell sorting. For example, viable neural cells may be isolated and selected from a heterogeneous cell suspension by positive selection using cell- specific surface antigens or by negative selection by virtue of depletion of unwanted cells. Also envisaged are separation methods based on the use of antibodies or fragments thereof or any other suitable binding molecule e.g. aptamers as described herein which may be coupled to beads or a matrix for conducting cell separation. It is also conceivable that the selective isolation of a particular cell type may be advantageously conducted by e.g. using antibodies directed against a specific cell surface protein. Such primary antibodies may be monoclonal or polyclonal. These antibodies may also be unconjugated, biotinylated or conjugated to a fluorochrome. It is also conceivable that neural cells may be isolated and selected from a heterogeneous cell suspension through depletion of unwanted cells by treating the cells with a chemical compound that specifically kills the unwanted cell type. Alternatively, a chemical compound/culture medium may be applied that favors the survival of the neural cell type over the survival of the unwanted cell type.
Without being limited thereto, the present disclosure describes one way of providing an isolated neural cell such as a primary astrocyte, a neuron and a neuronal stem cell (NSC). As described in Example 1 cortical neurons are prepared from embryonic rat brains (Bayatti et al, 2003). The preparation of NSCs is described in Example 1. Preparation of astrocytic cultures have been described in the art (Franke et al., 1998). Typically, for cultivation and maintenance neural cells are kept in a suitable medium, e.g. B27-Neurobasal medium
(neurons) or N2-NEM/F12-medium (astrocytes). It is to be understood that each cell type may require a specific medium which offer the conditions for maintenance and growth of the cells. Suitable media are commercially available and have been described in the art. The skilled person would know how to select a medium suitable for maintenance and growth of a neural cell.
Also envisaged is the use of immortalized neural cells (cell lines). Immortalized neural cells may be generated from primary neural cells by transformation with a telomerase, the tumor antigene of the Simian Virus 40 and mutated Ras. Alternatively, immortalized neural cells may be isolated from tumor tissue. Moreover immortalized cells may be purchased at ATCC or ECACC. Each cell line may require a specific medium. The skilled person would know how to select a medium suitable for maintenance and growth of the cell line. It is to be understood that neural cells can be practically isolated from any higher eukaryotic organism where neural cells as defined herein are present. Without being limited thereto a neural cell may be isolated from a mammal such as a human, a primate, a horse, a dog, a cat, or a rodent such as mouse or rat, or a fish. Optionally, the organism is an animal model suitable for studying depression or behaviourism. Specific animal models for studying depression include rodents. In some embodiments, the neural cell is isolated from the group consisting of mouse, rat, and human. In further specific embodiments the neural cell is isolated from Mus musculus, Rattus norvegicus, or Homo sapiens. The term "compound" as used herein refers to a pure, macroscopically homogeneous substance such as a molecule polymer consisting of atoms or ions of two or more different elements in definite proportions that cannot be separated by physical means. Compounds include biological or chemical compounds. A "biological compound" comprises any compound already preexisting in nature such as biomolecules. A "biomolecule" may be any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as lipids, phospholipids, glyco lipids, sterols,
glyero lipids, vitamins, hormones, neurotransmitters, metabolites such as primary or secondary metabolites, or natural products.
A "chemical compound" as used herein refers to a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reaction. Chemical compounds are typically described by a unique and defined chemical structure and consist of a fixed ratio of atoms that are held together in a defined spatial arrangement by chemical bonds. Chemical compounds can be molecular compounds held together by covalent bonds, salts held together by ionic bonds, intermetallic compounds which are held together by metallic bonds, or complexes which are held together by coordinate covalent bonds. The chemical compound can be a preexisting anorganic or organic molecule or compound or any newly synthesized molecule or compound not previously described. The chemical compound may, for instance, be a "drug".
The term "drug" as used herein refers to a chemical or biological substance used in the treatment, cure, prevention, or diagnosis of disease or used to otherwise enhance physical or mental well-being. Drugs may be prescribed for a limited duration, or on a regular basis for chronic disorders.
The term "contacting the compound with a neural cell under conditions suitable for cellular uptake of the compound" means that the neural cell is isolated and cultivated before bringing the cell in contact with the compound as defined herein. It is to be understood that the therapeutic activity in the treatment of a stress-related disorder, e.g. an antidepressant activity and/or a therapeutic activity in the treatment of a disorder caused by the experience of a traumatic event of a compound as described herein on a neural cell may be measured in vitro, i.e. using one or more isolated neural cells separated from other unrelated cells.
In typical embodiments the isolated neural cells are kept in a medium suitable for
maintenance of the isolated cell. In other specific embodiments neural cells as described herein may for this purpose be isolated from tissues for ex vivo culture.
Also envisaged by the present invention is the isolation and culturing of glial neuronal and neural stem cells as described in Shivachar et al. (Topics in Tissue Engineering, Vol. 4. Eds. N Ashammakhi, R Reis, & F Chiellini, 2008). Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out may be available for culture. Such a method is also referred to as explant culture. Typically, such cells that are cultured directly from a subject are termed primary cells.
The neural cells as defined herein may be grown and maintained at an appropriate
temperature and gas mixture. Typically, the cells may be grown on conditions which resemble the natural habitat e.g. at 37°C, 5% C02 for mammalian cells. Also contemplated are slight variations from these conditions. The cell may be grown and kept in a cell incubator. Culture conditions may vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in e.g. pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from animal blood e.g. calf serum. Within the context of the present invention "conditions suitable for cellular uptake of the compound" include conditions under which the cells are usually cultivated and maintained. The medium for this purpose may comprise suitable salts, nutrients, and/or supplements such as vitamins in order to support growth and long-term viability of the cultured cells. Usually, the medium offers or mimics the conditions of their natural habitat of the neural cell so that cellular uptake of molecules or compounds in the surrounding medium is favored. Suitable media for culturing of neural cells are commercially available and described in the art. In typical embodiments, a suitable medium comprises a biological buffer to maintain a constant pH or other important physiological conditions e.g. a certain salt concentration. Suitable biological buffers are e.g. phosphate buffers, acetate buffer, HEPES, Tris, Bicine, TAPS, TAPSO, TES, MOPS, PIPES, Cacodylate, SSC, MES, or the like. The medium may be supplemented with a substance selected from the group consisting of sodium pyruvate, antibiotics, transferrin, hormones such as progesterone, selenite, putrescine, insulin and growth factors such as basic fibroblast growth factor or epidermal growth factor.
In specific embodiments it may be advantageous to use specific conditions, where cellular uptake of the compound as described herein above is accelerated or favored. For instance, such a substance added to the medium may increase the permeability of the cell for a compound. Alternatively, such a substance may decrease the cellular efflux of a compound by inhibiting efflux pumps, such as P-glycoprotein, monocarboxylate transporters (MCTs), multidrug resistance-associated proteins (MRPs), peptide transporters (PEPTs), and Na+ phosphate transporters (NPTs). There are several inhibitors of efflux pumps known, e.g. verapamil, cyclosporine A, quinidine (Nobili et al., 2006). In other embodiments the medium may comprise certain growth factors for enhancing growth of neural cell.
A "value indicative for the cellular level of histone methyltransferase G9a in the neural cell" refers to any cell parameter useful for quantitation of the G9a protein level in a given neural cell. It is to be understood that the value can be derived from the cellular status of G9a on the transcriptional, post-transcriptional, translational or posttranslational level. In one
embodiment of the present invention, the cellular level can be determined by measuring the expression level or protein level.
The term "histone methyltransferase (HMT)" as used herein refers to a histone modifying enzyme such as histone-lysine N-methyltransferase and histone-arginine N-methyltransferase, which catalyzes the transfer of one to three methyl groups from the cofactor S-Adenosyl methionine to lysine and arginine residues of histone proteins. Such a histone methylation may serve in epigenetic gene regulation. The term "histone" refers to the main protein component of chromatin. Histones form the unit around which DNA is coiled in the nucleosomes of eukaryotic chromosomes. Histone H3 is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Histones may be modified by posttranslational modifications such as e.g. lysine acetylation, lysine methylation, lysine ubiquitylation, arginine methylation or serine, threonine or tyrosine phosphorylation.
The term "histone methyltransferase G9a", also known as euchromatic histone-lysine N- methyltransferase 2 (EHMT2), RP23-349B4.3, Bat8, D17Ertd710e, KMT1C, or NG36, refers to a specific histone methyltransferase which has been reported to play a dominant role in euchromatic histone H3 lysine 9 (H3K9) methylation. It has been previously reported that G9a provokes transcriptional silencing by executing mono-, di- and trimethylation of euchromatic H3K9 as well as by promoting DNA methylation (Collins and Cheng, 2010).
The amino acid sequences and the nucleotide sequences of the histone methyltransferase G9a polypeptides as well as the splice isoforms thereof may be retrieved from any suitable public database such as e.g. the NCBI database (e.g.http://www.ncbi.nlm.nih.gov/pubmed/). The present invention exemplary provides the sequences of histone methyltransferase G9a from Homo sapiens, Mus musculus, and Rattus norvegicus. Described herein below are the the mRNA and amino acid sequences of the isoforms as well as the genomic DNA sequences.
It is however to be understood that the method according to the present invention is not restricted to determining a value indicative for the cellular level of histone methyltransferase G9a in a particular organism. The present invention contemplates measuring the level of histone methyltransferase G9a or orthologues thereof in practically any organisms where neural cells as defined herein are present.
In a preferred embodiment the histone methyltransferase G9a protein in Homo sapiens is encoded by the genomic nucleic acid sequence according to SEQ ID NO: 5 (Accession number: NT_167244.1). In a preferred embodiment histone methyltransferase G9a protein has the amino acid sequence according to SEQ ID NO: 2 (Accession number: NP 006700.3) and the corresponding histone methyltransferase G9a mRNA transcript has a sequence according to SEQ ID NO: 1 ( Accession number: NM 006709), which corresponds to the isoform a in Homo sapiens.
In a further preferred embodiment histone methyltransferase G9a protein has the amino acid sequence according to SEQ ID NO: 4 (Accession number: NP 079532.5) and the corresponding histone methyltransferase G9a mRNA transcript has a sequence according to SEQ ID NO: 3 (Accession number: NM 025256.5), which corresponds to the isoform b in Homos sapiens.
In a preferred embodiment the histone methyltransferase G9a protein in Mus musculus is encoded by the genomic nucleic acid sequence according to SEQ ID NO: 10 (Accession number: NC 000083.5).
In a further preferred embodiment histone methyltransferase G9a protein has the amino acid sequence according to SEQ ID NO: 7 (Accession number: NP 665829.1) and the corresponding histone methyltransferase G9a mRNA transcript has a sequence according to SEQ ID NO: 6 (Accession number: NM 145830.1), which corresponds to the isoform a in Mus musculus.
In a further preferred embodiment histone methyltransferase G9a protein has the amino acid sequence according to SEQ ID NO: 9 (Accession number: NP 671493.1) and the corresponding histone methyltransferase G9a mRNA transcript has a sequence according to SEQ ID NO: 8 (Accession number: NM 147151.1), which corresponds to the isoform b in Mus musculus.
In a further preferred embodiment the histone methyltransferase G9a protein in Rattus norvegicus is encoded by the genomic nucleic acid sequence according to SEQ ID NO: 13 (Accession number: NC 005119.2).
In a further preferred embodiment histone methyltransferase G9a protein has the amino acid sequence according to SEQ ID NO: 12 (Accession number: NP 997628.1) and the corresponding histone methyltransferase G9a mRNA transcript has a sequence according to SEQ ID NO: 11 (Accession number: NM 212463.1), which corresponds to an isoform Rattus norvegicus.
The histone methyltransferase G9a protein may also comprise or consist of an amino acid sequence being at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence according to SEQ ID NO: 2, 4, 7, 9, or 12 and the histone methyltransferase G9a mRNA transcript may comprise or consist of a sequences being at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence according to SEQ ID NO: 1. 3, 6, 8, or 11.
A "gene", "genetic unit" or a "nucleotide sequence" is a nucleic acid sequence which may be transcribed under certain physiological or biochemical conditions. The transcribed nucleic acid may further (but must not necessarily) be translated under certain physiological or biochemical conditions into a polypeptide, e.g. when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence may be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
The terms "protein" or "polypeptide" are used herein to designate a produced or naturally occurring polypeptide or a recombinant polypeptide corresponding to histone
methyltransferase G9a protein as shown by any of the amino acid sequences according to SEQ ID NO: 2, 4, 7, 9, or 12. The term "protein" according to the present invention is to be seen as being interchangeably with the term "polypeptide". The polypeptides or proteins may be encoded by any of the above mentioned nucleic acid molecules.
An "isoform" refers to any of several different forms of the same protein. Different forms of the same protein may be produced from related genes, or may arise from the same gene by alternative splicing. For example, the Homo sapiens isoforms a and b, which correspond to the amino acid sequences as depicted in SEQ ID NO: 2 and 4, respectively are products of alternative splicing of the genomic nucleic acid sequence as depicted in SEQ ID NO: 5. In this example, the alternative splicing generates the mRNA transcripts as shown in SEQ ID NO: 1 (isoform a) and 3 (isoform b), respectively. Isoforms may also be generated by single nucleotide polymorphisms (SNPs), which are small genetic differences between alleles of the same gene. SNPs occur at specific individual nucleotide positions within a gene. It is to be understood that such a quantitation can be based on a direct or indirect measurement. In some embodiments of the present invention, the cellular level of histone methyltransferase G9a is directly determined by measuring the amount of histone
methyltransferase G9a protein in the neural cell. It will be immediately appreciated by the skilled person that the protein level means a steady state within a given cell which may be seen as the result of the balance between expression of the protein and its stability or degradation. A compound as defined herein may act on the G9a protein level by various pathways. It has now been found that compounds having an antidepressive or antidepressive- like activity reduce the cellular level of histone methyltransferase G9a.
In some embodiments of the invention reduction of the cellular level comprises inhibiting histone methyltransferase G9a by directly binding to histone methyltransferase G9a protein. Such a binding may result in a reduced stability or enhanced degradation of the protein. In other embodiments of the invention reduction of the cellular level comprises decrease or inhibition of histone methyltransferase G9a protein expression. In this embodiment, the compound as described herein above thus does not act on the protein directly but rather on one or more cellular factors responsible for proper expression of G9a protein. In a yet further preferred embodiment of the invention reduction of the cellular level comprises enhancing degradation of histone methyltransferase G9a protein in the neural cell.
The skilled person knows the respective methods for determining the level of a certain protein within a cell. In some embodiments, the neural cell may be subjected to protein extraction and the amount of the specific protein is determined in the extract. In other embodiments it may be useful to use total cell extract for extraction. In specific embodiments protein level of G9a is determined in a nuclear extract.
Numerous methods for detecting a specific protein in a sample have been described in the art. The skilled person knows how to use these methods. Without being limited thereto suitable methods for measuring the level of a specific protein in a cell comprise are immunostaining techniques such as immunohisto chemistry, immunostaining of cells, flow cytometry, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), In-Cell Western, In-cell ELISA or immuno-electron microscopy. "Immunostaining techniques" as used herein describe methods, where a detection molecule is used to detect a specific protein or protein epitope. A suitable detecting molecule may be an antibody or an aptamer. In specific embodiment of the present invention the detecting molecule is an antibody or a fragment thereof. Optionally, the antibody as defined herein is a monoclonal or polyclonal antibody. In some embodiments the detecting molecule may also be selected from antibody variants or fragments such as e.g. single chain antibodies, diabodies, minibodies, single chain Fv fragments (sc(Fv)), sc(Fv)2 antibodies, Fab fragments or a F(ab')2 fragments. In one embodiment commercially available antibodies specific for histone methyltransferase G9a may be used. Examples of commercially available antibodies are described herein below in the Examples section.
Antibodies may be produced according to any suitable method known to the person skilled in the art. Polyclonal antibodies may e.g. be produced by immunization of animals with the antigen of choice, whereas monoclonal antibodies of defined specificity may e.g. be produced using the hybridoma technology developed by Kohler and Milstein (Kohler and Milstein, 1976, Eur. J. Immunol, 6:511-519).
In a one embodiment, a detecting molecule as described herein above may comprise a detectable label. Any suitable label, which can be attached to the detecting molecule may be used. In one preferred embodiment the detectable label is covalently or non-covalently attached to the detecting agent. Examples of labels that may be attached to the detecting agent include e.g. fluorescent dyes such as e.g. Cyanine dyes, e.g. Cyanine 3, Cyanine 5 or Cyanine 7, Alexa Fluor dyes, e.g. Alexa 594, Alexa 488 or Alexa 532, fluorescein family dyes, R- Phycoerythrin, Texas Red and rhodamine. Detecting molecules may also be labeled with enzymes such as e.g. horseradish peroxidase, alkaline phosphatase or beta-lactamase,
3 14 32 33 35 125
radioisotopes such as e.g. H, C, P, P, S or I or metal such as e.g. gold.
The term "aptamer" as used herein refers to a polynucleotide that has a specific binding affinity for a target compound or molecule of interest, e.g. a protein. Aptamers may e.g. be RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (reference may e.g. be made to Gold (1995), Ann. Rev. Biochem 64, 763-797). In another embodiment the detecting molecule may also be detected by a secondary detecting molecule comprising a label as described above. Such a secondary detecting molecule may be capable of specifically binding to the above described primary detecting molecule. In an examplary embodiment a secondary detecting molecule is an antibody.
The term "Western blot" also referred to as "immunoblot" is a commonly used analytical technique suitable for detecting specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide under denaturing conditions or by the 3-D structure of the protein under non- denaturing conditions. The proteins are then transferred to a membrane such as nitrocellulose or PVDF, where they are probed (detected) using antibodies specific to the target protein.
In specific embodiments the protein level of histone methyltransferase G9a in the neural cell is determined by Western blot analysis as described previously in Perisic et al, 2009. In particularly preferred embodiments the Western blot analysis is conducted using specific antibodies directed against G9a for detection. It is conceivable that the signal obtained from the western blot analysis may be quantified using suitable methods known in the art. The signal from the treatment with a compound as described herein may be compared with the signal resulting from the untreated neural cell (negative control) and/or with the signal of cells being treated with an antidepressant which is known to exhibit the desired antidepressant activity (positive control) and/or with a compound having a therapeutic activity in the treatment of other stress-related diseases, e.g. disorders caused by the experience of a traumatic event (positive control).
An exemplary measurement of the G9a protein level is shown in Figure 3. Without being bound to a theory, the inventors have surprisingly found that G9a protein level is significantly reduced in a neural cell that has been treated with the structurally unrelated antidepressants AMI or PAR. These results thus for the first time reveal the mode of action of such antidepressants as described herein above, which exert their antidepressive activity via the decrease of the histone methyltransferase G9a protein level.
Another envisaged technique using antibodies to detect proteins in tissues and cells is the detection via enzyme-linked immunosorbent assay (ELISA). Typically, in such a detection method a unknown amount of antigen is bound to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. The antibody is typically linked to an enzyme, and subsequently a substance containing the substrate for the linked enzyme is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Alternatively, the antibody may be coupled to a fluorophore that produces a fluorescent signal.
In other embodiments, an antibody against the target of interest is bound to a surface.
Subsequently, the cell lysate is incubated with the antibody and the proteins that do not bind to the antibody are removed through washing steps. A second antibody that recognizes an isotope different from the first antibody is applied. This antibody may be linked to an enzyme, alternatively a secondary antibody that is linked to an enzyme may be used. Subsequently a substance containing the substrate for the linked enzyme is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Alternatively, the antibody may be coupled to a fluorophore that produces a fluorescent signal. This method is known as "Sandwich ELISA". Alternatively, a competitive ELISA may be applied. In this method, the antigen is bound to a surface. An unlabeled antibody is incubated with the antigen. Subsequently, a labeled competitor antigen is incubated with the complex. The label may be an enzyme or a fluorophore (see above).
The term ,,Ιη-Cell Western" refers to an immunocytochemical assay typically performed in a microplate format. Cells are grown in a microplate and fixed. Target-specific primary antibodies and fluorescently-labeled secondary antibodies may be used for the detection of a specific protein. The amount of protein is quantified by measuring the fluorescent signal from each well. The methology has the advantage that accuracy is enhanced and data are more meaningful because the proteins can be detected and measured in their cellular context.
An ,,Ιη-Cell ELISA" relies on the same principle as the ,,Ιη-Cell Western". However, instead of using antibody that is coupled to a fluorophore an antibody that is coupled to an enzyme is used. A substance containing the substrate for the linked enzyme is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Other suitable methods envisaged by the present invention are mass spectrometry based methods for measuring the protein level such as stable isotope labeling in cell culture
(SILAC), liquid chromatography, mass spectrometry (LC-MS), or the like. The term "stable isotope labeling with amino acids in cell culture (SILAC)" as used herein describes a common application of stable isotope metabolic labeling, mostly using single cell organisms or mammalian cell in culture, thus limited to cell culture. Typically, the proteins are labeled in vivo by introduction of label in the growth medium. SILAC basically relies on metabolic incorporation of a given "light" (unlabeled) or "heavy" (labeled) form of the amino acid into the proteins. When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a certain number of cell divisions, each particular amino acid will be replaced by the isotopic labeled analog. Labeled and unlabeled cells are subsequently mixed, followed by the analysis of the proteome where the intensity of light and heavy variants are compared to obtain relative quantification.
Beside the direct determination of G9a protein level the present invention contemplates measuring the decrease of expression level of G9a protein or the increased degradation of the G9a protein level as a value indicative for the reduction of cellular level.
In specific embodiments of the present invention, the cellular level of histone
methyltransferase G9 is determined by measuring the expression level of G9a protein. It will be immediately appreciated by the skilled person that a reduced expression level may lead to a reduced level or amount of G9a protein. In a further embodiment of the invention reduction of the cellular level comprises decrease or inhibition of histone methyltransferase G9a protein expression. This is typically the case when the compound as described herein above specifically acts on components of the cellular expression machinery or specific transcription factors leading to a reduced or inhibited expression of G9a protein. For instance, such compounds may act on the promoter region of the G9a gene or on specific regulators thereof. The compound may act on stability of the transcript. The compound may also inhibit translation of the G9a mRNA transcript e.g. by blocking the translational machinery of the cell by inhibiting specific factors which play a role for translation of G9a protein such as factors required for translation initiation. It is also conceivable that the substance acts on the post-transcriptional level e.g. on the stability of the G9a mRNA transcript, which in turn leads to an enhanced degradation of the transcript while transcription level is not affected.
The term "expression level" as used herein shall mean the level or amount of G9a protein and/or mRNA. In some embodiments, the level of G9a pre-mRNA may be determined. The expression level of G9a protein may be determined by any suitable technique known in the art. Determining the amount of mRNA may e.g. be achieved by in situ hybridization, Northern blot analysis, RNAse protection assays and PCR-based methods, such as e.g. reverse transcription PCR (RT-PCR) and real time quantitative PCR. The skilled person will know how to perform these methods. In some embodiments total RNA may be isolated from the sample from the subject prior to determining the amount of mRNA.
In some embodiments the amount of G9a mRNA may be detected using a detecting molecule specific for G9a mRNA. For example, said detecting molecule is an oligonucleotide probe. In some embodiments, the oligonucleotide probe is a single stranded RNA molecule. Typically, the oligonucleotide probe is specific for G9a mRNA if it is capable of hybridizing to G9a mRNA under highly stringent conditions.
As used herein the term "hybridize" or "hybridizes" refers to the hybridization of a first to a second polynucleotide. To determine, whether two polynucleotides hybridize to each other, the skilled person may conduct hybridization experiments in vitro under moderate or stringent hybridization conditions. Hybridization assays and conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1-6.3.6, 1991. Stringent conditions may e.g. be conditions in which hybridization takes place in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
Cellular levels, in particular cellular levels of histone methyltransferase G9a, are deemed to be "reduced" or "down-regulated" if the cellular level of histone methyltransferase G9a decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more of a control level or if the cellular level of histone methyltransferase G9a to a level of histone methyltransferase G9a is decreased to a level of less then 10%>, less then 20%>, less then 30%>, less then 40%>, less then 50%, less then 60%, less then 70%, less then 80%, less then 90%, less then 95%, or less then 98% of the control level. A control level may be a cellular level of an untreated neural cell (e.g. a neural cell not treated or contacted with any exogenous compound) or a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of stress-related disorders or a neural cell treated with a control compound not having a therapeutic activity in the treatment of stress-related disorders (negative control). A control level may e.g. be a cellular level of an untreated neural cell (e.g. a neural cell not treated or contacted with any exogenous compound) or a neural cell not contacted or treated with a compound having an anti-depressant activity or a neural cell treated with a control compound not having an anti-depressive effect (negative control). A control level may also be a cellular level of a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event or a cellular level of a neural cell treated with a control compound not having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event.
Expression levels, in particular expression levels of histone methyltransferase G9a, are deemed to be "reduced" or "down-regulated" if the gene expression level of histone methyltransferase G9a decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more of a control level or if the expression level of histone methyltransferase G9a to a level of histone methyltransferase G9a is decreased to a level of less then 10%>, less then 20%, less then 30%, less then 40%, less then 50%, less then 60%, less then 70%, less then 80%), less then 90%>, less then 95%, or less then 98%> of the control level. A control level may be an expression level of an untreated neural cell (e.g. a neural cell not treated or contacted with any exogenous compound) or a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of stress-related disorders or a neural cell treated with a control compound not having a therapeutic activity in the treatment of stress-related disorders (negative control). A control level may e.g. be an expression level of an untreated neural cell (e.g. a neural cell not treated or contacted with any exogenous compound) or a neural cell not contacted or treated with a compound having an antidepressant activity or a neural cell treated with a control compound not having an anti- depressive effect (negative control). A control level may also be an expression level of a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event or an expression level of a neural cell treated with a control compound not having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event. mRNA levels, in particular mRNA levels of histone methyltransferase G9a, are deemed to be "reduced" or "down-regulated" if the mRNA level of mRNA coding for histone
methyltransferase G9a decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more of a control level or if the mRNA level of histone methyltransferase G9a to a level of mRNA coding for histone methyltransferase G9a is decreased to a level of less then 10%, less then 20%, less then 30%, less then 40%, less then 50%, less then 60%, less then 70%, less then 80%, less then 90%, less then 95%, or less then 98% of the control level. A control level may be an mRNA level of an untreated neural cell (e.g. a neural cell not treated or contacted with any exogenous compound) or a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of stress-related disorders or a neural cell treated with a control compound not having a therapeutic activity in the treatment of stress-related disorders (negative control). A control level may e.g. be an mRNA level of an untreated neural cell (e.g. a neural cell not treated or contacted with any exogenous compound) or a neural cell not contacted or treated with a compound having an antidepressant activity or a neural cell treated with a control compound not having an anti- depressive effect (negative control). A control level may also be a mRNA level of a neural cell not contacted or treated with a compound having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event or a mRNA level of a neural cell treated with a control compound not having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event.
In a further preferred embodiment of the present invention reduction of the cellular level of G9a protein level to less than 80%>, optionally less than 75%, 70%, or 65%, specifically less than 60%, 55%, or 50%, more specifically less than 45%, 40%, or 35%, even more specifically less than 30%, 25%, 20%, 15%, 10%, or 5% of a control level is indicative of a therapeutic activity in the treatment of stress-related disorders, e.g. of an antidepressant activity of the compound and/or of a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event. In a further preferred embodiment of the present invention reduction of the expression level of G9a protein to less than 80%, optionally less than 75%, 70%>, or 65%, specifically less than 60%), 55%), or 50%), more specifically less than 45%, 40%>, or 35%, even more specifically less than 30%>, 25%, 20%, 15%, 10%, or 5% of a control level is indicative of a therapeutic activity in the treatment of stress-related disorders, e.g. of an antidepressant activity of the compound and/or of a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event. In a particularly preferred embodiment of the present invention reduction of the G9a mRNA level to less than 80%, optionally less than 75%, 70%, or 65%, specifically less than 60%, 55%), or 50%), more specifically less than 45%, 40%, or 35%, even more specifically less than 30%), 25%), 20%), 15%), 10%), or 5% of a control level is indicative of a therapeutic activity in the treatment of stress-related disorders, e.g. of an antidepressant activity of the compound and/or of a therapeutic activity of the compound in the treatment of disorders caused by the experience of a traumatic event.
In another embodiment of the invention reduction of the cellular level comprises enhancing degradation of histone methyltransferase G9a protein in the neural cell. It is conceivable that the level of G9a protein is regulated by a compound as described herein on the
posttranslational level. In such a case the G9a protein is expressed normally but may be targeted for degradation or be instable thus leading to an increased degradation of the protein. For instance, proteins which may undergo increased protein degradation by the ubiquitine- proteasome pathway are previously marked for degradation by ubiquitylation. Another reason for increased degradation may be misfolding of the protein after protein expression. The skilled person knows means and methods to measure increased degradation leading to a reduced protein level of a protein.
The "antidepressant" or "antidepressant-like activity" can be measured by any means and methods described in the art. The "antidepressant" activity can be measured in vitro or in vivo.
As mentioned herein above, it has been reported previously that DNA methylation has a crucial role in the regulation of stress-related disorders such as depression and anxiety disorders including PTSD on the molecular level. Without being bound to a theory, a reduction of DNMT activity may result in slower or insufficient remethylation, and this may ultimately lead to altered gene expression. It has been reported that antidepressive-like behavior in rodents triggered by administration of DNMT inhibitors or antidepressants is accompanied by reduced methylation and increased expression of genes that are considered to play a crucial role in depression, including the neurotrophic factors GDNF and BDNF
(Uchida et al., 2011; Sales et al., 2011). Further support for a role of DNA methylation in depression comes from a recent study in bonnet macaques, where high levels of whole- genome 5-methlycytosine were demonstrated to be associated with increased behavioral stress-reactivity subsequent to early life stress, an experimental paradigm often used to investigate depression like effects (Kinnally et al., 2011). Hence, the pathogenesis of MDD and related disorders such as anxiety disorders seems to be accompanied by the establishment of aberrant methylation patterns which might contribute to consolidate the disease state.
In another embodiment of the invention the method of identifying a compound having an antidepressant activity further comprises the step of determining whether the compound is capable of decreasing the activity of a DNA methyltransferase. In this embodiment, the compound's capability of decreasing the activity of a DNA methyltransferase is also indicative of an anti-depressant activity of the compound.
In another embodiment of the invention the method of identifying a compound useful in the treatment of other stress-related disorders such as disorders caused by the experience of trauma further comprises the step of determining whether the compound is capable of decreasing the activity of a DNA methyltransferase. In this embodiment, the compound's capability of decreasing the activity of a DNA methyltransferase is also indicative of a therapeutic activity in the treatment of other stress-related disorders such as disorders caused by the experience of a trauma of the compound.
The term "DNA methyltransferase (DNMT)" as used herein refers to a family of enzymes that catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All DNA methyltransferases known in the art use S-adenosyl methionine (SAM) as the methyl group donor. In mammals, three active DNA methyltransferases have been identified. They are named DNMT1, DNMT3A, and DNMT3B. A fourth enzyme previously known as DNMT2 is in fact not a DNA methyltransferase. DNA methyltransferases work cooperatively in perpetuating and establishing DNA methylation, with DNMT1 being the major DNMT that executes the maintenance of DNA methylation during the S-phase of replication, and DNMT3a /3b being mainly implicated in de novo methylation (Cheng and Blumenthal, 2008).
In yet another embodiment of the method of identifying a compound having an antidepressant activity further comprises the step of determining whether the compound is capable of decreasing the activity of DNA methyltransferase DNMT1. In typical embodiments of the present invention, the activity of DNA methyltransferase DNMT1 is measured in vitro.
In another embodiment of the method of identifying a compound having a therapeutic activity in the treatment of other stress-related disorders such as disorders caused by the experience of a traumatic event further comprises the step of determining whether the compound is capable of decreasing the activity of DNA methyltransferase DNMT1. In typical embodiments of the present invention, the activity of DNA methyltransferase DNMT1 is measured in vitro.
In some embodiments of the present invention the activity of the DNA methyltransferase is determined by i) contacting a DNA substrate with a methyl group donor and a total or nuclear extract of said neural cell and ii) detecting DNA methylation at cytosine residues.
Within the meaning of the present invention "DNA substrate" refers to any single or double stranded DNA molecule which can be methylated by DNA methyltransferase as described herein. In a preferred embodiment of the present invention the DNA substrate is Poly(dI- dC)-Poly(dl-dC) or hemi/unmethylated DNA substrate.
It is to be understood that a methyl group is transferred from the methyl group donor to the DNA substrate via the enzymatic activity of a DNA methyltransferase as described herein above. For this purpose, any suitable methyl group donor may be used. For the detection of enzymatic transfer, the DNA substrate may be subjected to Bisulfite sequencing (Zhang et al, 2009) or mass spectrometry analysis. The methyl group donor may advantageously be labeled for tracing of the transferred methyl group and incorporation of the label in the DNA substrate. Suitable means and methods for the labeling of the methyl group donor are known to the skilled person. In particular preferred embodiments, the label is a fluorophore or a radionucleotide (isotopic tracing). Suitable radionuclides are described in the art. Envisaged by the present invention is the use of a stable isotope. Suitable stable isotopes for the isotopic labeling of bio molecules are e.g. 13C, 15N, or 3H (tritium). In preferred embodiments, the methyl group donor is labeled with tritium. In further specific embodiments of the present invention the methyl group donor is S- adenosyl methionine (SAM). In specific embodiments of the present invention, S-adenosyl- methionine (SAM) that is labeled with tritium and is thus provided as S-adenosyl-L-[methyl- 3H]methionine (3H-SAM). The skilled person knows the various means and methods that may be advantageously used for tracing of a label. Examples are, for instance, any nuclear magnetic resonance based or mass spectrometry based methods described in the art. In specific embodiments the incorporation of a isotope label e.g tritium is detected by virtue of scintillation counting. The term "scintillation counter" as used herein refers to measurement of ionizing radiation. The sensor also termed scintillator consists of a transparent signal, usually phosphor, plastic or organic liquid that fluoresces when hit by ionizing radiation. Suitable scintillation counting methods are e.g. Gamma spectroscopy, Geiger counter, liquid scintillation counting, or Lucas cell.
Measurement of DNMT activity may be conducted using the DNA methyltransferase assay as described herein in the Examples section. DNMT activity may be determined by incubating total or nuclear extracts as defined herein in a buffer. In specific embodiments of the present invention the assay buffer may thus comprise a DNA substrate and a radionuclide label such as tritium.ln the Examples of the present invention, SAM is labeled with tritium and provided as S-adenosyl-L-[methyl-3H]methionine (3H-SAM). After incubation for a certain time period the DNMT transferase activity is determined by purifying the DNA substrate as used herein and measuring the incorporation of tritium. Typically, the incorporation of of the nuclides e.g. tritium is measured by scintillation counting as described herein above and in the Examples section.
Methods for purification of the (Poly(dl-dC)-Poly(dl-dC) or hemi/unmethylated DNA substrate are known in the art. For instance, such a purification may may involve the use of commercially available purification kits, e.g. GeneClean kit (MP Biomedicals). Alternatively, proteins may be precipitated and DNA may be separated by absorption to special membranes such as ion exchange papers (e.g. DE81 paper (Whatman)). The preparation of double stranded hemimethylated DNA substrate may occur via annealing of a single-stranded, unmethylated oligonucleotide with a complementary, methylated oligonucleotide. Unmethylated DNA substrate may be prepared accordingly (by annealing two unmethylated oligonucleotides). Alternatively, unmethylated DNA (e.g. plasmid DNA) may be isolated from an organism which lacks an enzyme capable of catalyzing the transfer of a methyl-group to cytosins leading to the generation of 5-methylcytosin. In order to be able to purify hemi/unmethylated oligonucleotides, oligonucleotides may contain a tag, e.g. a biotin-tag. The skilled person knows how to purify DNA that contains a distinct tag. Purification of biotinylated DNA may be achieved by using streptavidin coated beads, e.g. streptavidin coated dynabeads. It will be immediately appreciated by the skilled person that also any other DNA extraction or purification protocol can be used to purify the DNA substrate within the context of the present invention.
In a further embodiment of the present invention the decrease of DNA methyltransferase activity is at least about 10%, 15%, 20%, 25%, or 30%, optionally of at least about 35%, 40%, 45%), or 50%), specifically of at least about 55%, 60%>, 65%, or 70%>, more specifically of at least 75%), 80%>, 85%, or 90%>, and even more specifically of at least about 95%.
In another embodiment of the invention the method of identifying a compound having an antidepressant activity and/or a compound having a therapeutic activity in the treatment of other stress-related disorders such as disorders caused by the experience of a traumatic event further comprises the step of measuring whether the decrease of the activity of the DNA methyltransferase can be restored by the addition of purified histone methyltransferase G9a protein. Such restoration of DNMT activity by the addition of histone methyltransferase G9a protein is a crucial test, which allows the conclusion that the absence of G9a is indeed responsible for decrease of DNMT activity. "Purified" within the context of the present invention means a purity of a at least 50%, 55%, 60%, 65%, optionally at least 70%, 75%, 80%, or 85%, specifically of at least 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, or 99%). The skilled person knows the various means and methods for purifying a specific protein to a certain degree.
In specific embodiments the antidepressant activity and/or the therapeutic activity in the treatment of other stress-related disorders such as disorders caused by the experience of a traumatic event of a compound as described herein may be measured in vivo. To test whether a given compound may confer an antidepressant or antidepressant-like effect or not or whether a given compound has a therapeutic activity in the treatment of other stress-related disorders such as disorders caused by the experience of a traumatic event, the compound may be administered into an animal model e.g. mouse or rat model. In particularly preferred embodiments the behavior of the mouse is observed in a "tail suspension test", a "forced swimming test", a "learned helplessness test", a "sucrose preference test", a test for "marble- burying behaviour" or a "social interaction test" (Cryan et al, 2002; Dekeyne, 2005).
The term "tail suspension test (TST)" as used herein refers to a test method originally proposed by Steru et al (1985) as a primary screening test of anti-depressant drugs (Steru, L. et al. The tail suspension test: a new method for screening antidepressants in mice.
Psychopharmacology (Berl), 1985;85(3):367-70). In such a test, it is shown that tail suspension-induced immobility of mice is specifically antagonized by such drugs. The mouse is individually suspended to the shelf by the tail with an adhesive tape for 6 min with the head 80 cm to the floor. The test is typically carried out in a darkened room with minimal background noise. Mice are considered as being immobile when they are passive and completely motionless. The duration of immobility is recorded during the final of 4 min of the test.
In some embodiments, the antidepressant activity and/or the therapeutic activity of a compound in the treatment of other stress-related disorders such as disorders caused by the experience of a traumatic event of a compound as described herein is measured in vivo using the forced swimming test. The term "forced swimming test (FST)" as used herein refers to a method that estimates the behavioral despair in stressful and inescapable situations (for a review see: Petit-Demouliere et al, Forced swimming test in mice: a review of antidepressant activity. 2005 Jan;177(3):245-55). In FST, the mouse is placed in a glass cylinder that is filled with water. The time of immobility, swimming and struggling is recorded.
Anxiety occurs frequently as a comorbid disorder with depression. Moreover, the compound as described herein may be used for the treatment of anxiety disorders including disorders caused by the experience of a traumatic event, e.g. posttraumatic stress disorder. In specific embodiments the anxiolytic activity of a compound as described herein may be measured in vivo. In specific embodiments the behavior of the mouse is observed in an "elevated plus maze test", a "light-dark transition test", an "open field test", and/or a "novelty induced hypophagia test" (Imaizumi et al, 1994; Hunsberger and Duman, 2007). As used herein "therapeutic activity in the treatment of stress-related disorders" relates to any activity of the compound which alleviates and/or prevents any symptom related to stress- related disorders. Symptoms of stress-related disorders may be cognitive, emotional, physical, or behavioral. Cognitive symptoms may include memory problems, inability to concentrate, poor judgment, pessimistic approach or thoughts, anxious or racing thoughts, and constant worrying. Emotional symptoms may include moodiness, irritability or short temper, agitation, inability to relax, feeling overwhelmed, sense of loneliness and isolation, depression and general unhappiness. Physical symptoms may include aches and pains, diarrhea or constipation, increased frequency of urination, indigestion, low blood sugar, nausea, dizziness, chest pain, rapid heartbeat, loss of sex drive, frequent colds, and irregular periods. Behavioral symptoms may include eating more or less, sleeping too much or too little, isolating oneself from others, procrastinating or neglecting responsibilities, using alcohol, cigarettes, or drugs to relax, nervous habits (e.g. nail biting, pacing), In particular, an alleviation and/or prevention of the symptoms related to stress-related disorders as described above is achieved by compounds having a therapeutic activity in the treatment of stress- related disorders.
As used herein "therapeutic activity in the treatment of disorders caused by the experience of a traumatic event" relates to any activity of the compound which alleviates and/or prevents any symptom related to disorders caused by the experience of a traumatic event. In particular, an alleviation and/or prevention of the symptoms selected from the group consisting of excessive alertness, exhaustion, sleeping disorders, poor concentration and/or memory, social withdrawal, loss of interest in normal activities, acute stress reaction, fear, depression, anxiety and/or panic is achieved by compounds having a therapeutic activity in the treatment of disorders caused by the experience of a traumatic event.
In one embodiment, the therapeutic activity of the compound leads to an alleviation and/or reduction of symptoms associated with PTSD. Examplary symptoms of PTSD include any of the above mentioned symptoms such as re-experiencing symptoms, avoidance symptoms and/or hyperarousal symptoms.
It is to be understood that the definitions and preferred embodiments as set out for first aspect of the invention also apply to the second and further aspects of the present invention. In a second aspect, the present invention describes a method of identifying a compound having an anti-depressant activity and/or a therapeutic activity in the treatment of other stress- related disorders, e.g. disorders caused by the experience of a traumatic event, comprising the steps of contacting said compound with a neural cell under conditions suitable for cellular uptake of the chemical compound, and determining a value indicative for the loss of interaction of the histone methyltransferase G9a with DNA methyltransferase DNMT1, wherein said loss of interaction is indicative of an antidepressant activity and/or a therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event of the compound.
It will be immediately appreciated by the person skilled in the art that the histone
methyltransferase G9a is a regulator of DNA methyltransferase DNMT1. Within the context of the present invention, "interaction" can mean any form of association between G9a and DNMT1. Such an interaction may be a direct or indirect binding. The two proteins may interact for a period of time sufficient to form a protein complex. Also envisaged is that G9a may interact with DNMT1 only briefly so as to carry out a modification of DNMT1 protein. For this interaction the proteins typically require a specific binding affinity for each other. The KD is usually considered to be a measure for the affinity of an interaction between two molecules. Affinity may describe the strength of binding of a molecule to another molecule at a single site. "Indirect binding" means a binding that is mediated by a further molecule e.g. a protein or peptide. "Loss of interaction" thus means an interruption of any binding between the two interacting proteins. The skilled person is aware of the various means and methods for measuring the loss of interaction of two proteins that have been described in the art.
In a further embodiment of the invention determining the loss of interaction comprises a method selected from the group consisting of co-immunoprecipitation, pulldown-assay, fluorescence resonance energy transfer (FRET), protein complementation assay, enzyme complementation assay, yeast two hybrid, mammalian two hybrid, fluorescent polarization, and surface plasmon resonance.
The terms "pulldown assay" or "co-immunoprecipitation (Co-IP)" as used herein generally refers to the immunoprecipitation of intact protein complexes. Typically, a specific protein of interest is precipitated together with its interacting protein along with any other proteins or ligands that are bound to it. By targeting the specific protein with an antibody or aptamer as described herein it is possible to "pull" the entire protein complex out of the solution and thereby identify the interacting partners or proteins of the complex. This concept of pulling protein complexes out of solution is also referred to as a "pull-down".
Another envisaged technique for measuring the interaction of two proteins is fluorescence resonance energy transfer (FRET). The term "fluorescence resonance energy transfer
(FRET)" as used herein refers to a methodology for studying protein-protein interaction and basically relies on the distance-dependent transfer of energy from a donor molecule to an acceptor molecule. Typically, the interacting proteins are coupled to a fluorophore e.g. a fluorescent protein. The adsorption and emission spectra are chosen in a way that energy from the excited donor fluorophore is transferred to the acceptor fluorophore in close proximity. Excitation of the donor will produce sensitized emission from the acceptor. The emission of the acceptor fluorophore can be measured and indicates that two molecules are in close proximity (less than 10 nm) which points to an interaction of the two molecules. The skilled person would know how to select suitable donor/acceptor pairs in order to conduct a FRET experiment as described herein.
Examples of suitable fluorophores are the genetically encoded fluorescent dyes, such as Green Fluorescent Protein (GFP) and related molecules such as blue (BFP), cyan (CFP), yellow (YFP) and red (RFP). These dyes have been reported to be suitable for FRET in vitro and particularly in living cells (Tsien, R. (1998) Ann. Review Biochem, 67:509-544). Typically, these proteins may form FRET pairs with each other as well as with other conventional dyes. For instance, they can be attached to other proteins genetically or covalently while retaining their ability to function as a fluorophore. Suitable dyes have the utility of being genetic elements that can be linked with other genes to form chimeric proteins. Such chimeric proteins may contain a GFP (or related fluorescent protein element) and a putative binding domain. Protein-protein interactions may be investigated using different chimeric proteins (one donor and one acceptor). FRET can be observed when the donor/acceptor pairs interact through protein-protein interactions. Other suitable dyes are organic cyanine dyes such as Cy3, Cy5, Cy5.5 and Cy7, which emit in the red range (>550 nm). The present invention envisages the measurement of FRET signals in vitro and in vivo.
A further method suitable for studying protein-protein interaction is termed "Protein
Complementation Assays", which refers to a technique based on the division of a monomeric reporter protein into two separate, inactive components. For example, the green fluorescent protein (GFP) may be divided into two non-functional, e.g. non fluorescent, components, each of which may be coupled to the interacting proteins. The two components may be structurally reconstituted and thus become active when they are in close proximity indicating a protein- protein interaction. The reporter protein may be also an enzyme e.g., Luciferase, B- Galactosidase, dihydro folate reductase, B-lactamase or a fluorescent protein such as GFP, BFP, CFP, YFP, or RFP, or the like.
A further method suitable for measuring the interaction two protein is known as "Two hybrid screening". Examples for two hybrid systems are "Yeast Two Hybrid" or "Mammalian Two Hybrid". Two-hybrid screening is a molecular biology technique used to determining protein- protein interactions and protein-DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively. The underlying principle is similar to the "Protein Complementation Assay" as defined herein above. Typically, such a test is based on the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). Commonly a transcription factor such as GAL4 is split into two separate fragments, called the binding domain (BD) and activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription. If the bait and prey proteins interact e.g. by binding then the AD and BD of the transcription factor are indirectly connected, bringing the AD in proximity to the transcription start site and transcription of reporter gene(s) may occur. If the two proteins do not interact, there is no transcription of the reporter gene. In this way, a successful interaction between the fused protein is linked to a change in the cell phenotype. A further method envisaged by the present invention is known as "Fluorescent polarization". The term "Fluorescent polarization" as used herein refers to a technique applied to study molecular interactions. The method measures whether a tracer molecule e.g. fluorophore is in a bound or free state (bound/free ratio). The difference can be explained by the observation that small molecules rotate quickly during the excited state, and upon emission, have low polarization values, whereas large molecules, caused by binding of a second molecule, rotate little during the excited state, and therefore have high polarization values. One envisaged example of how to use this methodology within the context of the present invention is to fluorescently label a short, truncated version of DNMT1/G9a protein that contains the interaction domain and incubate this peptide together with G9a/DNMT1 protein. In specific embodiments of the present invention only peptides corresponding to the interacting domains may be used and couple to a fluorophore. The skilled person knows how to determine the interacting domains of the DNMT1-G9a couple. Examples for interacting domains in the histone methyl transferase G9a are the amino acid sequences as depicted in SEQ ID NO: 14, 16, 18, and the corresponding sequences of the G9a mRNA transcript as depicted in SEQ ID NO: 15, 17, or 19.
In one embodiment the interacting domain of Homo sapiens G9a protein may comprise or consist of the sequence as depicted in SEQ ID NO: 14 (amino acid 1-300) which is encoded by the corresponding sequence of the mRNA transcript as depicted in SEQ ID NO: 15 (nt 11- 910).
In another embodiment the interacting domain of Mus Musculus G9a protein may comprise or consist of the sequence as depicted in SEQ ID NO: 16 (amino acids 1-300) which is encoded by the corresponding sequence of the mRNA transcript as depicted in SEQ ID NO: 17 (nt 1- 900).
In another embodiment the interacting domain of Rattus norvegicus G9a protein may comprise or consist of the sequence as depicted in SEQ ID NO: 18 (amino acids 1-300) which is encoded by the corresponding sequence of the mRNA transcript as depicted in SEQ ID NO: 19 (nt 1-900).
The interacting domain of histone methyltransferase G9a protein may also comprise or consist of an amino acid sequence being at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence according to SEQ ID NO: 14, 16, or 18 and the histone methyltransferase G9a mRNA transcript may comprise or consist of a sequences being at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence according to SEQ ID NO: 15, 17, or 19. When the fluorescently labeled molecule, which can be either a peptide of DNMT1 or G9a, is bound to the interacting molecule, the rotational movement in solution decreases thus leading to an enhanced emission of the polarized light. The binding of such fluorescently labelled peptide may thus be measured by a change in polarized light. The present invention also contemplates the use of "Surface Plasmon Resonance". "Surface Plasmon Resonance" as used herein refers to a method based on the excitation of surface plasmons. Typically, surface plasmons can be generated in conducting materials by polarized light. The plasmons reduce the intensity of the reflected light. The resonance angle at which this reduction of intensity occurs depends on the refractive index of the solution close to the conducting material. The molecules of interest, e.g. DNMT1 or G9a protein, may be immobilized on a sensor surface. The refractive index at the interface between the surface and a solution flowing over the surface changes when the molecules on the surface and molecules in the solution interact. Therefore the angle at which reduced- intensity polarized light is reflected alters. The measurement of this angle can thus be used to determine protein interaction.
It is to be understood that any other method suitable for determining the loss of interaction of two interacting proteins may be advantageously used for measuring whether a compound is capable of interfering with the interaction of histone methyltransferase G9a with DNA methyltransferase DNMTlin neural cells.
In a third aspect, the present invention relates to a method of providing a compound having antidepressant activity and/or therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event comprising the steps of conducting the method of identifying a compound having antidepressant activity and/or having a therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event as described herein above; and synthesizing the compound identified by this method.
By conducting the method of identifying a compound having antidepressant activity or a therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event, as described herein, screening for compounds may be performed, which are not previously reported to have antidepressant activity or a therapeutic activity in the treatment of other stress-related disorders. In particular, the compound screened by the method according to the present invention is capable of reducing the level of histone methyltransferase G9a protein so as to result in an antidepressive or antidepressive like effect and/or in a therapeutic activity in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event. It will be immediately appreciated by the skilled artisan that such compounds are bona fide compounds for use in the treatment of depression and/or in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event. It is to be understood that once a candidate compound has been screened and verified by the tests as described herein above, the skilled person would know how to synthesize the compound as defined herein. Synthesis of anorganic and organic molecules are within the routine of the skilled person.
In a further aspect, the present invention describes an antibody or a binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein for use as a medicament.
In one embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of stress- related disorders.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of depression.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of disorders caused by the experience of a traumatic event.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of anxiety disorders.
In another embodiment, the antibody or the binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein is for use in the treatment of PTSD. In one embodiment, such an antibody may specifically bind to a protein or polypeptide having or comprising the amino acid sequence as shown in SEQ ID NO: 2, 4, 7, 9, or 12 or any derivative, fragment etc. thereof. Such antibodies are contemplated for any application, use, method, composition, immunoassay, screening method and pharmaceutical compositions as defined in the present application. Usually, such antibodies are bona fide compounds for use in the treatment of depression.
The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e. g., IgG, IgE, IgM, IgD, IgA and IgY), class (e. g., IgGl, IgG2, IgG3, lgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. Preferred epitopes according to the present invention are amino acids 1-10, 11-20, 21-30, 31- 40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141- 150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210 etc. or any other specific stretch of amino acids of a protein of histone methyltransferase G9a as described herein above.
Further envisaged are all other suitable epitopes, which can be recognized, determined, described and subsequently be employed according to methods known to the person skilled in the art.
The term "capable of specifically binding to the histone methyltransferase G9a" as used herein refers to the immunospecific binding of an antibody to an antigenic epitope as defined herein above. The term "specifically binding" excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens, in particular with antigens comprising the same antigenic epitope detected by the present antibody. In a one embodiment, antibodies of the invention include polyclonal, monoclonal, multispecific, human, humanized, murine, xenogeneic or chimeric antibodies, single chain antibodies, Fab fragments, Fab' fragments, fragments produced by a Fab expression library, F(ab')2, Fv, disulfide linked Fv, minibodies, diabodies, scFv, sc(Fv)2, whole immunoglobulin molecules, small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins, camelized antibodies, VHH containing antibodies, anti-idiotypic (anti-Id) antibodies (including, e. g., anti-Id antibodies to antibodies of the invention) and epitope- binding fragments of any of the above. The antibodies according to the invention may be from any animal origin including birds and mammals. Optionally, the antibodies are human, murine (e. g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig, camel, horse, or chicken. The antibodies according to the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
Antibodies of the present invention may also be described or specified in terms of their cross- reactivity. In a particularly preferred embodiment the present invention relates to antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention. However, also antibodies that bind polypeptides with at least 95%, at least 90%>, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are included in the present invention.
In a further preferred embodiment of the invention the antibody comprises a human, humanized, murine, xenogeneic or chimeric human-murine antibody or binding fragment thereof.
Antibodies or binding fragments thereof, capable of specifically binding to the histone methyltransferase G9a may have an equilibrium dissociation constant (KD) for the binding of the antibody (or the binding fragment thereof) to its antigen in the low nanomolar to low picomolar or even in the subpicomolar range (avidity). Thus the KD may be in the range of about 0.1 * 10~12 to about 1 * 10~8, optionally in the range of about 0.1 * 10~12 to about 0.1 * 10"7, specifically in the range of about 0.1 * 10~12 to about 10* 10"9, more specifically in the range of about 0.1 * 10~12 to about 1 * 10~9. The most specific KDs may be in the range of about 0.1 * 10~12 to about 0.1 * 10~9, in the range of about 0.1 * 10~12 to about 10* 10~12 or in the range of about 0.1 * 10~12 to about 1 * 10~12 such as about 0.9* 10~12, about 0.8* 10"12, about 0.7* 10"12, about 0.6* 10"12 or about 0.5* 10"12. Thus G9a binding antibodies or binding fragments thereof as described hereinafter may have a KD of about 300 pM or less, about 200 pM or less, about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less about 20 pM or less.
The KD is usually considered to be a measure for the affinity of an interaction between two molecules. Usually, affinity describes the strength of binding of a molecule to another molecule at a single site. However, an antibody usually has two binding sites for an antigen. The strength of this interaction is usually considered to be the avidity.
In the context of the present invention, the term "affinity" is used to describe both the strength of the interaction of e.g. a monovalent scFv to its antigen as well as the binding of a typical divalent antibody to its antigen.
KD values and thus the affinity/avidity of the antibodies or binding fragments thereof can be determined making use of established approaches in the art.
Another measure of the affinity of an antibody such as the G9a binding antibodies or binding fragments described herein towards their antigen is the EC50 concentration. The antibodies or binding fragments thereof as described herein may have an EC50 for the binding of the antibody (or the binding fragment thereof) to its antigen in the low nanomolar to low picomolar or even in the subpicomolar range. Thus, the EC50 may be in the range of about 0.1 * 10~12 to about 1 * 10~8, optionally in the range of about 0.1 * 10~12 to about 0.1 * 10"7, specifically in the range of about 0.1 * 10~12 to about 10* 10"9, even more specifically in the range of about 0.1 * 10~12 to about 1 * 10~9. The most specific EC50S may be in the range of about 0.1 * 10~12 to about 0.1 * 10~9, in the range of about 0.1 * 10~12 to about 10* 10~12 or in the range of about 0.1 * 10~12 to about 1 * 10~12 such as about 0.9* 10~12, about 0.8* 10~12, about 0.7* 10~12, about 0.6* 10"12 or about 0.5* 10"12. Thus G9a binding antibodies or binding fragments thereof as described hereinafter may have an EC50 of about 300 pM or less, about 200 pM or less, about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less about 20 pM or less. Even lower ECso's may be achievable by optimization of CDRs.
The EC50 is determined as the concentration at which half-maximal binding of the antibody to its antigen in ELISA was observed. In a further embodiment of the invention, the antibody is a monoclonal antibody.
The antibodies and binding fragments thereof as they are used in the context of the present invention may be monoclonal chimeric, humanized or human antibodies. In some
embodiments, these antibodies are of the IgG class.
In a further embodiment the antibodies of the invention include derivatives which are modified, for instance by the covalent attachment of any type of molecule to the antibody such that said covalent attachment does not prevent the antibody from specifically binding to the epitope or from generating an anti-idiotypic response. Typical examples of such modifications are glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Chemical modifications may be carried out by known techniques, including specific chemical cleavage, acetylation, formylation etc. Additionally, the derivative may contain one or more non-classical amino acids.
Antibodies may be produced according to any suitable method known to the person skilled in the art. Monoclonal antibodies of defined specificity may be produced using, for instance, the hybridoma technology developed by Kohler and Milstein (Kohler and Milstein, 1976, Eur. J. Immunol, 6: 511-519).
In a further embodiment of the present invention the antibody or fragment thereof as defined herein above may be biotinylated or labeled. In a particularly preferred embodiment said label is a radioactive label, an enzymatic label, a fluorescent label, a chemiluminescent or a bio luminescent label. Alternatively, antibodies may also be labeled or combined with fluorescent polypeptides, e.g. green fluorescent protein (GFP) as well as derivates thereof known to the person skilled in the art.
Alternatively, a polynucleotide encoding an antibody may be generated from a nucleic acid from a suitable source.
In a further embodiment of the present invention a nucleic acid molecule encoding the antibody or fragment thereof as defined herein above may be used for recombinant antibody expression. Optionally, such expression vectors contain the antibody coding sequences and appropriate transcriptional and trans lational control signals. The vectors may either comprise coding sequences for the variable heavy chain or the variable light chain or for both. Such vectors may also include the nucleotide sequence encoding the constant regions of the antibody molecule. In oneembodiment of the present invention mammalian cells, optionally Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus may be used as an effective expression system for antibodies.
In some embodiments, the antibodies according to the present invention may be delivered across the blood brain barrier by methods known to the skilled person. For instance, antibodies may be delivered into the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. The antibodies according to the present invention may thus enter the neural cell by receptor mediated delivery (RMD) technology as described in Tezel et al, 2000. In other embodiment, the antibodies may be delivered by conjugation to cell penetrating peptides as described in (Kerkis et al, 2006). In some embodiments of the present the antibody may be expressed in the neural cell using suitable vectors as described herein above. Suitable transfection techniques and vectors have been described previously (Karra and Dahm (2010). The Journal of Neuroscience, 30(18):6171-6177). Without being limited thereto suitable vectors include viral vectors e.g. Adenoviruses, Adeno-associated viruses, Lentiviral vectors, or Herpes Simplex viruses. In preferred embodiments such vectors may be brought into the neural cell by injection. In other specific embodiments the antibody according to the present invention may be directly injected into the CNS.
In a further aspect, the present invention describes an inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein comprising
a) an antisense oligonucleotide specific for histone methyltransferase G9a, or b) a small interfering RNA specific for histone methyltransferase G9a, or
c) a microRNA specific for histone methyltransferase G9a
for use as a medicament, in particular for use in the treatment of depression.
In one embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of stress- related disorders. In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of depression.
In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of disorders caused by the experience of a traumatic event. In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of anxiety disorders.
In another embodiment, the inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein is for use in the treatment of PTSD.
The terms "antisense oligonucleotide specific for histone methyltransferase G9a" or
"antisense oligonucleotide specific for histone methyltransferase G9a expression product" refer to nucleic acids corresponding to complementary strand of the G9a mR A. The antisense oligonucleotide of the invention may thus comprise a sequence complementary to at least a portion of the histone methyltransferase G9a expression product according to the present invention. While antisense oligonucleotides complementary to the coding region sequence of histone methyltransferase G9a expression products may be used, those complementary to the transcribed and untranslated region are preferred. Generally, antisense technology can be used to control, i.e. reduce or abolish gene expression through antisense DNA or R A, or through triple-helix formation. In one embodiment, an antisense molecule may be generated internally by the organism, for example intracellularly by transcription from an exogenous sequence. A vector or a portion thereof may be transcribed, producing an antisense nucleic acid of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense molecule. Corresponding vectors can be constructed by recombinant DNA technology by methods known to the person skilled in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells, e.g. vectors as defined herein above.
In another embodiment, the antisense oligonucleotides may be administered separately. For an example, the 5' coding portion of the G9a nucleic acid according to the present invention, e.g. of the sequence as indicated in SEQ ID NO: 1, 3, 6, 8, or 11 may be used to design an antisense RNA or DNA oligonucleotide of from about 6 to 50 nucleotides in length.
Optionally, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides in length.
The antisense oligonucleotides of the invention typically comprise a sequence complementary to at least a portion of the mRNA transcript of G9a. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA transcript" as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex or triplex formation in the case of double stranded antisense nucleic acids. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex or triplex. A person skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
Optionally antisense oligonucleotides complementary to the 5' end of the transcript, e.g., the 5' untranslated sequence up to and including the AUG initiation codon may be used for the inhibition of translation. In a further embodiment, sequences complementary to the 3' untranslated sequences of mRNAs may also be used.
An antisense oligonucleotide according to the present invention may be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. Such antisense oligonucleotides may be modified. For example, stability of the antisense oligonucleotide may be increased by modification with a phosphorothioate backbone. Such a phosphorothioate modification may also increase the uptake of the antisense oligonucleotide as described herein over cell membranes. In specific embodiments, the antisense oligonucleotide according to the present invention may bind to the mR A transcript which results in the activation of RNase H-dependent pathway. The RNA: RNA or RNA: DNA hybrid may then be cleaved by RNase H thereby preventing translation of the mRNA transcript.
In some embodiments, the antisense oligonucleotide according to the present invention may target the translation start site for preventing translation and elongation. In other
embodiments, the antisense oligonucleotides may bind to the AUG intitation site or downstream in order to prevent binding of the ribosomal units or to form a steric blockage. Such antisense oligonucleotides may be modified to render them RNAse H-resistant by addition of a methyl or methoxy-ethyl group to the 2Ό sugar ribose, which is the target cleavage site of the RNase H enzyme.
In other embodiments of the present invention, the antisense oligonucleotide may act on alternative splicing as described herein above. For example 2ΌΗ modified RNase H-resistant or alternatively modified antisense oligonucleotides complementary to the target pre-mRNA may be used. Such an approach may e.g. result in inclusion of an exon by binding to the exonic splicing silencers (ESEs) or intronic splicing silencers (ISSs). Another effect may be the exclusion of an exon by binding to the 3 ' or 5 ' splice sites or exon-internal sequences so as to result in an in- frame transcript and translation of a shorter partly functional protein. This form of "exon skipping" may thus result in the expression of a truncated and/or nonfunctional or partially non-funtional histone methyltransferase G9a protein leading to a more differentiated effect as compared to the complete down-regulation of the G9a protein. For instance, it could be advantageous to maintain the enzymatic activity of histone
methyltransferase G9a protein, namely histone methylation, while inhibiting its interaction with DNMT1 as described herein above.
In other specific embodiments the antisense oligonucleotides as described herein may be modified by using e.g. phosphorodiamidate morpholino oligomers (PMOs), peptide nucleic acids (PNA) or locked nucleic acids (LNA).
It is to be understood that an antisense oligonucleotide or any further antisense nucleic acid molecule according to the present invention or a siRNA molecule according to the present invention or any other small RNA molecules e.g. miRNA as defined herein can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The molecule may include other appended groups such as peptides (e. g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane or the blood-brain barrier hybridization triggered cleavage agents or intercalating agents. The molecule may accordingly be conjugated to another molecule, e. g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
One envisaged advantage of using antisense oligonucleotides according to present invention is that the once such antisense oligonucleotides reach the brain, i.e. cross the blood brain barrier, they are readily taken up by neurons and may thus be delivered to the central nervous system (CNS) without the use of viral transduction of neurons (for a review see Zalachoras et al., 2011). Increased brain uptake of antisense oligonucleotides after peripheral delivery may be achieved by increasing the permeability of the blood-brain barrier. Another envisaged way of delivering the antisense oligonucleotides according to the present invention is the delivery via the encapsulation of the antisense oligonucleotides in liposomes conjugated to monoclonal antibodies as described previously (Zhang,Y., JeongLee,H.,Boado,R. J.,and
Pardridge,W.M.(2002). Receptor-mediated delivery of an antisense gene to human brain cancer cells. J. GeneMed. 4, 183-194).
Another embodiment of delivering antisense oligonucleotides according to the present invention into the desired brain region is by local injection or injection in the cerebrospinal fluid, particularly if broad distribution in the brain is deemed more important. The present invention may also include the intraventricular or intrathecal delivery into the cerebrospinal fluid as described in Smith et al, 2006 (Smith, R.A. et al. (2006). Anti-sense oligonucleotide therapy for neurodegenerative disease. J. Clin. Investig. 116, 2290-2296.) The antisense molecule or antisense oligonucleotide, miRNA- or siRNA molecule, may comprise at least one modified base moiety which is selected from the group including 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-aminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyl guanine, 3-methyl cytosine, 5-methylcytosine, N6-adenine, 7- methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. The molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. In another embodiment, the molecule comprises alternatively or additionally at least one modified phosphate backbone, e.g. a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof. In another embodiment, the antisense oligonucleotide may be an alpha-anomeric
oligonucleotide, i.e. an oligonucleotide which forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other.
The term "siRNA specific for the histone methyltransferase G9a" as mentioned herein above refers to a particular type of small molecules, namely small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway to negatively regulate gene expression of the histone methyltransferase G9a.
The term "RNA interference" or "RNAi" as used herein refers to an RNA induced block of gene expression in a specific and post-transcriptional manner by degradation of a specific target mRNA.
These siRNA molecules can vary in length and may be between about 18-28 nucleotides in length, e.g. have a length of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.
Optionally, the molecule has a length of 21, 22 or 23 nucleotides. The siRNA molecule according to the present invention may contain varying degrees of complementarity to their target G9a mRNA, optionally in the antisense strand. siRNAs may have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region. In one embodiment, the siRNA may be double- stranded wherein the double-stranded siRNA molecule comprises a first and a second strand, each strand of the siRNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siRNA molecule comprises nucleotide sequence having sufficient
complementarity to the target RNA via RNA interference, and the second strand of said siRNA molecule comprises nucleotide sequence that is complementary to the first strand.
Methods for designing suitable siRNAs directed to a given target nucleic acid are known to person skilled in the art. Furthermore, antagonistic siRNA molecules may be obtained according to methods of identifying antagonists as described herein.
The terms "miRNA specific for the histone methyltransferase G9a" or "micro RNA specific for the histone methyltransferase G9a" as used herein refer to a short single-stranded RNA molecule of typically 18-27 nucleotides in length, which regulate gene expression of histone methyltransferase G9a. miRNAs are encoded by genes from whose DNA they are transcribed but are not translated into a protein. In a natural context miRNAs are first transcribed as primary transcripts or pre-miRNA with a cap and poly-A tail and processed to short, 70- nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre- miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). After integration into an active RISC complex, miRNAs may base pair with their complementary mRNA molecules and inhibit translation or may induce mRNA degradation by the catalytically active members of the RISC complex, e.g. argonaute proteins. Mature miRNA molecules are typically at least partially complementary to mRNA molecules corresponding to the expression product of the present invention, and fully or partially down- regulate gene expression. Optionally, miRNAs according to the present invention may be 100% complementary to their target sequences. Alternatively, they may have 1 , 2 or 3 mismatches, e.g. at the terminal residues or in the central portion of the molecule. miRNA molecules according to the present invention may have a length of between about 18 to 27 nucleotides, e.g. 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides. In one embodiment, the miRNA molecules are 21 to 23 mers. miRNAs having 100% complementarity may optionally be used for the degradation of nucleic acids according to the present invention, whereas miR As showing less than 100% complementarity may optionally be used for the blocking of translational processes.
In another aspect, the present invention relates to a vector comprising such inhibitory polynucleotide molecules as described herein. It is to be understood that any of the inhibitory polynucleotide molecules as described herein above may be expressed from a vector. In specific embodiments, such vectors according to the present invention may be delivered to the neural cell for expression of the inhibitory molecules of the invention. Suitable vectors and methods for delivery of such vectors have been described in the art and belong to the routine of the skilled person.
In yet another aspect, the present invention relates to a pharmaceutical composition comprising the antibody or binding fragment thereof as described herein and/or the inhibitory polynucleotide as described herein. In particular, the pharmaceutical composition and/or the inhibitory polynucleotide is for use in the treatment of depression. Alternatively or additionally, the pharmaceutical composition and/or the inhibitory polynucleotide may be for use in the treatment of other stress-related disorders, e.g. disorders caused by the experience of a traumatic event such as PTSD. A pharmaceutical composition according to the invention can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example in the form of suppositories. Administration can also be carried out parenterally, for example subcutaneously, intramuscularly or intravenously in the form of solutions for injection or infusion. Other suitable administration forms are, for example, percutaneous or topical administration, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or the inhalative administration in the form of nasal sprays or aerosol mixtures.
One embodiment relates to administration forms suitable for delivering a pharmaceutical composition to the brain, i.e. administration forms capable of overcoming the blood brain barrier (BBB). For example, increased brain uptake may be achieved by peripheral delivery and subsequent increase of the permeability of the BBB. The lipid solubility of the pharmaceutical composition may be increased to facilitate entry into the brain region. Other administration forms envisaged by the present invention comprise encapsulating the compound e.g. antibody or inhibiting polynucleotide molecule according to the present invention in liposomes. In some embodiments the pharmaceutical composition as described herein is locally injected, i.e. directly injected into the brain region of interest. The skilled person would know which methodology to use.
For the production of pills, tablets, sugar-coated tablets and hard gelatin capsules for example, lactose, starch, for example maize starch, or starch derivatives, talc, stearic acid or its salts, etc may be used. Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc. Suitable carriers for the preparation of solutions, for example of solutions for injection, or of emulsions or syrups are, for example, water, physiological sodium chloride solution, alcohols such as ethanol, glycerol, polyols, sucrose, invert sugar, glucose, mannitol, vegetable oils, etc.
In some embodiments the pharmaceutical compositions can also contain additives, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
Examples of suitable excipients for the various different forms of pharmaceutical
compositions described herein may e.g. be found in the "Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), Edited by A Wade and PJ Weller.
In some embodiments the pharmaceutical compositions may be sustained release
formulations.
Administration of the antibody, pharmaceutical composition and/or the inhibitory
polynucleotide molecule described herein for use in the treatment of disorders caused by the experience of a traumatic event may occur immediately after the traumatic event, e.g. as prophylaxis of such disorders. However, as disorders caused by the experience of a traumatic event may only occur after a certain time span after the event (e.g. days, weeks or years), the pharmaceutical composition and/or inhibitory polynucleotide molecule may also be administered days, weeks or years after the traumatic event, i.e. when the symptoms of the disorder are diagnosed.
Further exemplary embodiments of the invention are described in the following items:
Item 1. A method of identifying a compound having an anti-depressant activity, comprising the steps of
a) contacting said compound with a neural cell under conditions suitable for cellular uptake of the compound, and
b) determining a value indicative for the cellular level of histone methyltransferase
G9a in said neural cell, wherein reduction of the cellular level is indicative of an
antidepressant activity of the compound.
Item 2. The method of item 1, wherein determining the cellular level histone
methyltransferase G9a is determined by measuring the expression level and/or the amount of histone methyltransferase G9a protein in said neural cell.
Item 3. The method of item 1 or 2, wherein said neural cell is an astrocyte. Item 4. The method of any one of items 1 to 3, wherein the neural cell is derived from a mammal, the neural cell being optionally a human neural cell.
Item 5. The method of any one of items 1 to 4, wherein determining the cellular level histone methyltransferase G9a is measured by using a total or nuclear extract of said neural cell.
Item 6. The method of any one of items 1 to 5, wherein reduction of the cellular level to less than 80%, optionally less than 70%, is indicative of an antidepressant activity of the compound.
Item 7. The method of any one of items 1 to 6, further comprising the step of determining whether said compound is capable of decreasing the activity of a DNA methyltransferase, optionally of DNA methyltransferase DNMT1. Item 8. The method of item 7, wherein the activity of the DNA methyltransferase is determined by i) contacting a DNA substrate with a methyl group donor and a total or nuclear extract of said neural cell and ii) detecting DNA methylation at cytosine residues. Item 9. The method of item 8, wherein the DNA substrate is Poly(dI-dC)-Poly(dI-dC) or hemi/unmethylated DNA substrate.
Item 10. The method of any one of items 7 to 9, wherein the decrease DNA methyltransferase activity is at least about 30%, optionally 40%.
Item 11. The method of any one of items 7 to 10, further comprising the step of measuring whether the decrease of the activity of the DNA methyltransferase can be restored by the addition of purified histone methyltransferase G9a protein. Item 12. The method of any one of items 1 to 11, wherein said reduction of the cellular level comprises inhibiting histone methyltransferase G9a by directly binding to histone
methyltransferase G9a protein.
Item 13. The method of any one of items 1 to 12, wherein said reduction of the cellular level comprises decrease or inhibition of histone methyltransferase G9a protein expression.
Item 14. The method of any one of items 1 to 13, wherein said reduction of the cellular level comprises enhancing degradation of histone methyltransferase G9a protein in said neural cell. Item 15. A method of identifying a compound having an anti-depressant activity, comprising the steps of
a) contacting said compound with a neural cell under conditions suitable for uptake of the chemical compound, and
b) determining a value indicative for the loss of interaction of the histone methyltransferase G9a with DNA methyltransferase DNMT1 ,
wherein said loss of interaction is indicative is indicative of an antidepressant activity of the compound. Item 16. The method of item 15, wherein the loss of interaction is detected by determining the loss of binding of histone methyltransferase G9a to DNA methyltransferase DNMT1.
Item 17. The method of item 15 or 16, wherein determining the loss of interaction comprises a method selected from the group consisting of co-immunoprecipitation, pulldown-assay, resonance energy transfer (FRET), protein complementation assay, enzyme complementation assay, yeast two hybrid, mammalian two hybrid, fluorescent polarization, and surface plasmon resonance.
Item 18. A method of providing a compound having antidepressant activity comprising the steps of
a) conducting the method of identifying a compound having antidepressant activity according to item 1 to 17, and
b) synthesizing the compound identified in step a).
Item 19. An antibody or a binding fragment thereof capable of specifically binding to the histone methyltransferase G9a protein for use in the treatment of depression.
Item 20. The antibody or a binding fragment thereof of item 19, wherein the antibody comprises a human, humanized, murine, xenogeneic or chimeric human-murine antibody or binding fragment thereof.
Item 21. The antibody or a binding fragment thereof of item 19 or 20, wherein the antibody is a monoclonal antibody.
Item 22. An inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein comprising
a) an antisense nucleic acid specific for histone methyltransferase G9a,
b) a small interfering RNA specific for histone methyltransferase G9a , or c) a microRNA specific for histone methyltransferase G9a,
for use in the treatment of depression.
Item 23. A vector comprising the nucleic acid molecule according to item 22. Item 24. A pharmaceutical composition comprising the antibody of binding fragment thereof according to items 19 to 21 and/or the inhibitory polynucleotide according to items 21 to 22 for use in the treatment of depression. Item 25. The pharmaceutical composition according to item 24 further comprising pharmaceutically acceptable excipients.
EXAMPLES Example 1 - Materials and Methods
Drugs
Antidepressants and mood stabilizers were purchased from Sigma, except for Venlafaxine (VEN) (donation from Wyeth Pharma GmbH, Munster, Germany). Akt inhibitor (1L6- Hydroxymethyl-chiro-inositol-2(R)-2-0-methyl-3-0-octadecyl-sn-glycerocarbonate) and Go 6983 were from Calbiochem. Stock solutions were prepared by dissolving the substances in water or DMSO (Carbamazepine, Akt inhibitor, G5 6983)
Preparation of rat primary astrocytes, neurons and NSCs
Cortical neurons were prepared from embryonic day 18 rat brains (Sprague Dawley, Charles River) (Bayatti et al., 2003). Astrocytic cultures were prepared from postnatal dayl animals (Franke et al., 1998; Bayatti et al, 2003). Before commencement of the treatment, neurons or astrocytes were kept in B27-Neurobasal medium (neurons) or N2-MEM/F12-medium
(astrocytes) for 24 h. For NSC cultures, slices from the subventricular zone of postnatal day 4 rats were digested with 0.05% trypsin (Invitrogen), and the dissociated cells plated at a density of 200 cells/mm2. Cultures were maintained in DMEM/F12 (Invitrogen) with 2% B27 supplement (Invitrogen), 5mM Hepes (Sigma), lx Insulin-Transferrin-Sodium Selenite supplement (Roche), lOng/ml bFGF (Peprotech) and lOng/ml EGF (Peprotech). Culture medium was half-renewed every 3-5 days. NSCs were passaged every 2 weeks by direct pipetting and all the experiments were performed on P4-6 NSCs. MTT assay
Neurons were incubated with 0.5 mg/ml MTT reagent [tetrazole 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide] in culture medium for 6 h at 37 °C with 5% C02. Read-out was as described previously (Moosmann et al, 2001).
Protein extracts
In case of nuclear extract isolation, cells were lysed with hypotonic buffer (10 mM HEPES, 10 mM KC1, 0.5 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM DTT, protease inhibitor cocktail (Sigma)) followed by incubation of the nuclear pellet with nuclear extraction buffer (20 mM Tris pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM DTT, protease inhibitor cocktail (Sigma)). In case of salt extraction series, nuclear pellets were successively incubated with nuclear extraction buffer containing increasing salt
concentrations (100-400 mM NaCl). Total cell extracts were obtained by resuspending cells in nuclear extraction buffer followed by sonication. To remove endogenous DNA, samples were incubated with 0.2 bed vol. DEAE sepharose (Sigma) for 20 min at 4 °C.
Total cell extracts for Western blot analysis were obtained by lysing cells in a buffer containing 20 mM Tris/HCl pH 6.8, 0.66%> SDS and 3.33% sucrose, supplemented with protease inhibitor cocktail (Sigma). Samples were heated at 95 °C for 5 min and sonicated. Protein concentration was measured using the Pierce BCA Protein assay kit according to the manufacturer's instruction.
Western blot analysis
Western blot analysis was conducted as described previously (Perisic et al, 2010). G9a immuno -reactive bands were quantified using the FUSION FX7 system (Vilber Lourmat). The following primary antibodies were used: anti-DNMTl (1 :2000, no. IMG-261 A,
Imgenex), anti-G9a (1 :1000, no. 3306S, Cell Signaling), anti-NAA10 (1 :2000, no.
SAB2100142, Sigma), anti-actin (1 :2000, no. A2066, Santa Cruz), anti-hsc70 (1 :2000, no. sc- 7298, Santa Cruz). Real-time PCR analysis
Total RNA from neural cells was obtained using the NucleoSpin RNA II kit (Macherey- Nagel, Germany) according to the manufacturer's instructions. RNA was reverse transcribed with Omniscript Reverse Transcriptase (Qiagen) and random primers (Promega). The
LightCycler® System (Roche Applied Science, Germany) and SYBR green detection
(QuantiFast SYBR Green, Qiagen) served to carry out relative quantification of DNMTl/3a and 3b mRNA levels. HPRT mRNA was used for normalization. The efficiency of the PCR reaction was determined for each primer pair. Flow cytometric analysis of cell cycle phases
Cortical astrocytes were fixed in 2% formaldehyde followed by further permeabilisation in 66% ethanol. RNA was digested using RNAse A (100 μg/ml in PBS; Sigma) and DNA was stained using propidium iodide solution (100μg/ml in PBS; Sigma). Cells were analyzed using a Beckman Coulter Epics XL.
DNA methyltransferase assay
DNMT activity was determined by incubating total/nuclear extracts in 25 μΐ assay buffer (20 mM Tris pH 7.8, 10% glycerol, 5 mM EDTA, 1 mM DTT, 0.2 mM PMSF) containing 0.5 μg Poly (deoxyinosinicdeoxycytidylic) acid (Poly(dl-dC)-Poly(dl-dC); Sigma) or 1.25 μΜ of hemi/unmethylated DNA substrate and 3 μΜ S-adenosyl-L-[methyl-3H]methionine (3H-SAM, specific activity: 10.0 Ci/mmol; PerkinElmer) for 2 h at 37 °C. When Poly(dI-dC)-Poly(dI- dC) was used, 4 μg protein extract was applied, and 10 μg when hemi/unmethylated DNA was used. DNA substrate was purified and tritium incorporation was measured by scintillation counting using a Beckman LS 6500. In case of DNMT assays supplemented with purified G9a, nuclear extracts (0.7 μg/μl total proteins in 90 μΐ) were pre-incubated with vehicle or G9a (no. M0235S, New England Bio labs) and 160 μΜ unlabelled SAM for 30 min at 37 °C. Unreacted SAM was removed using BioSpin 6 columns.
Purification of Poly(dl-dC) -Poly(dl-dC) substrate - The DNA substrate Poly(dl-dC)- Poly(dI-dC) was isolated using the GeneClean kit (MP Biomedicals).
Preparation and purification of hemi/unmethylated substrate DNA - Equal amounts (20 μΜ) of complementary single-stranded oligonucleotides were mixed, heated for 5 min at 95 °C and gradually cooled to room temperature (1 h). To generate un- or hemi-methylated DNA, the oligonucleotides S'-gatctcgtcgtcgcgcgcgcgtcg tcgtcggatc-3 ' and biotin-5'- gatccgacgacgacgcgcgcgcgacgacgagatc-3 ' or biotin-5 '- gatcmgamgamgamgmgmgmgmgamgamgagatc-3' (5-methyl-2-deoxycytosine is abbreviated by m) were mixed. Subsequently to the DNA methyl transfer reaction, biotinylated hemi/unmethylated DNA was isolated using streptavidin coated magnetic beads (Dynabeads M-280 Streptavidin, lOmg/ml; Invitrogen).
Co-Immunoprecipitation
Protein extracts were obtained by incubating cells in lysis buffer (10 mM Tris/HCl, pH 7.4, 250 mM NaCl, 0.5 mM EDTA, 0.5% NP40, protease inhibitor cocktail [Sigma]) for 20 min at 4 °C with constant mixing. Lysates were cleared by centrifugation (12000 g, 15 min, 4 °C). 1 mg of protein extract was diluted in lysis buffer containing 120 mM NaCl to a final volume of 1 ml and incubated with 4 μg anti-G9a antibody (no. PP-A8620A-00, Perseus Proteomics) for 6 h at 4 °C with constant mixing. Subsequently, protein G-coated magnetic beads
(Dynabeads Protein G, 30 mg/ml, Invitrogen) were added and incubated for 2 h at 4 °C.
Beads were washed four times with PBS (Invitrogen), and bound proteins were eluted by incubation with 50 μΐ elution buffer (80mM Tris/HCl, pH 6.8, 20% glycerin, 2.5 % SDS, 2.5% β-mercaptethanol) for 15 min at 70 °C.
Statistics
Generally, three independent experiments were conducted. Significance between control and treatment groups was analyzed using Student's t-test.
Example 2- Psychoactive drugs differentially affect DNMT activity. The observation that AMI reduces DNMT activity in primary cortical astrocytes (Perisic et al, 2010) prompted us to screen additional ADs, including imipramine (IMI), venlafaxine (VEN), citalopram (CIT) and paroxetine (PAR), as well as the mood stabilizers (MSs) carbamazepine (CBZ) and valproic acid (VP A), for their effect on DNMT activity. Cortical astrocytes were treated with the respective drugs for 72 h. Nuclear proteins were extracted and DNMT activity was assayed using Poly(dI-dC)-Poly(dI-dC) as DNA substrate. In addition to AMI, the TCA IMI, as well as the selective serotonin re-uptake inhibitor (SSRI) PAR decreased DNMT activity by -50%, while CIT showed a trend in the same direction (Figure 1 A). In contrast, the selective serotonin/noradrenalin re-uptake inhibitor (SSNRI) YEN did not impact DNMT activity, neither did CBZ or VPA
Example 3 - Inhibition of DNMT activity is indirect and cell-type specific.
To investigate whether AMI inhibits DNMT activity directly, nuclear extracts from primary astrocytes were treated with 10 μΜ AMI for 0.5 h and then assayed for DNMT activity. The effect of AMI does not involve a direct hindrance of the enzyme as DNMT activity was unchanged in these extracts (Figure IB). Consistently, a time-course analysis revealed a significant inhibition of DNMT activity after 72 h of AMI treatment, but not after 24 and 48 h (Figure 1C). To obtain a dose-response relationship, astrocytes were treated with different concentrations of AMI. While 1 μΜ AMI exerted no effect and a trend was observed with 5 μΜ AMI, DNMT activity was significantly diminished upon treatment with 10 μΜ AMI (Figure ID).
Neurons and presumably NSCs are primary targets of AD action. We therefore determined DNMT activity in cortical neurons and subventricular NSCs, which were exposed to AMI or PAR for 72 h at 10 μΜ (in case of NSCs) or at 5 μΜ (in case of neurons). The lower concentration in case of neurons was chosen since 10 μΜ AMI and PAR exhibited cytotoxic effects as determined by MTT assay (data not shown). In contrast to astrocytes, DNMT activity was not altered in neurons and NSCs on AD treatment (Figure IE).
Example 4 - ADs target the DNMTl subtype. Since cell-type specific differences were monitored (Figure IE), Real-time PCR analysis of the expression profiles of DNMTl, 3a and 3b in the respective cells may point to the DNMT subtype targeted by ADs. DNMTl and DNMT3a were transcribed at similar levels in neurons and astrocytes, whereas mRNA levels in NSCs were about 2.5 times higher. In neurons and NSCs, DNMT3b was expressed at 10-20 fold lower levels than DNMTl/3a. The difference was even more pronounced in astrocytes, with DNMT3b constituting around 0.25% of
DNMTl (Figure 2 A). Therefore, it is conducted that DNMT3b contributed only marginally to total DNMT activity in astrocytes. DNMTl and 3a were reported to possess different affinities to chromatin (Jeong et al, 2009). To test whether DNMTl, 3a or both were present in nuclear extracts used for the DNMT activity assay, we isolated nuclei from cortical astrocytes and successively extracted proteins with a buffer containing increasing salt concentrations (100-400 mM NaCl). A lysate fraction containing the remaining proteins past the last extraction step was also included (> 400 mM NaCl). Whereas the majority of DNMTl was already extracted with 250 mM NaCl, an immunoreactive signal representing DNMT3a was exclusively detected in the fraction > 400 mM NaCl (Figure 2B). Hence previously measured DNMT activity mainly originated from DNMTl as nuclear extracts used in the DNMT activity assay were obtained with the buffer containing 250 mM NaCl.
To further corroborate this conclusion, we compared DNMT activity on different DNA substrates. DNMTl clearly prefers hemi- over unmethylated DNA in vitro, whereas DNMT3a favors unmethylated DNA (Yokochi and Robertson, 2002). As expected, DNMT activity in 250 mM NaCl nuclear extracts was high on hemimethylated, and low on unmethylated DNA (Figure 2C). AMI treatment of astrocytes resulted in an inhibition of DNMT activity only on hemimethylated, but not on unmethylated DNA substrate (Figure 2D). This further suggested that predominantly maintenance DNMTl activity is affected by AD treatment. Furthermore, DNMT activity assays of total cell extracts from AMI treated astrocytes provided no evidence for DNMT3a as target for ADs. When Poly(dI-dC)-Poly(dI-dC) as substrate for DNMTl and 3a was used, DNMT activity was reduced to 46% (± 1 %) of control extracts (Figure 2E), which is similar to the reduction in nuclear extracts (52% ±6%). Also like in nuclear extracts, DNMT activity was unchanged on unmethylated DNA (Figure 2E). In conclusion, AD treatment inhibits DNMTl, while no evidence was found for a decrease of DNMT3a activity.
Example 5- DNMTl protein levels are unaffected by ADs. The decrease of DNMTl activity may be due to its diminished expression. As DNMTl expression is regulated during the cell cycle (Torrisani et al, 2007), we first analyzed whether AMI or PAR treatment alters cell cycle parameters in astrocytes using PI/FACS analysis. The percentage of cells in S+G2 was very low in control cells (~8%) and did not change in drug treated (10 μΜ, 3d) cells (-7-8%) (Figure 2F). In addition, DNMTl expression levels remained stable in both total and 250 mM NaCl nuclear extracts on AD treatment (10 μΜ, 3d) as determined by Western blot analysis (Figure 2G).
Example 6 -ADs reduce protein levels of G9a, and replenishing G9a restores DNMT1 activity.
Several factors are known to modulate DNMT1 activity apart from controlling gene expression. The N-a-acetyltransferase 10 (NAAIO) and the histone methyltransferase G9a are interaction partners of DNMT1 and were found to regulate its enzymatic activity in vitro (Esteve et al, 2006; Lee et al, 2010). Whereas NAAIO levels were not changed after treatment with 10 μΜ AMI/PAR for 3d, G9a levels were diminished in both total and nuclear extracts. In contrast, CBZ, which has a similar structure to AMI, but did not reduce DNMT activity, did not affect G9a levels (Figure 3 A). Of note, after 1 or 2d of AMI exposure, where no effect on DNMT activity was observed (Figure 1C), also no reduction in G9a levels was observed (Figure 3B). If the reduction of G9a levels was responsible for the effect of AMI on DNMT1, it should be reversed by adding G9a to the extracts. Therefore, we supplemented nuclear extracts from AMI treated astrocytes with recombinant G9a. When 500 pg of G9a was added, DNMT1 activity was recovered to levels of control cells (Figure 3C). From Western blot analysis including a standard curve with purified G9a, it is concluded that AMI- derived extracts complemented with purified G9a and control extracts contain levels of G9a at about the same order of magnitude (~ 7 fold higher compared to control).
To testwhether the reduced levels of G9a upon treatment with AMI also result in a reduction of G9a-DNMT1 interactions, co-immunoprecipitation experiments were conducted. Indeed, immunoprecipitation in extracts derived from AMI -treated cells yielded not only significantly less G9a, but also smaller amounts of co -precipitated DNMT1 (cf. Figure 4). In contrast, when extracts from AMI -treated cells were supplemented with recombinant G9a, a reduction in the amount of co -precipitated DNMT1 was not detectable.
Example 7 - G9a protein levels in BL6 mice treated with paroxetineNine BL6 mice were treated with paroxetine through drinking water for three weeks, whereby the estimated dose of paroxetine was 20 mg/kg/day. As a control, 9 BL6 mice were only treated with drinking water. After three weeks of treatment, total protein extracts were obtained from the following brain regions of the mice: midbrain, superior and inferior Colliculus, Cerebellum, Pons and Medulla.
Relative protein levels were determined by Western Blot analysis. Here, 100 μg of the protein extract were loaded on a polyacrylamid gel and electrotransferred onto nitrocellulose membrans at 100 V for three hours. G9a protein was detected by applying anti-G9a antibody (Cell Signaling, no. 3306S), followed by incubation with anti-rabbit ("secondary antibody") and anti-goat horseradish peroxidase (HRP) antibody ("tertiary antibody").
As can be derived from figure 5B, the G9a antibody detects three bands when applied on extracts from mouse brain antibodies. Western Blot analysis revealed a significant downregulation of G9a protein levels in brain extracts from animals treated with paroxetine.
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Sequences
1) Homo sapiens
SEQ ID NO:l
Homo sapiens euchromatic histone- lysine N-methyltransferase 2 (EHMT2), transcript variant NG36/G9a, isoform a, mRNA sequence
NM 006709
1 gcaagcggcg atggcggcgg cggcgggagc tgcagcggcg gcggccgccg agggggaggc 61 ccccgctgag atgggggcgc tgctgctgga gaaggaaacc agaggagcca ccgagagagt 121 tcatggctct ttgggggaca cccctcgtag tgaagaaacc ctgcccaagg ccacccccga 181 ctccctggag cctgctggcc cctcatctcc agcctctgtc actgtcactg ttggtgatga 241 gggggctgac acccctgtag gggctacacc actcattggg gatgaatctg agaatcttga 301 gggagatggg gacctccgtg ggggccggat cctgctgggc catgccacaa agtcattccc 361 ctcttccccc agcaaggggg gttcctgtcc tagccgggcc aagatgtcaa tgacaggggc 421 gggaaaatca cctccatctg tccagagttt ggctatgagg ctactgagta tgccaggagc 481 ccagggagct gcagcagcag ggtctgaacc ccctccagcc accacgagcc cagagggaca 541 gcccaaggtc caccgagccc gcaaaaccat gtccaaacca ggaaatggac agcccccggt 601 ccctgagaag cggccccctg aaatacagca tttccgcatg agtgatgatg tccactcact 661 gggaaaggtg acctcagatc tggccaaaag gaggaagctg aactcaggag gtggcctgtc 721 agaggagtta ggttctgccc ggcgttcagg agaagtgacc ctgacgaaag gggaccccgg 781 gtccctggag gagtgggaga cggtggtggg tgatgacttc agtctctact atgattccta 841 ctctgtggat gagcgcgtgg actccgacag caagtctgaa gttgaagctc taactgaaca 901 actaagtgaa gaggaggagg aggaagagga ggaagaagaa gaagaggaag aggaggagga 961 agaggaagaa gaagaggaag atgaggagtc agggaatcag tcagatagga gtggttccag 1021 tggccggcgc aaggccaaga agaaatggcg aaaagacagc ccatgggtga agccgtctcg 1081 gaaacggcgc aagcgggagc ctccgcgggc caaggagcca cgaggagtga atggtgtggg 1141 ctcctcaggc cccagtgagt acatggaggt ccctctgggg tccctggagc tgcccagcga 1201 ggggaccctc tcccccaacc acgctggggt gtccaatgac acatcttcgc tggagacaga 1261 gcgagggttt gaggagttgc ccctgtgcag ctgccgcatg gaggcaccca agattgaccg 1321 catcagcgag agggcggggc acaagtgcat ggccactgag agtgtggacg gagagctgtc 1381 aggctgcaat gccgccatcc tcaagcggga gaccatgagg ccatccagcc gtgtggccct 1441 gatggtgctc tgtgagaccc accgcgcccg catggtcaaa caccactgct gcccgggctg 1501 cggctacttc tgcacggcgg gcaccttcct ggagtgccac cctgacttcc gtgtggccca 1561 ccgcttccac aaggcctgtg tgtctcagct gaatgggatg gtcttctgtc cccactgtgg 1621 ggaggatgct tctgaagctc aagaggtgac catcccccgg ggtgacgggg tgaccccacc 1681 ggccggcact gcagctcctg cacccccacc cctgtcccag gatgtccccg ggagagcaga 1741 cacttctcag cccagtgccc ggatgcgagg gcatggggaa ccccggcgcc cgccctgcga 1801 tcccctggct gacaccattg acagctcagg gccctccctg accctgccca atgggggctg 1861 cctttcagcc gtggggctgc cactggggcc aggccgggag gccctggaaa aggccctggt 1921 catccaggag tcagagaggc ggaagaagct ccgtttccac cctcggcagt tgtacctgtc 1981 cgtgaagcag ggcgagctgc agaaggtgat cctgatgctg ttggacaacc tggaccccaa 2041 cttccagagc gaccagcaga gcaagcgcac gcccctgcat gcagccgccc agaagggctc 2101 cgtggagatc tgccatgtgc tgctgcaggc tggagccaac ataaatgcag tggacaaaca 2161 gcagcggacg ccactgatgg aggccgtggt gaacaaccac ctggaggtag cccgttacat 2221 ggtgcagcgt ggtggctgtg tctatagcaa ggaggaggac ggttccacct gcctccacca 2281 cgcagccaaa atcgggaact tggagatggt cagcctgctg ctgagcacag gacaggtgga 2341 cgtcaacgcc caggacagtg gggggtggac gcccatcatc tgggctgcag agcacaagca 2401 catcgaggtg atccgcatgc tactgacgcg gggcgccgac gtcaccctca ctgacaacga 2461 ggagaacatc tgcctgcact gggcctcctt cacgggcagc gccgccatcg ccgaagtcct 2521 tctgaatgcg cgctgtgacc tccatgctgt caactaccat ggggacaccc ccctgcacat 2581 cgcagctcgg gagagctacc atgactgcgt gctgttattc ctgtcacgtg gggccaaccc 2641 tgagctgcgg aacaaagagg gggacacagc atgggacctg actcccgagc gctccgacgt 2701 gtggtttgcg cttcaactca accgcaagct ccgacttggg gtgggaaatc gggccatccg 2761 cacagagaag atcatctgcc gggacgtggc tcggggctat gagaacgtgc ccattccctg 2821 tgtcaacggt gtggatgggg agccctgccc tgaggattac aagtacatct cagagaactg 2881 cgagacgtcc accatgaaca tcgatcgcaa catcacccac ctgcagcact gcacgtgtgt 2941 ggacgactgc tctagctcca actgcctgtg cggccagctc agcatccggt gctggtatga 3001 caaggatggg cgattgctcc aggaatttaa caagattgag cctccgctga ttttcgagtg 3061 taaccaggcg tgctcatgct ggagaaactg caagaaccgg gtcgtacaga gtggcatcaa 3121 ggtgcggcta cagctctacc gaacagccaa gatgggctgg ggggtccgcg ccctgcagac 3181 catcccacag gggaccttca tctgcgagta tgtcggggag ctgatctctg atgctgaggc 3241 tgatgtgaga gaggatgatt cttacctctt cgacttagac aacaaggatg gagaggtgta 3301 ctgcatagat gcccgttact atggcaacat cagccgcttc atcaaccacc tgtgtgaccc 3361 caacatcatt cccgtccggg tcttcatgct gcaccaagac ctgcgatttc cacgcatcgc 3421 cttcttcagt tcccgagaca tccggactgg ggaggagcta gggtttgact atggcgaccg 3481 cttctgggac atcaaaagca aatatttcac ctgccaatgt ggctctgaga agtgcaagca 3541 ctcagccgaa gccattgccc tggagcagag ccgtctggcc cgcctggacc cacaccctga 3601 gctgctgccc gagctcggct ccctgccccc tgtcaacaca tgagaacgga ccacaccctc 3661 tctccccagc atggatggcc acagctcagc cgcctcctct gccaccagct gctcgcagcc 3721 catgcctggg ggtgctgcca tcttctctcc ccaccaccct ttcacacatt cctgaccaga 3781 gatcccagcc aggccctgga ggtctgacag cccctccctc ccagagctgg ttcctccctg 3841 ggagggcaac ttcagggctg gccacccccc gtgttcccca tcctcagttg aagtttgatg 3901 aattgaagtc gggcctctat gccaactggt tccttttgtt ctcaataaat gttgggtttg 3961 gtaataaaaa aaaaaaaaaa aa
SEQ ID NO: 2
Homo sapiens histone- lysine N-methyltransferase EHMT2 isoform a, amino acid sequence
NP 006700.3
1 maaaagaaaa aaaegeapae mgallleket rgatervhgs lgdtprseet lpkatpdsle 61 pagpsspasv tvtvgdegad tpvgatplig desenlegdg dlrggrillg hatksfpssp 121 skggscpsra kmsmtgagks ppsvqslamr llsmpgaqga aaagsepppa ttspegqpkv 181 hrarktmskp gngqppvpek rppeiqhfrm sddvhslgkv tsdlakrrkl nsggglseel 241 gsarrsgevt ltkgdpgsle ewet vgddf slyydsysvd ervdsdskse vealteqlse 301 eeeeeeeeee eeeeeeeeee eeedeesgnq sdrsgssgrr kakkkwrkds pwvkpsrkrr 361 krepprakep rgvngvgssg pseymevplg slelpsegtl spnhagvsnd tssletergf 421 eelplcscrm eapkidrise raghkcmate svdgelsgcn aailkretmr pssrvalmvl 481 cethrarmvk hhccpgcgyf ctagtflech pdfrvahrfh kacvsqlngm vfcphcgeda 541 seaqevtipr gdgvtppagt aapappplsq dvpgradtsq psarmrghge prrppcdpla 601 dtidssgpsl tlpnggclsa vglplgpgre alekalviqe serrkklrfh prqlylsvkq 661 gelqkvilml ldnldpnfqs dqqskrtplh aaaqkgsvei chvllqagan inavdkqqrt 721 plmea vnnh levarymvqr ggcvyskeed gstclhhaak ignlemvsll lstgqvdvna 781 qdsggwtpii waaehkhiev irmlltrgad vtltdneeni clhwasftgs aaiaevllna 841 rcdlhavnyh gdtplhiaar esyhdcvllf lsrganpelr nkegdtawdl tpersdvwfa 901 lqlnrklrlg vgnrairtek iicrdvargy envpipcvng vdgepcpedy kyisencets 961 tmnidrnith lqhctcvddc sssnclcgql sircwydkdg rllqefnkie pplifecnqa 1021 cscwrncknr vqsgikvrl qlyrtakmgw gvralqtipq gtficeyvge lisdaeadvr 1081 eddsylfdld nkdgevycid aryygnisrf inhlcdpnii pvrvfmlhqd lrfpriaffs 1141 srdirtgeel gfdygdrfwd ikskyftcqc gsekckhsae aialeqsrla rldphpellp 1201 elgslppvnt
SEQ ID NO: 3
Homo sapiens euchromatic histone- lysine N-methyltransferase 2 (EHMT2), transcript variant NG36/G9a-SPI, isoform b, m NA sequence
NM_025256.5
1 gcaagcggcg atggcggcgg cggcgggagc tgcagcggcg gcggccgccg agggggaggc
61 ccccgctgag atgggggcgc tgctgctgga gaaggaaacc agaggagcca ccgagagagt
121 tcatggctct ttgggggaca cccctcgtag tgaagaaacc ctgcccaagg ccacccccga
181 ctccctggag cctgctggcc cctcatctcc agcctctgtc actgtcactg ttggtgatga
241 gggggctgac acccctgtag gggctacacc actcattggg gatgaatctg agaatcttga
301 gggagatggg gacctccgtg ggggccggat cctgctgggc catgccacaa agtcattccc 361 ctcttccccc agcaaggggg gttcctgtcc tagccgggcc aagatgtcaa tgacaggggc 421 gggaaaatca cctccatctg tccagagttt ggctatgagg ctactgagta tgccaggagc 481 ccagggagct gcagcagcag ggtctgaacc ccctccagcc accacgagcc cagagggaca 541 gcccaaggtc caccgagccc gcaaaaccat gtccaaacca ggaaatggac agcccccggt 601 ccctgagaag cggccccctg aaatacagca tttccgcatg agtgatgatg tccactcact 661 gggaaaggtg acctcagatc tggccaaaag gaggaagctg aactcaggag gtggcctgtc 721 agaggagtta ggttctgccc ggcgttcagg agaagtgacc ctgacgaaag gggaccccgg 781 gtccctggag gagtgggaga cggtggtggg tgatgacttc agtctctact atgattccta 841 ctctgtggat gagcgcgtgg actccgacag caagtctgaa gttgaagctc taactgaaca 901 actaagtgaa gaggaggagg aggaagagga ggaagaagaa gaagaggaag aggaggagga 961 agaggaagaa gaagaggaag atgaggagtc agggaatcag tcagatagga gtggttccag 1021 tggccggcgc aaggccaaga agaaatggcg aaaagacagc ccatgggtga agccgtctcg 1081 gaaacggcgc aagcgggagc ctccgcgggc caaggagcca cgaggggtgt ccaatgacac 1141 atcttcgctg gagacagagc gagggtttga ggagttgccc ctgtgcagct gccgcatgga 1201 ggcacccaag attgaccgca tcagcgagag ggcggggcac aagtgcatgg ccactgagag 1261 tgtggacgga gagctgtcag gctgcaatgc cgccatcctc aagcgggaga ccatgaggcc 1321 atccagccgt gtggccctga tggtgctctg tgagacccac cgcgcccgca tggtcaaaca 1381 ccactgctgc ccgggctgcg gctacttctg cacggcgggc accttcctgg agtgccaccc 1441 tgacttccgt gtggcccacc gcttccacaa ggcctgtgtg tctcagctga atgggatggt 1501 cttctgtccc cactgtgggg aggatgcttc tgaagctcaa gaggtgacca tcccccgggg 1561 tgacggggtg accccaccgg ccggcactgc agctcctgca cccccacccc tgtcccagga 1621 tgtccccggg agagcagaca cttctcagcc cagtgcccgg atgcgagggc atggggaacc 1681 ccggcgcccg ccctgcgatc ccctggctga caccattgac agctcagggc cctccctgac 1741 cctgcccaat gggggctgcc tttcagccgt ggggctgcca ctggggccag gccgggaggc 1801 cctggaaaag gccctggtca tccaggagtc agagaggcgg aagaagctcc gtttccaccc 1861 tcggcagttg tacctgtccg tgaagcaggg cgagctgcag aaggtgatcc tgatgctgtt 1921 ggacaacctg gaccccaact tccagagcga ccagcagagc aagcgcacgc ccctgcatgc 1981 agccgcccag aagggctccg tggagatctg ccatgtgctg ctgcaggctg gagccaacat 2041 aaatgcagtg gacaaacagc agcggacgcc actgatggag gccgtggtga acaaccacct 2101 ggaggtagcc cgttacatgg tgcagcgtgg tggctgtgtc tatagcaagg aggaggacgg 2161 ttccacctgc ctccaccacg cagccaaaat cgggaacttg gagatggtca gcctgctgct 2221 gagcacagga caggtggacg tcaacgccca ggacagtggg gggtggacgc ccatcatctg 2281 ggctgcagag cacaagcaca tcgaggtgat ccgcatgcta ctgacgcggg gcgccgacgt 2341 caccctcact gacaacgagg agaacatctg cctgcactgg gcctccttca cgggcagcgc 2401 cgccatcgcc gaagtccttc tgaatgcgcg ctgtgacctc catgctgtca actaccatgg 2461 ggacaccccc ctgcacatcg cagctcggga gagctaccat gactgcgtgc tgttattcct 2521 gtcacgtggg gccaaccctg agctgcggaa caaagagggg gacacagcat gggacctgac 2581 tcccgagcgc tccgacgtgt ggtttgcgct tcaactcaac cgcaagctcc gacttggggt 2641 gggaaatcgg gccatccgca cagagaagat catctgccgg gacgtggctc ggggctatga 2701 gaacgtgccc attccctgtg tcaacggtgt ggatggggag ccctgccctg aggattacaa 2761 gtacatctca gagaactgcg agacgtccac catgaacatc gatcgcaaca tcacccacct 2821 gcagcactgc acgtgtgtgg acgactgctc tagctccaac tgcctgtgcg gccagctcag 2881 catccggtgc tggtatgaca aggatgggcg attgctccag gaatttaaca agattgagcc 2941 tccgctgatt ttcgagtgta accaggcgtg ctcatgctgg agaaactgca agaaccgggt 3001 cgtacagagt ggcatcaagg tgcggctaca gctctaccga acagccaaga tgggctgggg 3061 ggtccgcgcc ctgcagacca tcccacaggg gaccttcatc tgcgagtatg tcggggagct 3121 gatctctgat gctgaggctg atgtgagaga ggatgattct tacctcttcg acttagacaa 3181 caaggatgga gaggtgtact gcatagatgc ccgttactat ggcaacatca gccgcttcat 3241 caaccacctg tgtgacccca acatcattcc cgtccgggtc ttcatgctgc accaagacct 3301 gcgatttcca cgcatcgcct tcttcagttc ccgagacatc cggactgggg aggagctagg 3361 gtttgactat ggcgaccgct tctgggacat caaaagcaaa tatttcacct gccaatgtgg 3421 ctctgagaag tgcaagcact cagccgaagc cattgccctg gagcagagcc gtctggcccg 3481 cctggaccca caccctgagc tgctgcccga gctcggctcc ctgccccctg tcaacacatg 3541 agaacggacc acaccctctc tccccagcat ggatggccac agctcagccg cctcctctgc 3601 caccagctgc tcgcagccca tgcctggggg tgctgccatc ttctctcccc accacccttt 3661 cacacattcc tgaccagaga tcccagccag gccctggagg tctgacagcc cctccctccc 3721 agagctggtt cctccctggg agggcaactt cagggctggc caccccccgt gttccccatc 3781 ctcagttgaa gtttgatgaa ttgaagtcgg gcctctatgc caactggttc cttttgttct 3841 caataaatgt tgggtttggt aataaaaaaa aaaaaaaaaa
SEQ ID NO: 4 Homo sapiens histone- lysine N-methyltransferase EHMT2 isoform b, amino acid sequence
NP 079532.5
1 maaaagaaaa aaaegeapae mgallleket rgatervhgs lgdtprseet lpkatpdsle
61 pagpsspasv tvtvgdegad tpvgatplig desenlegdg dlrggrillg hatksfpssp
121 skggscpsra kmsmtgagks ppsvqslamr llsmpgaqga aaagsepppa ttspegqpkv
181 hrarktmskp gngqppvpek rppeiqhfrm sddvhslgkv tsdlakrrkl nsggglseel
241 gsarrsgevt ltkgdpgsle ewet vgddf slyydsysvd ervdsdskse vealteqlse
301 eeeeeeeeee eeeeeeeeee eeedeesgnq sdrsgssgrr kakkkwrkds pwvkpsrkrr
361 krepprakep rgvsndtssl etergfeelp lcscrmeapk idriseragh kcmatesvdg
421 elsgcnaail kretmrpssr valmvlceth rarmvkhhcc pgcgyfctag tflechpdfr
481 vahrfhkacv sqlngmvfcp hcgedaseaq evtiprgdgv tppagtaapa ppplsqdvpg
541 radtsqpsar mrghgeprrp pcdpladtid ssgpsltlpn ggclsavglp lgpgrealek
601 alviqeserr kklrfhprql ylsvkqgelq kvilmlldnl dpnfqsdqqs krtplhaaaq
661 kgsveichvl lqaganinav dkqqrtplme a vnnhleva rymvqrggcv yskeedgstc
721 lhhaakignl emvslllstg qvdvnaqdsg gwtpiiwaae hkhievirml ltrgadvtlt
781 dneeniclhw asftgsaaia evllnarcdl havnyhgdtp lhiaaresyh dcvllflsrg
841 anpelrnkeg dtawdltper sdvwfalqln rklrlgvgnr airtekiicr dvargyenvp
901 ipcvngvdge pcpedykyis encetstmni drnithlqhc tcvddcsssn clcgqlsirc
961 wydkdgrllq efnkieppli fecnqacscw rncknr vqs gikvrlqlyr takmgwgvra
1021 lqtipqgtfi ceyvgelisd aeadvredds ylfdldnkdg evycidaryy gnisrfinhl
1081 cdpniipvrv fmlhqdlrfp riaffssrdi rtgeelgfdy gdrfwdiksk yftcqcgsek
1141 ckhsaeaial eqsrlarldp hpellpelgs lppvnt
SEQ ID NO: 5
Homo sapiens chromosome 6 genomic contig, GRCh37.p5 alternate locus group
ALT REF LOCI l, G9a genomic sequence
NT 167244.1
1 tttattacca aacccaacat ttattgagaa caaaaggaac cagttggcat agaggcccga 61 cttcaattca tcaaacttca actgaggatg gggaacacgg ggggtggcca gccctgaagt 121 tgccctccca gggaggaacc agctctggga gggaggggct gtcagacctc cagggcctgg 181 ctgggatctc tggtcaggaa tgtgtgaaag ggtggtgggg agagaagatg gcagcacccc 241 caggcatggg ctgcgagcag ctggtggcag aggaggcggc tgagctgtgg ccatccatgc 301 tggggagaga gggtgtggtc cgttctcatg tgttgacagg gggcagggag ccgagctcgg 361 gcagcagctc agggtgtggg tccaggcggg ccagacggct ctgctccagg gcaatggctt 421 cggctgagtg cttgcacttc tcagagccac attggcaggt gaaatatttg cttttgatgt 481 cccagaagcg gtcgccatag tcaaacctgt cagaggaaaa caggagcttg tgggacctgg 541 acccagccac caagagccca ccccgaagac cctgtggatc ctgctccctg agagggaccc 601 gacacccaac ctatcttctc cagatgggat ctgagcccct tgtatgttct atggactttc 661 agcatcagca ttgcctgggg acttgttaga aatgcagaat cctgggcccc atcccaagcc 721 tactgattca aaatctctct gggaggcaca ggactgtttc cccaagtcct ccaggaaata 781 cttatgtaca ctgaaatctg agaagctctg cactactcca tgcctggaca ccaggtacat 841 gccagccttc aggtcccagg tttgctgcat ctcccacccc ctggcagagc ccctagagac 901 ccctagagtc tcaccctagc tcctccccag tccggatgtc tcgggaactg aagaaggcga 961 tgcgtggaaa tcgcaggtct tggtgcagca tgaagacccg gacgggaatg atgttggggt 1021 cacacaggtg gttgatgaag cggctgatgt tgccatagta acgggcatct atgcagtaca 1081 cctctccatc ctggggcagg gggatggcac tcttcacatc tcccccgacc ctgcttgccc 1141 tccccaccca ctgactcccc agtccctcct ccccaggttt ccatttgctg acttcccaga 1201 ggctcctgaa agccagccct ggggagcagc agggtaagga gggtctcctg ctcaccttgt 1261 tgtctaagtc gaagaggtaa gaatcatcct ctctcacatc agcctcagca tcagagatca 1321 gctccccgac atacctgtgg gacaggaatc catggttctg aaggtgagtg tgggctatta 1381 ggaggtggct ccaggcccca tctctcttca caagcctgtg gaatctggaa tgggcagggc 1441 tggcaggtgt ggggaaggga aggcctggag cagcagtggt gggcaagtga aagggcagca 1501 ttccagcctt gacagaggaa gccttcagtc agcacagaga cagacaacaa gctctgtggt 1561 taaggggatt aatgtgtagg ggcagttggc ctgggtgggg aagttcgggt ttggacacag 1621 agaggtttgt gttccaggag ccacccggca ggaatgggcg atatggaaca ggagaggggc 1681 caggactgca ggaagagcca gaggtacagg agtggcaagg aactcaaggc atgattcggg 1741 gcaagagcac ccacacatat ctggacacca gagggaggag aggagccagc tatctaagga 1801 gggtgagcag acatgggaga ttcagacaca cggagaggac gtgggtggga agtgactgtc 1861 aagagacagc ttcagcagag tgggaagggc aaaggccgat tttggcaggg acaggcagtg 1921 agtggatggt ggggaaactg aggcccagca ggaaggggct gcttgccaga gaagttgaga 1981 gatgacatga tggaaagaaa ctggatggtc tgttgaacag gcaagtatgg ttagaggact 2041 atctttttta aaggccaaag aatggtcagg cacggtggct cacgcctgta atcccagcac 2101 tttgggaggc cgaggtgggc ggatcatctg aggtcaggag ttggagacca gcctggctaa 2161 catggtgaaa ctccgtttct actaaaaata caaaaaatta gccgggtgtg gtggtgcgca 2221 cctgtaatcc cagctacttg ggaggctgag gcaggagaat cgcttgaacc tgggaggtgg 2281 agactgcagt gagccaagat tgtgccattg cactccagct tgggcaacaa gagtgaaact 2341 ccgtctcaaa aaataaatta aaaaaaaaaa aaaaaaagag ccaaaggaga ctaaagtaag 2401 attgagggtt gtgggatggc agccaagaga aagggggaga ttacagatgc tgggcagaga 2461 aagaactgat ggagagggac aggcccctga ggaggtggac agataggtag ctgttatcac 2521 ctccactcta cagacaagaa aaataaggct caaagaggtt aagtaacttg gccaagaaca 2581 tccagaagca gagaggggct caaacccaag tctgtttgtc tcccaaactg gcactttctc 2641 cagctaggaa gggcgaggag ggggtggagg ggaaggtaga gggtggaggt ggaggggagg 2701 gaagacaagc tctgtggtct gggcagagtg gaggcaggtg ccattctcag ctggggggat 2761 gggggtcaga ggcggctggc tgctcagctg caggaatagg ggtcagagga ggctggctgg 2821 agagtggcca gatggagaca tgtgactcat cagggcagat ggctgagagg gaggcctggc 2881 agtcagcagt ggccatgtat ccccttccca ccaggtgtta aggtgctccc ggtgacttac 2941 tcgcagatga aggtcccctg tgggatggtc tgcagggcgc ggacccccca gcccatcttg 3001 gctgttcggt agagctgtag ccgcaccctg ggggtaggag agatggcgct gttgggtgga 3061 ggccctggaa aagccccagg ggcagggagg aaagggtgag gtggggagag ggtgggctgt 3121 ggagcagggc ctcacttgat gccactctgt acgacccggt tcttgcagtt tctccagcat 3181 gagcacgcct ggttacactc gaaaatcagc ggaggctcaa tcttgttaaa ttcctggagc 3241 aatcgcccat cctagggtgc ggaggggagg atagtggttt ctctgtgggg cccacctcag 3301 ctgcccaccc aggaacccca agactctaca gagacaggga agttggggtt ggggaggtca 3361 cacaggctct gagatccgag agcacgaaat gcaggagcat catccctggt ttgcatagac 3421 ctgggcacac gcccatcgct gtcccagcca catcccagga ttcccaggcc ttgcccagtc 3481 ctctcagtca cttcccccac agggtaggag gtgagggaca tggtcccagg gagctggttt 3541 attggaggct ggctcctctg aaggaggggc cgggtgtctg tggccaaggc aaggggcacg 3601 caccttgtca taccagcacc ggatgctgag ctggccgcac aggcagttgg agctagagca 3661 gtcgtccaca cacgtgcagt gctggggcga ggaggcagag gtcagctcaa ccccatgatc 3721 ggtctgggcc cctctactct tgatgccccc tgacccccta accactgtcc tttctttggg 3781 gtccatgtgt tacaacagtg ggtggtgatg gtcctagggt gacgggtaat cagtatggtg 3841 gtgtccccag ggctactggg agctcatatg ataccttgct gtgacctagg aaaaggatcc 3901 ctcccctggt ggggatgcga ccccacacca gggatccctt tcagccaacc cttccttggc 3961 caggtgcctt tgctggtttg aagcttgtcc aactgtactt ggcagctctc ggtgtccttt 4021 tggggaggcc ccgggccccc tactcacctg caggtgggtg atgttgcgat cgatgttcat 4081 ggtggacgtc tcgcagttct ctgagatgta cttgtaatcc tcagggcagg gctccccatc 4141 cacaccgttg acacagggaa tgggcacgtt ctcatagccc cgagccacgt ccctgcagaa 4201 gacgggaaga aggggctggg aagctggaaa agggggtgag gagctactcc aggtataagg 4261 aagagagttg gggaggttcc tggggctggg ggcaggggag taaggttgcc aggtaagatg 4321 caggacagcg agttaacata gaattttaga taaacaagaa atagcttttt agtatgtccc 4381 aaaaattaca caggacattc tcacactaaa aaagtatgca tctgtgcatc tgaaattcca 4441 gtttaactgg gtgtcttcta tttttatttg ctgtatctgg caaccctagt ggggaggggg 4501 cctgtgggtg gttctgggga ttcagtggtg catggggagg ggttggggaa tgttgtgagg 4561 atgcaatgga gcctggggag ggtatgggtg gggaggaggt ggtcttgggt gcagagaggg 4621 gcccagggct caccggcaga tgatcttctc tgtgcggatg gcccgatttc ccaccccaag 4681 tcggagcttg cggttgagtt gaagcgcaaa ccacacgtcg gagcgctcgg gagtcaggtc 4741 ccatgctgtg tccccctctt tgttccgcag ctcagggttg gccccacgtg acaggaataa 4801 cctgaagagg ggacaggatg cccaatgcag ggtctgaggc tgcaagaagt gggggcaggg 4861 gcatcaaggg cggggcaggg gctcacagca cgcagtcatg gtagctctcc cgagctgcga 4921 tgtgcagggg ggtgtcccca tggtagttga cagcatggag gtcacagcgc gcattcagaa 4981 ggacttcggc gatggcggcg ctgcccgtga aggaggccca gtgcaggcag atgttctcct 5041 cctgtggagg taggagggga acagatgagg tgcaggcagc tgggcccttg aatccagcct 5101 ccaccttgct caggggcctg gggctgccct acctcaacca aacgctcact cacgttgtca 5161 gtgagggtga cgtcggcgcc ccgcgtcagt agcatgcgga tcacctcgat gtgcttgtgc 5221 tctgcagccc agatgatggg cgtccacccc ccactgtcct gtgggtggga agggagtgag 5281 ggtgggggca gctggccctg ctcaccaaag cagcaaatgg tcaagattgg ctgtgtgtgt 5341 gaatcccagc tccaccattc acaagctgtg ggaccctggg taagtcactt aacgtctctg 5401 ggtcgcagtt tcttcatcta aaaaatggga ctagtagggt cgggcgcggt ggctcatgcc 5461 tgtaatccca gcactttggg aggccgaggc gggcggatca cgaggtcagg agatggaggc 5521 cattgtggcc aacacggtga aaccctgtct ctactaaaaa atagaaaaaa ttagctgggc 5581 gtggtggcag gcgcctgtag tcccagctac tagggaggct gaggcagaat ggcgtgaacc 5641 cgggaggcgg agcttgcagt gagccaagat cgtgccactg cactccagcc tgggcgacag 5701 agcaagactc cgtctcaaaa aacaaacaaa caaaaatggg actagtagcg tctaccatct 5761 gatgccagag agaaaataaa gtaattgttc tctttccaaa aaatacagcc aggagctggt 5821 catggaggtg catgcctgta gtcccagcta ctcatgtgac tgagatggga gggttgcttg 5881 agcccaggat ttcgaggctg cagagagcta tgactgtctg tgaactgcta ctgtacttca 5941 gcctgggtga catagcaaga ccctgtctct taaaagaaaa aacgaacaaa aatttcctaa 6001 gtctgcccac tcaaaagtcc tagaagcagc gacaacccaa taacaataaa cactcctagg 6061 aacatagatt gtattctcta aaaaatgctt ctggccgggc gctgtggctc acgaggtcag 6121 gagttcaaga tcagcctggc caatatggtg aaaccccgtc tctactaaaa atacaaaaat 6181 tagccgggca tggtggtggg cgcctgtaat cccagctact cgggaggctg aggcaggaga 6241 atggcgtgaa cctgggaggc ggagcttgca gtaagctgtg atcacgccat tgcactccag 6301 cctgggcaac agagtgagac tccgtctcaa aaaaaaaaaa aaaagtttcc cataaaggaa 6361 gcagagtttc ttagagaaat ggtggattct gagttggggg caggaaatgt gctgaaaggt 6421 caggaggctc tcaaaggcca ctgggccact gggtcatgtc acagccacag aggcctctta 6481 aaggggcttc ttctggacaa tgatggaata attcaaagac tgagaagaat gccaataaat 6541 gactaaaaca catccaatgt atgacaaccc aagagttaat aaaaagcctc actggacact 6601 ttcagagatt aagacaggaa ctgattattc tgaaacttga taaagagaaa gaaacgagaa 6661 agaaaagaat gaagagaaat acaaatgagg aagaagaaag caatgaggac agacacgagc 6721 agtgtgaggt cagatgtagg aaaggcggcc caaagcctga ggccaagcca aggaacccag 6781 gcaccaggga cccagagggg ctgggctggg tgggccgctg acctgggcgt tgacgtccac 6841 ctgtcctgtg ctcagcagca ggctgaccat ctccaagttc ccgattttgg ctgcgtggtg 6901 gaggcaggtg gaaccgtcct cctcctgagg gagacacggg caaatgagcc tttgggctgg 6961 caccccaaac ctggtccctg actccggggg ccacgccctg ctgcctgcgc gcacaccttg 7021 ctatagacac agccaccacg ctgcaccatg taacgggcta cctccaggtg gttgttcacc 7081 acggcctcca tcagtggcgt ccgctgctgt ttgtccactg catttatgtt ggctccagcc 7141 tgtgaggggg caggagggct ggcaccaggg aggcatgggg caggggaggg gcctaagggc 7201 ctggtgaatg aggcatgggg ccgggcccgt gctgacctgc agcagcacat ggcagatctc 7261 cacggagccc ttctgggcgg ctgcatgcag gggcgtgcgc ttgctctgct ggtcgctctg 7321 gaagttgggg tccaggttgt ccactgcggg gagagcccgc cacaccggga gagggaggga 7381 caagtggtaa gcaagctagg gggcaggtgg cacttctttc aggaaggctt ctcagggccc 7441 caagctggat cagggcccct cctggcattc tccgagcttg cctccaccac agcatttatc 7501 agaataagga gtcaaaggca tcagctctgc ctgaattcaa accctgcctt gcttctcagt 7561 accactgtgc actgtgcaag gtccctaacc tctctgtgca agccaaggct aacaggtata 7621 agcactcaga acaggaccca gcacctatga gtcaccacat ccccatcgtt atgggttaca 7681 tgtgtctttt ccccaccaca ctaagtcctt cagggcaagg actgtgtcct tcacgactgt 7741 actcctggcc ctgtacccag tgcctggtat atacatgaag cttggtcaag gtctgctgaa 7801 ggaatgggtg gcactcacac agcatcagga tcaccttctg cagctcgccc tgcttcacgg 7861 acaggtacaa ctgccgaggg tggaaacgga gcttcttccg cctgccaagg gagcacggga 7921 gcggggagag aaggggagct cctcagattc cagcatcagc ctcgacacca ctcctctggc 7981 ctcagcccca gttgctgtgc ctgagcaact ccccactcac ctctctgact cctggatgac 8041 cagggccttt tccagggcct cccggcctgg ccccagtggc agccccacgg ctgaaaggca 8101 gcccccattg ggcagggtca gggagggccc tgagctgtca atggtgtcag ccaggggatc 8161 gcagggcggg cgccggggtt ccccatgccc tcgcatccgg gcactgtgga agaaggagct 8221 catgtccagg agcaataggg gtgggggagg gaacagacag tacagaaggg ggaggccagt 8281 acctgggctg agaagtgtct gctctcccgg ggacatcctg ggacaggggt gggggtgcag 8341 gagctgcagt gccggccggt ggggtcaccc cgtcaccccg ggggatggtc acctcttgag 8401 cttcagaagc atcctcccca cagtggggac agaagaccat cccattcagc tgagacacac 8461 aggccttgtg gaagcggtgg gccacacgga agtcagggtg gcactccagg aaggtgccct 8521 gggagcaggg aaacaacatg gtcaggttac tggggccccc tctgccacag ggcatgctac 8581 ctgtctgccc cactggtcac tcaccgccgt gcagaagtag ccgcagcccg ggcagcagtg 8641 gtgtttgacc atgcgggcgc ggtgggtctc acagagcacc atcagggcca cacggctgga 8701 tggcctcatg gtctcccgct tgaggatggc ggcattgcag cctgacagct gtgcgcagtg 8761 aggatgggtg agaagagagc gtgaggctgg ggccggggac tggacgccct ggcacctctc 8821 ccaccagccc acggccccac ctctccgtcc acactctcag tggccatgca cttgtgcccc 8881 gccctctcgc tgatgcggtc aatcttgggt gcctccatgc ggcagctgca caggggcaac 8941 tcctcaaacc ctcgctctgt ctccagcgaa gatgtgtcat tggacacccc ttggatggag 9001 gaaaagagga gctgagggag gctctgcacc tcacctactg ggacccctgg cgggtcctct 9061 cactccctcc ctaccccacc ccgccatgcc ccagaacccc taaagcctgg ccatggacac 9121 cccggctctg gcgtggttcc cctccttccc tttccctcct gccctgaggt cgccccctag 9181 tggctccctg tcccggcaat tggcaattac cagcgtggtt gggggagagg gtcccctcgc 9241 tgggcagctc cagggacccc agagggacct ccatgtactc actggggcct gaggagccca 9301 caccattcac tcctgacaca gagacagaga gagtgagagt gcgagctcac aggtgcctgg 9361 acgcgtgggt acatgcaggt ggacatgcga gagcgtgtgt gtgcgtgcac acactctggg 9421 gggccgggcg ggggctggag ggcacccaaa agcagcagag cctcctcacc tcgtggctcc 9481 ttggcccgcg gaggctcccg cttgcgccgt ttccgagacg gcttcaccca tgggctgtct 9541 tttcgccatt tcttcttggc cttgcgccgg ccactggaac cactctggga agggggagga 9601 ggaggagtta ggaaccctca cccccagggg cccccccaac accttcagga ccagacctcc 9661 agccccatag tctcccactc ctctggagat atcagcctcc gtctcttacc ctatctgact 9721 gattccctga ctcctcatct tcctcttctt cttcctcttc ctcctcctct tcctcttctt 9781 cttcttcctc ctcttcctcc tcctcctctt cacttagttg ttcagttaga gcttcaactt 9841 cagactggga gagaggcaga acagacatat ccaaccccca ggactcagac aatgaggtga 9901 gtaaagaaaa ccaccaccac cattgccccc cgccactacc cacggatggc tgctggggat 9961 aagtgtgggt agcagaggag acaaagggcc acataaagag agggtgcatg gaatattaca 10021 cagcagtgaa aaagttacag acagcaatgt gcacagatct tggtaatgtg atattaagtt 10081 aaaaaacaaa aagcaagtac cagaagataa acatactttg ataccccttt tatgatgttc 10141 ataaacaggc aagaccacca atggttgcta aaaacactag acacaaagct catgagaaac 10201 tttatatgaa aggttcaggc tgacatcacc tgaacccact ggtcaatctt atcactaaca 10261 agaaaaatga ccagattaga tgttccatgc atcctgatgt gatgtggcca gaagcacttg 10321 cacccactgt caagtcttct tggcacctga agctgattcc gcctctagat ctatcagttt 10381 acaagaaata tgggcagaga ggatgtgtca atctccaccc aatcagccaa ctcctaaatg 10441 tgaaaaattc tgtaggacaa ctgagctggt ttctttgaca aataaatggc aaaaaaaaaa 10501 aatctttctt atttatttat tgagttttgc tcttgttgcc caggctgcat tgcaatggtg 10561 tgatctcagc tcactgcaac ctccacctcc tggattcaag caattctctt gcctcagcct 10621 cctgagtagc tgggattata ggcacccgcc accacaccca gctaattttc gtatttttat 10681 tagagatgtg ttttcaccat gttggctagg ctggtctcaa actcctgacc tcaggtgatc 10741 cacctgcctc ccaaagtgct gggattacag gcgtgagcca ccacgcctgg gccaaaaaat 10801 ttttttttag aagatgagga aatcaggcca ggtgtggtgg ctcacgcctg taatcccagc 10861 gctttgggag gccgaggtgg gcagatcacg agatcaggag tttgagacca gcctggccaa 10921 catagtgaaa ccctgcctct actaaaaata caaaaaatta gctgggcatg gtggtgggtg 10981 cctgtaatcc cagccacttg ggagactgag gcaggagaat tgcttgaact caggaggtgg 11041 agcttgcagt gagccaagat cacgccactg cactccagcc tgggtgacag tgtgagactc 11101 catctcaaaa aacaaaaaca aacaaacaaa caaacaaaca caaagaagat ggggaaacct 11161 aaatactgag agagacctaa gacaaaaaaa aatttttttt tttttgagac ggagtttcgc 11221 tcttgttgcc gaggctggag tgcaatggta cgatcttggc tcactgcaac ctccacctcc 11281 caggttcaag cgattctcct gcctcagcct cccgagtagc tggaattaca ggcacgtacc 11341 actacgtcca gctaattttg tatttttttc agtagagacg gggtttctcc atgttgatca 11401 ggctggtctc gaactcccaa cctcacgtga tctgcccgcc ttggcctccc aaagtattgg 11461 gattacaggc gtgagccact gtgcctggct gacctaagac aaatgttaat caaatcaagg 11521 tgtgggcctc atttggatct tgacaaaaac catttgtgag agctgaggaa atgtgaagac 11581 tgacaggata tttgatggta ttaagaaatc ggtaagtttt tttaggtgtg aaaacagtag 11641 tgtaatgatg ttgaacgaca aaaagaggcc ttatatttac aaatctatat ggatatatgt 11701 ttaggtaaaa tgatatgagg tctgggattt gctttaaaat aacctagtag gtgtgtgtgc 11761 tgggagatgt acagatgggt caagatggac tgtgtactga taatggctgg agctgtgtat 11821 tgggtacatg ggggctccct attctactct tttgattatg cttgcaagtt ttcatgataa 11881 aatgttaaat aaaaggcaaa atcagagaga ctaaacattc tactgtgtag gcaaacatat 11941 aagataaaac cagacaaaga gcaacgaaat aagcaaataa atgacaatgc aatgcttttg 12001 aattttatat aaacagcata acgtatgttt taaaaaagtg ctttctggtc atttcttttt 12061 ttgttttctt tttttaaaca gtacatgtct gttaaatggt catttcatta gctgattaaa 12121 aaaaaaagaa tactaaatcc catgtgcagg gtggtggcca cctttgtgga tgaaacgggc 12181 agaatacaca ttgaaaatga gttacagctg ggcgcggtgg ctcacagctg taatcccagc 12241 actttgggag gccaaggtgg gtggatcaac taaggtcagg agttagagac cagcctggcc 12301 aacacaggga aaccccgtct ctactaaaaa tacaaaaatt agccgggcgt ggtggcaggt 12361 gcctgtaatc ccagctactc gggaggctga ggcaggagaa ttgctttaac cctggagaca 12421 gaggttgcag tgagcccaga tcgtgatatt gcgctccagc ctgggcgaca gaatgagatt 12481 ccgtctcccc ccacaaaaaa aaggagttat agacagcatg gggcaatgac ttagtggata 12541 ttcagaagat aaaaaggaca gaaagcagaa aaacagggaa caaggaggac tggacagtga 12601 gccccagccc tgggggagca ccggcgggga gggcagacca gctctgtctc accttgctgt 12661 cggagtccac gcgctcatcc acagagtagg aatcatagta gagactgaag tcatcaccca 12721 ccaccgtctc ccactcctcc agggacccgg ggtccccttt cgtcagggtc acttctcctg 12781 aacgccgggc agaacctaac tcctccgact agaaaaagat cagaaaaatt gaggccactg 12841 acaccctgcg catttctact gaggatggga tgcagcccca cctctgaccc tccctcagag 12901 cagcccccga ggggtagagg ctctgcctct gctgcttacc aggccacctc ctgagttcag 12961 cttcctcctt ttggccagat ctggaagaag agagagaatg gtgtggggcc tatcaccgaa 13021 accttcagaa cagaccacat caagccaccg ggggtggggg atgggactga cctgaggtca 13081 cctttcccag tgagtggaca tcatcactca tgcggaaatg ctgtatttca gggggccgct 13141 tctcagggac cgggggctgt gggccgagag ggagcacact gagggtcaga gagcacctac 13201 agttttgcct gggttagcct ggagccccag gcgggggtgg ggtagtgagc cacacctcca 13261 aatgccatgt gaggctccag tagccacaaa ctggcaacca cgggtgctat ttcctcagag 13321 gaagagtgtc aagcacacta acactcactc atctctgcaa ccatgcagag caggcccttt 13381 tccattttac agatgagaaa acaaagctta ataaagttaa aagacctttt atatgtggcc 13441 atatacacag caggactgtt tacaacagct gaggtgcgga agcaactcaa gtgccactga 13501 cagatgaatg gataagcaaa atgtggcatt tatacacaat ggaataacat tcagccataa 13561 aaaggaaaga tatacttttt ttaagagata aggtctcatt ctgttaccct ggatggagtg 13621 cagtggcatg actatggctc acttcagcct cgaactggac tcaagccatt ctcctgcctc 13681 agcttcctgg gaagctggga ttacaggcac atgtcacaat gcctaactaa tgtcttctta 13741 attttttttt ttggtagaga agaggtcttg ccatgttgcc caggctggtc ttgaactcct 13801 ggtgtcaagt gatcctcccc agaaagtacg ggattacagg cgtgagtcac tgggcctggc 13861 ctttgaaaca ttcttttaaa cttcttttag agatggggtc ttggtatgct gcccaggtga 13921 aaggaaagaa attctgacat ggtacaacat agatgaacct tgaggacatt atgctaagtg 13981 aaataagcca gtcacaaaag gataaatact gtatgattac acttagataa agtacttact 14041 caaatttata gagaaagaaa ggacagtggt ccttgccagg ggctaggggg tggagggaat 14101 ggagagttat gttttaatgg gtacagagtt tcagttttac aagatgagtt atggtgactg 14161 atgattgcac atgatgaaag tatttaatac cattaaatta tatacttaaa aatgtttttt 14221 attttatttt taaattttta gatggagtct cactctgttg cccaagctgg agtgcagtgg 14281 cgcaatctca gttcactgca gcctctacct cccaggttca agcgtttctc tcacctctgc 14341 ctcctgagta gctggaacta caggcacatg ccaccacgcc cggctaattt ttgttttgtt 14401 tttttttttg agacagagtt ttgctcttgt tgtccaggct ggagtgcaat ggcaggatct 14461 cggctaacta caacctctgc ctcctggatt caagcgattc tcctgcctca gcctcccaag 14521 tagcggactg ttacaggcat gtaccaccat gcccggctaa ttttgtattt ttaatagaga 14581 tggggtttca ccatgttcgt ccggctggtc tcgaactcct gacctcaggt gatccacctg 14641 ccttggcctc ccaaagtgct gggattacat gcgtgaggca ccccgcctgg cctaattttt 14701 gtatttttag tagagacagg gtttcactat gttggccagg ctggtctcaa actcctgacc 14761 tcaggtgatc ctcccgcctc ggcctccaaa atgctgagat tacaggcgtg agccactgcg 14821 cctggcctaa aattgttttt tagatggtaa attttacgtg acactagtcc cctcttatcc 14881 agttcatcag cagtgatggt ggcatattgt tagaattgtg ctattttttt tgagtctcgc 14941 tctgttgccc aggctgaacc gcagtggcgc gatcttggct cactgcaagt gattctcctg 15001 cctcagcctc tcgactagct gggattacag gcgcacgcca ccacacctgg ctaatttatt 15061 attattatta ttattatttt agttagagac ggggtttgga catgtttacc aggctggtct 15121 cgaactcctg acctcaagtg attgccggcc ttggcctctg aaagtgttga gattataggc 15181 aagccacgcc tggcctactg ttaggattac gctattatgt tattattgtt gttaatctct 15241 cactgtacct aatttataaa ttcaattttc cttttcttcc ctattcttta caaaatgaat 15301 tgcaactata aaaattaatg tttatcatag tgagaaagga aaggtagctc atagcaacct 15361 gtgctatgtg aagcaggcaa aattgatcag gctcagcgag aagtcagcat ggaacggtta 15421 gggcccatgc ctggaggcaa ctgcttaaag gcattttgta cctgactagg gtgctgcttc 15481 acccattatc ttcatgtgcc taatatctgt gagacaaaga acaatgtata gcagatcaat 15541 agcttgttat tctaatgtaa actggtaaac aatttaggaa ctgcctcttc ttttcctttg 15601 ttatttcttc aatcttttaa aaaattttta tctttttttt tttctttttg cggctccttc 15661 cagagcaggg ctaactccta cgcagtgtgc ccagagtcag cctgtttttt ttcaatatct 15721 tcacgtcatc caatcttctt ttcctttaaa aacctacttg tgggctggtt gtggtggctt 15781 gcacctgtaa tcccagcact ttgcgaggtc aaggcaggaa gattgctgaa gcccagcagt 15841 ttgagaccag cctgggcaac atagtgaaac tgtcttcaaa aacaaaacaa aacaaacaaa 15901 aaaaacccta cttataactg ctgctaatca gagtgtattt tcacggcaac ttgaatcttt 15961 gctcctaaag gctgtcctca aaacctgacc aaatatactt tacttaatgt taagtttgcc 16021 tcagtttttt cctttaggtc aacaataggt atgacccaag aaccctagaa cttggtcata 16081 aagcttctgg tgcccttgtc acttccctcc tctattattt ctgtggccct catctccttt 16141 cccactggga ttcccaggaa aaactttaca aatagagcag tgacagatga gttccccaag 16201 ggcttgcttt gaggtagaaa ggaagagtgg tttgaaattc ccttaccttg tcattatcat 16261 aagagtaatt aagacattaa ctatataatt gactctttaa catcaaactt tcaccaccca 16321 agaatgtaaa ctgcaggaag agaggaacct gtctgttggt tcacagatca agcacagcct 16381 aatatttgac acacagcagc cccttgctta aatatgtgaa tgagtaaatg gagtagaagc 16441 cttaagtgaa actgtaaaag agctcaccaa aggtttatgg ttgattatcc catctctccc 16501 atcccactca cctgtccatt tcctggtttg gacatggttt tgcgggctcg gtggaccttg 16561 ggctgtccct ctgggctcgt ggtggctgga gggggttcag accctgctgc tgcagctccc 16621 tgggctcctg gcatactcag tagcctcata gccaaactct ggacagatgg aggtgatttt 16681 cccgcccctg tcattgacat cttggcccgg ctaggacagg aacccccctt gctgggggaa 16741 gaggggaatg actttgtggc atggcctaga aaacaagcaa gcaaaaggca agataagaaa 16801 gaaggcaaga gtcagaaatt tcccaccaac cccccaggct acccagcctc tcacccagca 16861 ggatccggcc cccacggagg tccccatctc cctcaagatt ctcagattca tccccaatga 16921 gtggtgtagc ccctacaggg gtgtcagccc cctcatcacc aacagtgaca gtgacagagg 16981 ctggagatga ggggccagca ggctccaggg agtcggggtt ggccttgggc agggtttctt 17041 cactacgagg ggtgtccccc aaagagccat gaactgtaga ggaagagaaa aagttcagag 17101 ctaagggctc aggagatcct gtgtttaggg aaggtgacgg tccaattggg gcccgtttta 17161 gctgcactca cctctctcgg tggctcctct ggtttccttc tccagcagca gcgcccccat 17221 ctcagcgggg gcctccccct gggaggggag acaagggaca ggagggctgg tcagcccagt 17281 agagagttgg ggggtccagg atgcctgggc cctgggaaga gagagtaggc tccggggcct 17341 acctcttcct ctgtggggcc ccccccttcc gcggcctcgg ctgcccggag gggccgcacg 17401 acccctcccc cgggcccgca tcaaccccct ccctctcggt agaccccgca tctctggggc 17461 cgagagaaga ggagggggag ggggcggggc ctccgcgccc cggccccgcc ccctcctccc 17521 ggctgcacgc gccgctcccc ctttgtcccc caggccgcgg ggaccccggg caccaacccc 17581 tccagcaccc gctgcccccc agcccggtgg acggcccctc gtgcccctca cgcgtgctcc 17641 tggggccccg gcgcccgtcg cccactcagg ggcagccggc ggctgcacgc gcgcctccgt 17701 gcccactccc cccacctccc acaccctggt cccctcatcc gcccccggtg ctggccccct 17761 ggattgctgc aagtcccgcc cgggcccccc ggccccgttg cacccccgga gcattgcacg 17821 ggcgcgcgct tcccccgggc gcgcgcgcgg gcatgcaccc gcctctcccc ctccccttcc 17881 gcacctcggc ggccgccgcc gctgcagctc ccgccgccgc cgccatcgcc gcttgc
2) Mus Musculus
SEQ ID NO: 6
Mus musculus euchromatic histone lysine N-methyltransferase 2 (Ehmt2), transcript variant long, isoform a, mRNA sequence
NM 145830.1
1 atgcggggtc tgccgagagg gagggggctg atgcgggccc gggggcgggg gcgtgcggcc 61 cccacgggcg gccgcggccg cggtcggggg ggcgcccacc gagggcgagg taggccccga 121 agcctgctct cgctgcccag ggcccaggcg tcttgggccc cccagctgcc tgccgggctg 181 accggccccc cggttccttg tctcccctcc cagggggagg cccccgctga gatgggggcg 241 ctgctgctgg agaaggagcc ccgaggagcc gccgagagag ttcatagctc tttgggggac 301 acccctcaga gtgaggagac ccttcccaag gccaaccccg actccttgga gcctgccggc 361 ccctcctctc cggcctctgt cactgtcacc gtcggcgatg agggggctga cacccctgtc 421 ggggccgcat cactcatcgg ggacgaaccc gagagcctgg agggagatgg gggtcgcatc 481 gtgctgggcc atgccacaaa gtcgttcccc tcttccccca gcaagggggg tgcctgtccc 541 agtcgggcca aaatgtcaat gacaggggca ggaaagtcgc ccccctcggt ccagagtttg 601 gccatgaggc tgttgagcat gcccggggcc cagggagctg caactgctgg gcctgaaccc 661 tctccggcaa caactgccgc ccaggagggg cagcccaaag tgcaccgagc ccggaaaacc 721 atgtccaaac ctagcaacgg acagcctcca atccctgaga agcggccccc tgaagtccag 781 catttccgca tgagtgatga catgcatctg gggaaggtga cttcagatgt ggccaaaagg 841 aggaagctga actctggtag cctgtccgag gacttgggct ctgccggggg ctcaggagat 901 ataatcctgg agaagggaga gcccaggccc ctggaggagt gggagacggt ggtgggcgat 961 gacttcagcc tgtactatga tgcgtactct gtggatgagc gggtggactc tgacagcaag 1021 tctgaagtcg aagctctagc tgaacagttg agtgaggagg aggaggagga agaggaggaa 1081 gaagaagaag aggaggagga ggaggaagag gaggaggagg aagaagagga cgaggagtcg 1141 ggcaatcagt cagacaggag cggttctagt ggccggcgca aggccaagaa gaaatggcgg 1201 aaagacagcc cgtgggtgaa gccatctaga aaacggcgga aacgagagcc tccgagggcc 1261 aaggagccaa gaggagtgaa tggtgtgggt tcctcagggc ccagtgagta catggaggtt 1321 cctctggggt ccctggagct gcccagcgag gggaccctct cccccaacca cgctggggtc 1381 tccaatgaca cgtcttcact ggagacagaa cgcgggtttg aggagctgcc cctctgcagc 1441 tgccgcatgg aggctcccaa gattgaccgc atcagcgaga gagcagggca caagtgcatg 1501 gccacagaga gtgtggatgg agagctcctg ggctgcaatg ctgccatcct taagcgggag 1561 accatgcggc cgtctagccg cgtggcgctg atggtgctct gtgaggccca tcgagcccgc 1621 atggtcaagc accattgctg cccgggctgc ggctacttct gcacagcggg caccttcctg 1681 gaatgccacc ccgactttcg tgtagctcac cgcttccata aggcctgcgt atcccagctc 1741 aatgggatgg tcttctgtcc ccactgtgga gaggatgcct cagaggccca ggaggtgacc 1801 attcctcggg gcgatggggg aacaccccca attggcaccg cagctcctgc tctgccaccc 1861 ctggcacatg atgccccagg gcgagcggat acctcccagc ctagcgcccg aatgcgaggg 1921 catggagagc cgcggcgccc gccctgtgat cccctggctg acaccatcga cagctcaggg 1981 ccttcactga ctctgcctaa tgggggctgc ctctccgctg tgggtctgcc cccagggccg 2041 ggcagggaag ccctggaaaa agccttggtc atccaggagt ctgagaggcg gaagaagctg 2101 cgattccacc cacggcagct gtacctgtcg gtgaagcagg gggagctgca gaaggtgatc 2161 cttatgctgt tagacaacct ggaccccaac ttccagagcg accagcagag caagcgcacg 2221 cccctgcacg cggccgccca gaaggggtcg gtagagatct gtcatgtgct gctgcaggca 2281 ggagccaaca tcaatgccgt agataagcaa caacgcacgc cactaatgga ggccgtggtg 2341 aacaaccacc tggaggtggc acgctacatg gtgcagttag gtggctgtgt ctacagcaag 2401 gaagaggatg gctccacctg tctacatcat gcagccaaaa ttgggaactt ggaaatggtc 2461 agcctgctac tgagcacagg acaggtggac gtcaatgccc aggacagtgg gggctggacg 2521 cccatcatct gggcagccga gcacaagcac atcgatgtga ttcgtatgct gctgacccgg 2581 ggtgccgatg tcaccctgac tgacaatgag gaaaacatct gcctgcactg ggcctccttc 2641 acgggtagtg ccgccatcgc tgaggtcctt ctgaatgccc agtgtgatct ccatgctgtc 2701 aactaccatg gggacacgcc cctgcacata gccgccaggg agagctacca tgactgtgtt 2761 ctgttgttcc tgtctcgtgg agccaaccct gagcttcgga acaaagaagg agacacggca 2821 tgggatctga ccccagagcg ctctgatgtg tggtttgcac tgcagctcaa tcgaaagctt 2881 aggcttgggg tagggaaccg ggctgtccgc accgagaaga tcatctgccg ggacgtagcc 2941 cgaggctatg agaatgtacc catcccctgt gtcaatggtg tggatgggga gccgtgcccg 3001 gaggactaca agtacatctc tgagaactgc gagacatcga ccatgaacat cgaccgcaac 3061 atcacccatc tgcagcactg cacgtgtgtg gatgactgct ccagctccaa ttgcctatgt 3121 ggtcagctca gtatccgatg ctggtatgac aaggacgggc ggctgctcca ggagtttaac 3181 aagatcgagc cccccctgat ctttgagtgt aaccaggcat gctcctgctg gagaagctgc 3241 aagaaccgcg tggtgcagag cggcatcaag gtacggctgc agctctaccg gactgccaag 3301 atgggctggg gggtccgagc cttgcagacc atcccccagg gcacgttcat ctgcgagtat 3361 gtaggagagc tgatctctga tgccgaggct gatgtgagag aggatgattc ttacctcttc 3421 gatttagata acaaggatgg cgaggtttac tgcattgatg cccgttacta tggcaacatc 3481 agccgattca ttaaccacct gtgtgacccc aacatcatcc ctgtccgggt tttcatgctg 3541 caccaagatc tacggttccc acgcattgcc ttcttcagct ccagggacat ccggactggg 3601 gaggagctgg gctttgacta cggtgaccga ttctgggaca tcaagagcaa gtatttcacc 3661 tgccagtgtg gctctgagaa gtgcaagcat tcagcggagg ccatcgccct ggagcagagc 3721 cgcctggccc ggctggaccc ccacccggag ctgctccctg acctcagctc cctgcccccc 3781 atcaacacct gaggactctt aaaatccagg ccgggcactg cccttcagac atttctccat 3841 cagagacccc agtaaggcct ggaaggtcga tggcccctct cccagagctg gtttctcact 3901 gggagtgcaa gtgacttcag ggctggcctt ccccactgag cctggcctca gttagctgat 3961 tgaagttggg cctctgccag ctgattttct gtgttctcaa taaatgttgg gtttggtaaa 4021 aaaaaa
SEQ ID NO: 7
Mus musculus histone- lysine N-methyltransferase EHMT2 isoform G9a long, isoform a, amino acid sequence
NP_665829.1
1 mrglprgrgl mrargrgraa ptggrgrgrg gahrgrgrpr sllslpraqa swapqlpagl
61 tgppvpclps qgeapaemga lllekeprga aervhsslgd tpqseetlpk anpdslepag
121 psspasvtvt vgdegadtpv gaasligdep eslegdggri vlghatksfp sspskggacp
181 srakmsmtga gksppsvqsl amrllsmpga qgaatagpep spattaaqeg qpkvhrarkt
241 mskpsngqpp ipekrppevq hfrmsddmhl gkvtsdvakr rklnsgslse dlgsaggsgd
301 iilekgeprp leewet vgd dfslyydays vdervdsdsk sevealaeql seeeeeeeee
361 eeeeeeeeee eeeeeedees gnqsdrsgss grrkakkkwr kdspwvkpsr krrkreppra
421 keprgvngvg ssgpseymev plgslelpse gtlspnhagv sndtsslete rgfeelplcs
481 crmeapkidr iseraghkcm atesvdgell gcnaailkre tmrpssrval mvlceahrar
541 mvkhhccpgc gyfctagtfl echpdfrvah rfhkacvsql ngmvfcphcg edaseaqevt
601 iprgdggtpp igtaapalpp lahdapgrad tsqpsarmrg hgeprrppcd pladtidssg
661 psltlpnggc lsavglppgp grealekalv iqeserrkkl rfhprqlyls vkqgelqkvi
721 lmlldnldpn fqsdqqskrt plhaaaqkgs veichvllqa ganinavdkq qrtplmea v
781 nnhlevarym vqlggcvysk eedgstclhh aakignlemv slllstgqvd vnaqdsggwt 841 piiwaaehkh idvirmlltr gadvtltdne eniclhwasf tgsaaiaevl lnaqcdlhav
901 nyhgdtplhi aaresyhdcv llflsrganp elrnkegdta wdltpersdv wfalqlnrkl
961 rlgvgnravr tekiicrdva rgyenvpipc vngvdgepcp edykyisenc etstmnidrn
1021 ithlqhctcv ddcsssnclc gqlsircwyd kdgrllqefn kiepplifec nqacscwrsc
1081 knr vqsgik vrlqlyrtak mgwgvralqt ipqgtficey vgelisdaea dvreddsylf
1141 dldnkdgevy cidaryygni srfinhlcdp niipvrvfml hqdlrfpria ffssrdirtg
1201 eelgfdygdr fwdikskyft cqcgsekckh saeaialeqs rlarldphpe llpdlsslpp 1261 int
SEQ ID NO: 8
Mus musculus euchromatic histone lysine N-methyltransferase 2 (Ehmt2), transcript variant short, mRNA, isoform b
NM 147151.1
1 atggcggcgg cggcgggagc tgctgcggcg gcggccgccg agggggaggc ccccgctgag 61 atgggggcgc tgctgctgga gaaggagccc cgaggagccg ccgagagagt tcatagctct 121 ttgggggaca cccctcagag tgaggagacc cttcccaagg ccaaccccga ctccttggag 181 cctgccggcc cctcctctcc ggcctctgtc actgtcaccg tcggcgatga gggggctgac 241 acccctgtcg gggccgcatc actcatcggg gacgaacccg agagcctgga gggagatggg 301 ggtcgcatcg tgctgggcca tgccacaaag tcgttcccct cttcccccag caaggggggt 361 gcctgtccca gtcgggccaa aatgtcaatg acaggggcag gaaagtcgcc cccctcggtc 421 cagagtttgg ccatgaggct gttgagcatg cccggggccc agggagctgc aactgctggg 481 cctgaaccct ctccggcaac aactgccgcc caggaggggc agcccaaagt gcaccgagcc 541 cggaaaacca tgtccaaacc tagcaacgga cagcctccaa tccctgagaa gcggccccct 601 gaagtccagc atttccgcat gagtgatgac atgcatctgg ggaaggtgac ttcagatgtg 661 gccaaaagga ggaagctgaa ctctggtagc ctgtccgagg acttgggctc tgccgggggc 721 tcaggagata taatcctgga gaagggagag cccaggcccc tggaggagtg ggagacggtg 781 gtgggcgatg acttcagcct gtactatgat gcgtactctg tggatgagcg ggtggactct 841 gacagcaagt ctgaagtcga agctctagct gaacagttga gtgaggagga ggaggaggaa 901 gaggaggaag aagaagaaga ggaggaggag gaggaagagg aggaggagga agaagaggac 961 gaggagtcgg gcaatcagtc agacaggagc ggttctagtg gccggcgcaa ggccaagaag 1021 aaatggcgga aagacagccc gtgggtgaag ccatctagaa aacggcggaa acgagagcct 1081 ccgagggcca aggagccaag aggggtctcc aatgacacgt cttcactgga gacagaacgc 1141 gggtttgagg agctgcccct ctgcagctgc cgcatggagg ctcccaagat tgaccgcatc 1201 agcgagagag cagggcacaa gtgcatggcc acagagagtg tggatggaga gctcctgggc 1261 tgcaatgctg ccatccttaa gcgggagacc atgcggccgt ctagccgcgt ggcgctgatg 1321 gtgctctgtg aggcccatcg agcccgcatg gtcaagcacc attgctgccc gggctgcggc 1381 tacttctgca cagcgggcac cttcctggaa tgccaccccg actttcgtgt agctcaccgc 1441 ttccataagg cctgcgtatc ccagctcaat gggatggtct tctgtcccca ctgtggagag 1501 gatgcctcag aggcccagga ggtgaccatt cctcggggcg atgggggaac acccccaatt 1561 ggcaccgcag ctcctgctct gccacccctg gcacatgatg ccccagggcg agcggatacc 1621 tcccagccta gcgcccgaat gcgagggcat ggagagccgc ggcgcccgcc ctgtgatccc 1681 ctggctgaca ccatcgacag ctcagggcct tcactgactc tgcctaatgg gggctgcctc 1741 tccgctgtgg gtctgccccc agggccgggc agggaagccc tggaaaaagc cttggtcatc 1801 caggagtctg agaggcggaa gaagctgcga ttccacccac ggcagctgta cctgtcggtg 1861 aagcaggggg agctgcagaa ggtgatcctt atgctgttag acaacctgga ccccaacttc 1921 cagagcgacc agcagagcaa gcgcacgccc ctgcacgcgg ccgcccagaa ggggtcggta 1981 gagatctgtc atgtgctgct gcaggcagga gccaacatca atgccgtaga taagcaacaa 2041 cgcacgccac taatggaggc cgtggtgaac aaccacctgg aggtggcacg ctacatggtg 2101 cagttaggtg gctgtgtcta cagcaaggaa gaggatggct ccacctgtct acatcatgca 2161 gccaaaattg ggaacttgga aatggtcagc ctgctactga gcacaggaca ggtggacgtc 2221 aatgcccagg acagtggggg ctggacgccc atcatctggg cagccgagca caagcacatc 2281 gatgtgattc gtatgctgct gacccggggt gccgatgtca ccctgactga caatgaggaa 2341 aacatctgcc tgcactgggc ctccttcacg ggtagtgccg ccatcgctga ggtccttctg 2401 aatgcccagt gtgatctcca tgctgtcaac taccatgggg acacgcccct gcacatagcc 2461 gccagggaga gctaccatga ctgtgttctg ttgttcctgt ctcgtggagc caaccctgag 2521 cttcggaaca aagaaggaga cacggcatgg gatctgaccc cagagcgctc tgatgtgtgg 2581 tttgcactgc agctcaatcg aaagcttagg cttggggtag ggaaccgggc tgtccgcacc 2641 gagaagatca tctgccggga cgtagcccga ggctatgaga atgtacccat cccctgtgtc 2701 aatggtgtgg atggggagcc gtgcccggag gactacaagt acatctctga gaactgcgag 2761 acatcgacca tgaacatcga ccgcaacatc acccatctgc agcactgcac gtgtgtggat 2821 gactgctcca gctccaattg cctatgtggt cagctcagta tccgatgctg gtatgacaag 2881 gacgggcggc tgctccagga gtttaacaag atcgagcccc ccctgatctt tgagtgtaac 2941 caggcatgct cctgctggag aagctgcaag aaccgcgtgg tgcagagcgg catcaaggta 3001 cggctgcagc tctaccggac tgccaagatg ggctgggggg tccgagcctt gcagaccatc 3061 ccccagggca cgttcatctg cgagtatgta ggagagctga tctctgatgc cgaggctgat 3121 gtgagagagg atgattctta cctcttcgat ttagataaca aggatggcga ggtttactgc 3181 attgatgccc gttactatgg caacatcagc cgattcatta accacctgtg tgaccccaac 3241 atcatccctg tccgggtttt catgctgcac caagatctac ggttcccacg cattgccttc 3301 ttcagctcca gggacatccg gactggggag gagctgggct ttgactacgg tgaccgattc 3361 tgggacatca agagcaagta tttcacctgc cagtgtggct ctgagaagtg caagcattca 3421 gcggaggcca tcgccctgga gcagagccgc ctggcccggc tggaccccca cccggagctg 3481 ctccctgacc tcagctccct gccccccatc aacacctgag gactcttaaa atccaggccg 3541 ggcactgccc ttcagacatt tctccatcag agaccccagt aaggcctgga aggtcgatgg 3601 cccctctccc agagctggtt tctcactggg agtgcaagtg acttcagggc tggccttccc 3661 cactgagcct ggcctcagtt agctgattga agttgggcct ctgccagctg attttctgtg 3721 ttctcaataa atgttgggtt tggtaaaaaa aaaaaaa
SEQ ID NO: 9
Mus musculus histone- lysine N-methyltransferase EHMT2 isoform G9a short, isoform b, amino acid sequence
NP 671493.1
1 maaaagaaaa aaaegeapae mgalllekep rgaaervhss lgdtpqseet lpkanpdsle
61 pagpsspasv tvtvgdegad tpvgaaslig depeslegdg grivlghatk sfpsspskgg 121 acpsrakmsm tgagksppsv qslamrllsm pgaqgaatag pepspattaa qegqpkvhra 181 rktmskpsng qppipekrpp evqhfrmsdd mhlgkvtsdv akrrklnsgs lsedlgsagg 241 sgdiilekge prpleewetv vgddfslyyd aysvdervds dskseveala eqlseeeeee 301 eeeeeeeeee eeeeeeeeed eesgnqsdrs gssgrrkakk kwrkdspwvk psrkrrkrep 361 prakeprgvs ndtssleter gfeelplcsc rmeapkidri seraghkcma tesvdgellg 421 cnaailkret mrpssrvalm vlceahrarm vkhhccpgcg yfctagtfle chpdfrvahr 481 fhkacvsqln gmvfcphcge daseaqevti prgdggtppi gtaapalppl ahdapgradt 541 sqpsarmrgh geprrppcdp ladtidssgp sltlpnggcl savglppgpg realekalvi 601 qeserrkklr fhprqlylsv kqgelqkvil mlldnldpnf qsdqqskrtp lhaaaqkgsv 661 eichvllqag aninavdkqq rtplmea vn nhlevarymv qlggcvyske edgstclhha 721 akignlemvs lllstgqvdv naqdsggwtp iiwaaehkhi dvirmlltrg advtltdnee 781 niclhwasft gsaaiaevll naqcdlhavn yhgdtplhia aresyhdcvl Iflsrganpe 841 lrnkegdtaw dltpersdvw falqlnrklr lgvgnravrt ekiicrdvar gyenvpipcv 901 ngvdgepcpe dykyisence tstmnidrni thlqhctcvd dcsssnclcg qlsircwydk 961 dgrllqefnk iepplifecn qacscwrsck nr vqsgikv rlqlyrtakm gwgvralqti 1021 pqgtficeyv gelisdaead vreddsylfd ldnkdgevyc idaryygnis rfinhlcdpn 1081 iipvrvfmlh qdlrfpriaf fssrdirtge elgfdygdrf wdikskyftc qcgsekckhs 1141 aeaialeqsr larldphpel lpdlsslppi nt SEQ ID NO: 10
Mus musculus strain C57BL/6J chromosome 17, MGSCv37 C57BL/6J, G9a genomic sequence
NC 000083.5
1 atggcggcgg cggcgggagc tgctgcggcg gcggccgccg aggtgcggaa ggggaggggg 61 agaggcgggt gcatgcccgc gcgcgcgccc gggggaagcg cgcgctcgtg caatgctccg 121 ggggtgcaac ggggccgggg ggcccgagcc gaacttacag caaaccaggg ggccaacacc 181 gggggcggat gaggggacca gggggtggga ggtgggggga gtgggcgccg agtcgcacgt 241 gccgccgccc gctgcccctg cgtgggcggt gggcaccgcg accccgggag cccgcgcgag 301 gggcgcgagg ggccgtccgc cgggctgggg cgcagcgggc gttgcagggg ttggtggccg 361 gggtccccgc ggcctggggg acaaaggggg agcggcgcgt gcagccggga ggagggggcg 421 gggccggggc gcggaggccc cgccccctcc ccctcctctg ctcccggccc cagagatgcg 481 gggtctgccg agagggaggg ggctgatgcg ggcccggggg cgggggcgtg cggcccccac 541 gggcggccgc ggccgcggtc gggggggcgc ccaccgaggg cgaggtaggc cccgaagcct 601 gctctcgctg cccagggccc aggcgtcttg ggccccccag ctgcctgccg ggctgaccgg 661 ccccccggtt ccttgtctcc cctcccaggg ggaggccccc gctgagatgg gggcgctgct 721 gctggagaag gagccccgag gagccgccga gagaggtgag tgcagcagga gcaggcccga 781 ccggacctcc accctcccca cccccaacac gggatcccgg gagctttcag ccctgaactt 841 tttcttttcc cctcccgaca gttcatagct ctttggggga cacccctcag agtgaggaga 901 cccttcccaa ggccaacccc gactccttgg agcctgccgg cccctcctct ccggcctctg 961 tcactgtcac cgtcggcgat gagggggctg acacccctgt cggggccgca tcactcatcg 1021 gggacgaacc cgagagcctg gagggagatg ggggtcgcat cgtgctgggt gagagactgg 1081 ggagtgaggg agacggggcg cagaggggct ttctgcatgc ctgctcaccc tgcaccccct 1141 ttctgcctgc ctcttttcta ggccatgcca caaagtcgtt cccctcttcc cccagcaagg 1201 ggggtgcctg tcccagtcgg gccaaaatgt caatgacagg ggcaggaaag tcgcccccct 1261 cggtccagag tttggccatg aggctgttga gcatgcccgg ggcccaggga gctgcaactg 1321 ctgggcctga accctctccg gcaacaactg ccgcccagga ggggcagccc aaagtgcacc 1381 gagcccggaa aaccatgtcc aaacctagca acggacaggt gagagtgtgg aagttggggt 1441 ggagagactt ggggagtctc tgggtcggat tttttgtttt gttttgtttt tgttggtggt 1501 ttgttctttt gggttttttt tttttttttt ttccttcttc cgggatttag ccttagacct 1561 cttgtattca gatttttttt ttcgaattta ccaataggct accaagtgtc agctgttggg 1621 ctgggcctct gtgagcagag taattgtcgg cttcctggag tttgtgttct ttggggaggg 1681 gtggtcagca gaggagactg acagatgtag aattaattat atagttattg tcctaattac 1741 acaaatgact gggcaggatc agccttccca agtttgtgtt ttttttgttt tgttttttgc 1801 ctctgaacaa atgcttaggg aggttggttt gttcgtttgt ttgtttgttt gtttgtttgt 1861 ttttcaaatc cctactttga gaccagtctc aaaccagata aagtttttac tgggaactct 1921 tccctgggaa ggccacataa tgacagaatg tgggcggagg cgtgtgtgtg tgtgtgtgtg 1981 tgtgtgtgtg tgacaagaac ctggaaatca aaattatgtc aaggttttaa gggctcttga 2041 ttcagcttaa gtatggcttg tgtagccttc ctctgggtct tgtttcgttt attttgttgt 2101 ttgttttgag ctttattttg ttttgagaga gtagtttttg ttttttgttg ttttgtgtgt 2161 gtgtgtgtgt gtaacagggt ctcactgtgt agccctggct ggctggctgt gtagaccagg 2221 ctggccttta actcacagag atctaccttt ttctgtctcc tacatcctgg ggctgaaggt 2281 gtacacacat gccttttatt tataatcttt tttttaaaaa agatttattt atttatttta 2341 tgtatatgaa tatactgtaa ctgtcttcag acacaccaga agagggaatc agatcccatt 2401 acagatggtt gtgagccaca ccatgtggtt gctgggattt gaactcaaga cctctggaag 2461 agcagtcagt gctcttaact gctgagccat atctctggcc cttgttttat ttcctctttt 2521 tgagatggtc tcatgtagac tgaacttgct atgatccacc ccacccagct tctacattta 2581 atttttttct ttcttttttt ttttctttac tctttctttt tttaaaaact tttaagcttc 2641 tgggtgtttt acctgcacat atgtctgtgt accaccttgt gtgtctggtg cccttcaagt 2701 caagggggca gtagaccttg agtttgaagg ggttgtgagc atcttgtcag tgctgggcaa 2761 ttggacctgg gtcttccaca agagcagcaa ccggctctta accactgcac tacctctccg 2821 acaatgcttg ttttcttttt ctgggaaagt gattacctca ccatggtgtg gctgttcttc 2881 tctggtacag aagttcgtgt ttgctcgggc tcctcatata aaattatgag agccaggtgg 2941 atctcttaag gtctgggtca gctaagaatc aacatttgca tatgccttac acgtatctgt 3001 cttccttgta gataccttat agatgattag ctaatgcctg tattagtagt ttaaagactc 3061 ctatatgggc acaactttta aaattttatc tgttgctggg tggtgctggc tcacgcctct 3121 aatcccagtt cttgggagac tgaggcaggc agatctctga gttcaaggcc agcctagtta 3181 gagtatacac aattccttag acagccaagt ctatatagtg agttccaggg cagccaaggc 3241 tacacaggga aatcctgtct tgaaagaaaa atattgatta taagtgtgta tgtgtaatgg 3301 gggaggttga ggggatggac gcagtagagg acatatctgt ggagcctggt ctcttccacc 3361 ttttcatgcc atctaggagt cagactcaag ttttagggtt tgcgcagcaa gcacccctgc 3421 ccactgagcc atcttgctgg ccactgttct ctttttaatt tttttttttt tttttttttt 3481 ggtttttcga gacagggttt ctctgtatag tcctggctgt cctggaactc actctgtaga 3541 ccaggctggc ctcgaactca gaaatccgcc tatctctgcc tcccaagtgc tgggattaaa 3601 ggcgtgcacc accacgcccg gctttcgggt agcatttttt taaaaactca tttctttttt 3661 tttttttttt taagatttat gggagttccc tgtcgctgtc ttcagacaca ccagaagagg 3721 gcatcagatc tcattacaca tggtcgtgag ccaccatgta gttgctggga tttgaactca 3781 ggacctctgg aagagcagtc agtgctctta accctgagcc atccctccag cccttcggat 3841 agctttgttt gtttgttttt tttttgtttg tttgtttttt aatgtatgtg aatacactgt 3901 acagatggtt gtgagccttc atgtggttgt tgggaatcga actcttagga cctctgctcc 3961 ctctggtcaa ccctactcac ttcagtcagc cccactagct ctggcccaaa gctttactta 4021 cttttatatg taagtacact gtagctgtct tcagacgaac cagaagaagg catcagatcc 4081 cattacacat ggtcatgagc caccatgtgg ttgctgggat ttgaactcag gaccttcaga 4141 agggcagtca gtgctcttaa ccactaagcc atcttgccag tcccccagat agcatttttg 4201 aggtacagcc agccacaata ctcccacctg ggccccaggc tgagggaaca gacccataac 4261 tttaatttgg tggctcactt atttcactta gcacagtgcc ttcaagtttc ctctatgttg 4321 tatatttcca ggctcccttt ttgtccttgg gtgttctgtt tgtttttgag atagggtctc 4381 actcactcta tagccctagc tggcctgggt ctcggatccg cctgcctccc ggagtgctgg 4441 aatgaaaggt gtagcccacc acacctagcc tgggtgagta accttctgac tgaaacgtgt 4501 tccttcctgc ttcagcctcc aatccctgag aagcggcccc ctgaagtcca gcatttccgc 4561 atgagtgatg acatgcatct ggggaaggtg acttcaggtc agtcacctct gctcctccca 4621 cacactccag gtggcctgcg tagtctatcc caggattgag agttcacttt tctgtctttc 4681 cagatgtggc caaaaggagg aagctgaact ctggtagcct ggtgagctgg agaggaagac 4741 ctacctggtg ggggtgggag cggaggggag gggcggattg gtgggctgga gccaggaacc 4801 tcagactctg tcatcttttt ctagtccgag gacttgggct ctgccggggg ctcaggagat 4861 ataatcctgg agaagggaga gcccaggccc ctggaggagt gggagacggt ggtgggcgat 4921 gacttcagcc tgtactatga tgcgtactct gtggatgagc gggtggactc tgacagcaag 4981 gtgtgctggt gtgctcttct caagcttccc tacccagtct gttctttccc tttaatagct 5041 ctagttcacc gccctgtgtt gtctgcaact gtttcttata tcccttatct catctctggt 5101 cctctcattc caacccagcc tcctccctgc cttgtttgct ccctttgtgt tgtttgctcc 5161 gttggctttt ggctagtttt gtttgtttga cagggtctca ctagtagacc aagctggcct 5221 caaattccta gaaatctgcc tgcctttgcc tctttagtgt tgggttgaaa ggcacatact 5281 gccacactgt gtgtgcgctc atcctggggt tagaggacag catctcgggc ttggttctct 5341 ttcgatgtag ctctggggat cttaatctct tacagtttgt gggtttgagc tttcagaatt 5401 ttcccccgtg tttgaggttg agcctggcgc tctgcaagtg cattgtaaca ggcctgtgct 5461 ttatttttgc tgattgaggg attaaactca aggcctcgca gggcgagcaa gcactgagct 5521 gctacttaaa tatttgattt caggcctcat tctctaccaa cttgcctggg aatggcccgg 5581 ccttaaccca ctcctgtagt ctgtctgcct cagcctcccg agtggctggg gttaaaggca 5641 tgctttttct aaatggggat ggggcttgag acagagtctc actttatttt tttaaagatt 5701 tatttatttt atgtatatga gtatactgta gctgtacagc tggttgtgag ccttcatgtg 5761 gttgttagga attgaatttt aggacctctg cttgctccgg tcaaccccgc tcactcagtc 5821 actgatcatt ctggcccaaa gatttattta ttattataca taagtacact gtagctaact 5881 tcagacacac cagaagaggg tgtcagatct cattacatga tggttgtgag ccaccatgtg 5941 gttgctggga tttgaactta ggacctgcag aagagcagtc agtactctta cccactgagc 6001 catctcgcca gccccagctc ctgagctgat cctcttgctt ctgtctccac gtttggacta 6061 caggcatgtg ccgccatgtc tgctgctgct tctgcttttt gtttttgatt ttttgggggg 6121 aagaaaatgt ggcagtggtt gcaggtgcct ccttcctatt gcctgtcttt ggaagctgtg 6181 tagggtgttt ggtcagttct tgtgctggtc agtgttgaca tgctccagag gcagaggcgt 6241 aggatctctg tgagttccag gtagtggcca ggactaccta gtgaggccct gtctccaaac 6301 aatcaatcaa aaagaaaact ttgacattgg gcctgtggtt tccggtgatg gcagcctgac 6361 cccttcgtta gagagcacct ccttagcttt tcgtggagtg tggttggttt tgttgccatg 6421 ggtagtcttg tcagagtgtt tcctaagacc ggcccttgtc actgctgtgt ggcattctgt 6481 gcctctgtcc ctatccaccc tttcctgcct cctgcccaca tcccccaccg ggagctttcc 6541 ttgggggggg ggggggtgat gatatggggc ctcagcagtc accgagagag ctgcctttgc 6601 tggtttccca gcctgatttt tctttttcag tctgaagtcg aagctctagc tgaacagttg 6661 agtgaggagg aggaggagga agaggaggaa gaagaagaag aggaggagga ggaggaagag 6721 gaggaggagg aagaagagga cgaggagtcg ggcaatcagt cagacagggt aagagcagag 6781 accggcctac ctggaggagt gggggagctg ggagcagagc tcttgagtct tcctcttcca 6841 cagagcggtt ctagtggccg gcgcaaggcc aagaagaaat ggcggaaaga cagcccgtgg 6901 gtgaagccat ctagaaaacg gcggaaacga gagcctccga gggccaagga gccaagaggt 6961 aagacggctc ctctgctctc aggtcccctc ctccggcccc agagagcagc acacactcgg 7021 atgctcgtgc atgttcactg tgtagaccgc atgtgcgcac gcgcccagga cctgtgagcc 7081 cccactctca ctctctctgt ctctgtgtca ggagtgaatg gtgtgggttc ctcagggccc 7141 agtgagtaca tggaggttcc tctggggtcc ctggagctgc ccagcgaggg gaccctctcc 7201 cccaaccacg ctggtaatta attgccaact gccgggacag ggagccacca gggggtgccc 7261 atgggggtgg agggaaaccc tgccacagct ggggtgtcca tgccaggctt tggggttcta 7321 gaacatggtg gggtggctgg gcggaaagga ggggaccctc ccagtgggtg aggggcaagg 7381 cctccatcat ctctgtctct tcctgtctaa ggggtctcca atgacacgtc ttcactggag 7441 acagaacgcg ggtttgagga gctgcccctc tgcagctgcc gcatggaggc tcccaagatt 7501 gaccgcatca gcgagagagc agggcacaag tgcatggcca cagagagtgt ggatggagag 7561 gtgggtctgg gtacggttgg cttagggggc ccagggtgag ctagcctggg ccctggcctc 7621 acccacatgc ccccaccttc cttcatccct agctcctggg ctgcaatgct gccatcctta 7681 agcgggagac catgcggccg tctagccgcg tggcgctgat ggtgctctgt gaggcccatc 7741 gagcccgcat ggtcaagcac cattgctgcc cgggctgcgg ctacttctgc acagcggtga 7801 gtgaccattg gggtggatgg gcggtctggc cagcaccgga gagctccggg gctgactgct 7861 gcctcttccc ttccccaggg caccttcctg gaatgccacc ccgactttcg tgtagctcac 7921 cgcttccata aggcctgcgt atcccagctc aatgggatgg tcttctgtcc ccactgtgga 7981 gaggatgcct cagaggccca ggaggtgacc attcctcggg gcgatggggg aacaccccca 8041 attggcaccg cagctcctgc tctgccaccc ctggcacatg atgccccagg gcgagcggat 8101 acctcccagc ctaggtattg gcctctccac caccaccacc acccggttat ccatcttatc 8161 tcccttgccc ccagacctca aaaccttttt ccacagcgcc cgaatgcgag ggcatggaga 8221 gccgcggcgc ccgccctgtg atcccctggc tgacaccatc gacagctcag ggccttcact 8281 gactctgcct aatgggggct gcctctccgc tgtgggtctg cccccagggc cgggcaggga 8341 agccctggaa aaagccttgg tcatccagga gtctgagagg tgagtttgtg ctgttttcag 8401 ggtgtgactg ggccagaaac gcaggtgttg ctgtcggtac ttaaatctga gcagcccacc 8461 ttctccccct ccatcccctg acaggcggaa gaagctgcga ttccacccac ggcagctgta 8521 cctgtcggtg aagcaggggg agctgcagaa ggtgatcctt atgctgtgtg agtgtctgac 8581 cccaccccga gggctgtgcg tgaaagggaa ggaccctcgg tggcggcagc agtggtaacc 8641 tgttggtaat gggcactgat ccagtgcaca acttataagc tttcccggcc tggaggctga 8701 gtgttcctct agtgcggggc agtactgagg ggctggcacg gcttgcctag tggggaatcc 8761 tgggaagtct ctcttggtac agggctgtcc cacccccagc tctcttacct cttgtctcat 8821 ctctgcctgc agtagacaac ctggacccca acttccagag cgaccagcag agcaagcgca 8881 cgcccctgca cgcggccgcc cagaaggggt cggtagagat ctgtcatgtg ctgctgcagg 8941 tcagtcctca tgacccttcc cactgtccaa cccctcccca gcatcccctc tccctggtgc 9001 cagccttcac actgtcctca caggcaggag ccaacatcaa tgccgtagat aagcaacaac 9061 gcacgccact aatggaggcc gtggtgaaca accacctgga ggtggcacgc tacatggtgc 9121 agttaggtgg ctgtgtctac agcaaggtgc gggatggcgc tggagagctg ggtggtggga 9181 ggaagaaggg atacctgggc acggggtgct agctgaaggg ttcactggct cgtgtttccc 9241 tcaggaagag gatggctcca cctgtctaca tcatgcagcc aaaattggga acttggaaat 9301 ggtcagcctg ctactgagca caggacaggt ggacgtcaat gcccaggtta gcaacctggc 9361 tgcccctcct gtacacagat tctttggcta tctgtccctt gggcatcagt cccgcttctt 9421 cctcagctga cctgaccctc ccccactacc tcccactgcc cacgttgatt ccctcatttt 9481 tttggttcat ggaactcgac tcccatctct tatctctgag tgcgtccagt gaggttccct 9541 gtgagcattg gggggcgggg gtgcatctgt ggatacccat tctctggctg tggttgtttc 9601 cagaggtgcc cctgagctcc tattttctgg ccagcaagtc tgggccctca ttcccttcct 9661 ccttccttcc ctcaggacag tgggggctgg acgcccatca tctgggcagc cgagcacaag 9721 cacatcgatg tgattcgtat gctgctgacc cggggtgccg atgtcaccct gactgacaat 9781 gtgagggaca ctcaaggggg ctcagagaac ttggggtggg tagcagcggg acccctatgc 9841 agagtggggg ctcagttcaa ggattgtgtc acctgcaacc aagtctctgc ctctcacctc 9901 cacaggagga aaacatctgc ctgcactggg cctccttcac gggtagtgcc gccatcgctg 9961 aggtccttct gaatgcccag tgtgatctcc atgctgtcaa ctaccatggg gacacgcccc 10021 tgcacatagc cgccagggag agctaccatg actgtgttct gtaagcccta gcttccccaa 10081 cccctggcac ccacacccca ctgtccaatc tctgacctag gccttatgtt cctcttcagg 10141 ttgttcctgt ctcgtggagc caaccctgag cttcggaaca aagaaggaga cacggcatgg 10201 gatctgaccc cagagcgctc tgatgtgtgg tttgcactgc agctcaatcg aaagcttagg 10261 cttggggtag ggaaccgggc tgtccgcacc gagaagatca tctgccggtg agtgcagtcc 10321 agacatccat gcccactgcc acggcccacg ttggccagtc ccaggcccct tcccaggcac 10381 aggacatccc gagaattgct agaatcatct tgccactagg ggaggtggaa ataagggact 10441 cagccagtga gaccctcagt ggacaaggga gcctgtggcc aagcctaaca gaggtcagtc 10501 cctggactca catggagaaa gtcaactcct ccaaattgtc ctctggctta caaacacacc 10561 tcatgacact ctcacacaca gttgaaaaaa gctgagggca gggtgtggtg acccacacct 10621 gtaatctcag catgaaggag gcagacagct gattgttttg aacttgaggc cagcctgctt 10681 tgtatctatg taataagttc caggccaaca agggccacat aatgagacct taatctttaa 10741 acaaaagaaa gaggagacgg gcattggtgg tgcacacctt taatcccagc acttgggacg 10801 caggggcagg tggatttctg agtttgagga cccctggtct acaaagtgag ttccaggaca 10861 gccagggctg cacagagaaa ccctgtctca gaaaacaaac aaaaaaggac aagagaacaa 10921 aaaggacatg ttttccaagc cttcgaggtg gtggaggctg gaggatttca agtttgaggc 10981 agacacccag atagataaga atttcagaga ggagctaggt gttacttgtg atcccttcta 11041 ttctgaaggc caaggaggaa gactacctct tgagcctgca tattggaggc tcaatgagac 11101 tcaaaactta aaaaaggaaa aacaaacaaa acaaaaacaa ccacaaagct agtgtgaggc 11161 ggcccaactg ctgaaagtgc gtgtggagtt taagccatga acccaccaga aggagagagc 11221 tgcctcccga ggactgccct ctgagctttc catctcattg tatcctgtgc acaccacact 11281 cagacatgag gctgctcggg ccaggcggag agtggtgtag acctttaatc tcgctctgga 11341 ggcagaggca gaggcagagg caggctgatt tctgggttcc agggctgtac agtgagatgc 11401 tgtctcaaag aaggaggtgg gttgccgggc gtggtggtgc actcctttaa tcccagcact 11461 caggaggcag atgcaggggg atttctgagt ttgaggccag cctggtctac aaagtgagtt 11521 ccaggacagc cagggctata cagagaaacc ctgtctcaaa aaaaccaacc acaaaaacca 11581 acaaaacaaa aagatatgta agtttgtaat ccagatgtgt tctgtgcaag ctgagtactt 11641 tggtgtttgt ctgtccttat agcatctgag gaggcagagg cgggaaaatc ataacttcac 11701 gtttaaggcc agctggggca ttgtagcaag actcagtctc cagctaaaat ttaaaagtcc 11761 agagtccatt ggggtggtga catatgcctc tgaccctagc actcgggagc cagggcagga 11821 gaatagactg tcgaagtcat tctctgctat ctttcagtct ggggccagcc ggggctatag 11881 gatgctgtat ctcaccaagg aatgagaaaa ggtcggggct gggaacatcg gtcagtgggc 11941 gagcatttat tggcctagta tgcaccgtgc tgttgggctc agtctgcatt ctcaccaaga 12001 aacagaaata acccagtcac tacttgggac ataactattt cttagccgag attgttctcc 12061 gtggtgtctt tgtttgtttt caagaacgtc tccctgcatg tgctgagata aaagtgccag 12121 cacccttaga tcaaggaatc ctcccctcag aactactctg ccccactttg accttatctg 12181 ccccttctgt agggacgtag cccgaggcta tgagaatgta cccatcccct gtgtcaatgg 12241 tgtggatggg gagccgtgcc cggaggacta caagtacatc tctgagaact gcgagacatc 12301 gaccatgaac atcgaccgca acatcaccca tctgcaggta aggcacagcc ccgctcacca 12361 tgctgtggga gcacctccac accaggatgg ggggcatgaa gactccccct cacactggga 12421 ccaccaccac tcactgtcct ggaacatgaa ccccactcac tcctactttt ctcccacagc 12481 actgcacgtg tgtggatgac tgctccagct ccaattgcct atgtggtcag ctcagtatcc 12541 gatgctggta tgacaaggta cgtgaccctc acctggcagg tctgccaagg agccaggacc 12601 ctgctgtgtt ggggaagaga cacagggtgt ggcaggaagc cctgccagga atgcaatggg 12661 cctctctgct ggagtatatg caaatgcgcg ctctagtgcc tgcgcgcaca cttcctggtc 12721 tctgacctca gcctgcagac tctgcacctt gctcttctgc tcttggctgg ggtcagagtg 12781 gagtttcctg tggagtcctc tgggtccagg gcagctgaaa tgggccccac agagaaacca 12841 ctatcccctc cctccgcacc ccaggacggg cggctgctcc aggagtttaa caagatcgag 12901 ccccccctga tctttgagtg taaccaggca tgctcctgct ggagaagctg caagaaccgc 12961 gtggtgcaga gcggcatcaa gtgaggcgcg cgccccggca ccctgcctcg cctccgcctc 13021 ccctcccctg gggccctccc aggtgtccca cactcagtgt gactccctct ccacctctag 13081 ggtacggctg cagctctacc ggactgccaa gatgggctgg ggggtccgag ccttgcagac 13141 catcccccag ggcacgttca tctgcgagta agttcactgt gaggccgtgg tgcatgaggg 13201 ctctgctgac cctagagctc catgtcagga tacatgtctc tgctcgccca ccgagtgtat 13261 ttccaaacat catgtccccc ctttacctgc acgctgccta gatggagcat gcctgtggca 13321 gacagatctg taaagtggag aatagctttt tcctgtgcac ttcctcccct ctgccgtcag 13381 actcacctag gttctctgtg gcctttagag cagagaagcc ccttcacctg ctccgaggac 13441 ctcccagttt ctctgttccc tcaacttctc agacacccac ttctcttggg caattcctct 13501 tccagccaca tggggcgacc tcatgactga tgtagctgcg cagagcctac cacctgtttg 13561 atagttcctg gtgcacacac ctcctaatgt ctaagcctgg caactttcca cccagccagc 13621 caccagggat gctaatcccc agaacacagg ctcacacaca cacacacaca cacacacaca 13681 cacacacacg tcatctccca ccatgcctgc tcaggtcttg caggctccac ctgtacagag 13741 cctcccacct gcctgtctac agctgcacag cgacccctag tggctggacc atgtcctgca 13801 tctctcccct gggctctgag aactgagtgg gaaggtgggg ccgggagccc tgctcactgt 13861 tcatgcagaa ctgaagtttc cgtccaacag gtatgtagga gagctgatct ctgatgccga 13921 ggctgatgtg agagaggatg attcttacct cttcgattta gataacaagg tgggatgcag 13981 cacctctgcc ctcaactacc aaaggctgct ttgtgcagcc acctgtaaca tctgggactg 14041 gaagcctggg tggagggact gggtgtcact gcaggaacac tagccaggca agtggataga 14101 agtggatatt aagaacactg tgtctccacc cccaggatgg cgaggtttac tgcattgatg 14161 cccgttacta tggcaacatc agccgattca ttaaccacct gtgtgacccc aacatcatcc 14221 ctgtccgggt tttcatgctg caccaagatc tacggttccc acgcattgcc ttcttcagct 14281 ccagggacat ccggactggg gaggagctgg ggtgaggagc cagcctgagg gcactgacca 14341 gcctttggtg ttcttttttt tttttttttt tttttcccgg tttttcggga cagggtttct 14401 ctgtgtagtc ctggctgtcc tggaactcac tctgtagacc aggctggcct cgaactcaga 14461 aatctgcctg cctctgcctc ccaagtgctg ggattaaagg tgtgtgctac catacccggc 14521 ttcctttggt gttcttaggt gtcccttgga aggcttgtta aaactgtgta aggcaggagt 14581 aggaacatac attcatgatc acagcactca ggaggctgag gcaggaggat cacaaagagt 14641 ctgggctaca gagtgagacc ctgtgtcaaa acaaaacaag agaagtccag gttgccctgg 14701 tatcacatgc ctgtcatgct agtgtttgaa agagcttgaa gcagaagtac ctacaaattc 14761 aaggccagcc tgggctaact acagagttca ggacagccag gcctatgtag agagacccta 14821 tcttaagcaa aaaacaagaa gggggatggg ggctagtgtg ggttggctgg atggttgagt 14881 atactaggcc ctgggcaggc tgcatcccac ttcccgcaga gcctcgggag agtctggtca 14941 caaggcagga aggctagaag cctgcattta tgacagttct ctgggagtgt gagtcaacag 15001 agccaacaag gacctggagt ccatctgcag gagtggttgg gtggaagaag gtggatccct 15061 caagagggct cctgggctct atgagatggg tttgaagtgt ctagatccaa gggccaagag 15121 ctcctgtgtt tcctctgcca gctttgacta cggtgaccga ttctgggaca tcaagagcaa 15181 gtatttcacc tgccagtgtg gctctgagaa gtgcaagcat tcagcggagg ccatcgccct 15241 ggagcagagc cgcctggccc ggctggaccc ccacccggag ctgctccctg acctcagctc 15301 cctgcccccc atcaacacct gaggactctt aaaatccagg ccgggcactg cccttcagac 15361 atttctccat cagagacccc agtaaggcct ggaaggtcga tggcccctct cccagagctg 15421 gtttctcact gggagtgcaa gtgacttcag ggctggcctt ccccactgag cctggcctca 15481 gttagctgat tgaagttggg cctctgccag ctgattttct gtgttctcaa taaatgttgg 15541 gtttggtaa
3) Rattus norvegicus
SEQ ID NO: 11
Rattus norvegicus euchromatic histone lysine N-methyltransferase 2 (Ehmt2), mRNA sequence
NM 212463.1
1 atgcggggtc tgccgagagg gagggggctg atgcgggccc gggggcgggg gcgtgcggcc 61 cccacgggcg gccgcggccg gggtcgcggg ggcgcccacc gtggacgagg taggccccga 121 agcctgctct cgctgcccag ggcccaggcg tcttgggccc cccagctgcc tgccgggctg 181 accggccctc cggtcccttg tctcccctcc cagggggagg cccccgctga gatgggggcg 241 ctgctgctgg agaaggagcc ccgaggagcc gccgagagag ttcatggctc tttgggggac 301 acctctcata gtgaggagac ccttcccaag gccaaccccg actccttgga gcctaccggc 361 ccctcctctc cggcctctgt cactgtcacc gtcggcgatg agggggctga cacccctgtc 421 ggggccacac cgctcatcgg ggaggaaccc gagaacctgg agggagatgg gggtcgaatc 481 ctgctgggcc atgccacaaa gtcattcccc tcttccccca gcaagggggg tgcctgtccc 541 agtcgggcca aaatgtcaat gacaggggca ggaaagtcgc ccccctcggt ccagagtttg 601 gccatgaggc tgttgagcat gcctggggcc cagggagctg caactgctgg gcctgagccc 661 cctccggcaa caactgccgg ccaggagggg cagcccaaag tacaccgagc tcggaaaacc 721 atgtccaagc ctagcaacgg acagcctcca gtccctgaga agcggccccc tgaagtccag 781 catttccgca tgagtgacga catgcatctg ggaaaggtga cttcagatgt ggccaaacgg 841 aggaagttga cctcgggcag cctgtcagag gacttgggct ctgctggggg ctcaggagaa 901 gtgatcctgg agaagggaga gcccaggcct ctggaggagt gggagacggt ggtgggcgat 961 gacttcagcc tgtactatga cgcgtactct gtggatgagc gggtggactc tgacagcaag 1021 tctgaagtcg aagctctagc tgaacagttg agtgaagagg aggaggagga agaggaggaa 1081 gaagaagagg aagaggagga agaggaggag gaggaagagg aggaggagga tgaggagtcg 1141 ggcaaccagt cagacaggag tggctctagc ggccgacgca aggccaagaa gaaatggcgg 1201 aaagacagcc catgggtgaa gccatccagg aaacggcgga agcgagagcc tccgagggcc 1261 aaggagccca gaggagtgaa tggtgtgggt tcctcagggc ccagtgagta catggaggtt 1321 cctctggggt ccctggagct gcccagcgag gggaccctct cccccaacca cgctggggtc 1381 tccaatgaca cgtcttcgct ggagacggag cgtgggtttg aggagctgcc cctctgcagc 1441 tgccgcatgg aggcgcccaa gattgaccgc atcagcgaga gagcaggaca caagtgtatg 1501 gccaccgaaa gtgtggacgg agagctgctg ggttgcaatg ccgccatctt gaagcgggag 1561 accatgcggc cgtctagccg tgtggcgctg atggtactgt gtgaggccca tcgagcccgc 1621 atggtcaagc accattgctg cccaggctgt ggctacttct gcacggcggg caccttcctg 1681 gagtgtcacc ccgacttccg tgtagctcac cgcttccata aggcctgcgt gtcccagctc 1741 aatgggatgg ttttctgtcc ccactgtggg gaggacgcct cagaggccca ggaggtgacc 1801 atccctcggg gcgatggggg aacacccccg gttggcactg tggcgcctgc cccgccaccc 1861 ctggcacacg acgccccagg gcgagcggat acctcccagc ccagtgcccg aatgcgagga 1921 cacggagagc caaggcgccc accctgtgat cccctggccg acaccattga cagctcgggg 1981 ccttctctga ctctgcctaa tggcggctgc ctctcagctg tgggtctgcc cccagggcca 2041 ggccgggaag ccctggaaaa agccttggtc atccaggagt ccgagaggag gaagaagctg 2101 cggttccacc cgcggcagct ctacctgtcg gtgaagcagg gggagctgca gaaggtgatc 2161 ctcatgctgc tcgacaacct ggaccccaac ttccagagcg accagcagag caagcgcacg 2221 cccctgcacg cagccgccca gaaggggtcc gtggagatct gtcacgtgct gctacaggca 2281 ggagccaaca tcaacgccgt ggataagcag cagcgcaccc cgctgatgga ggccgtggtg 2341 aacaaccacc tggaggtggc tcgctacatg gtgcagctgg gcggctgtgt gtacagcaag 2401 gaagaggatg gctctacctg tctccatcat gcggccaaaa ttgggaactt ggaaatggtc 2461 agcctgctgc tgagcacagg gcaggtggac gtcaatgccc aggacagtgg gggctggaca 2521 cccatcatct gggcagccga gcacaagcac atcgacgtga tccgtatgct gctgacccgg 2581 ggcgccgacg tcacccttac cgacaacgag gaaaacatct gcctgcactg ggcttccttc 2641 acgggtagtg ccgctattgc cgaggtcctc ctgaatgccc agtgcgacct ccatgctgtc 2701 aactaccacg gggacacgcc cctgcacata gccgccaggg agagctacca tgactgcgtt 2761 ctgttgttcc tgtcccgtgg agccaaccct gagcttcgga acaaagaagg ggacacggcg 2821 tgggatctga ccccagagcg ctccgatgtg tggtttgcac tgcagctcaa tcgcaagctt 2881 aggctcgggg tagggaaccg ggctgtccgc accgagaaga tcatctgccg ggatgtagcc 2941 cgaggctatg agaatgtgcc cattccctgt gtcaatggcg tggatgggga gccatgccca 3001 gaggactata agtacatctc cgagaactgt gaaacgtcca ccatgaacat cgaccgaaac 3061 atcactcacc tgcagcactg cacgtgtgtt gacgactgct ccagctccaa ctgcctgtgt 3121 ggccagctaa gcatccgatg ctggtatgat aaggacgggc ggctgctcca ggagtttaac 3181 aagatcgagc cccccctgat ctttgagtgt aaccaggcat gctcctgctg gagaagctgc 3241 aagaaccgcg tggtgcagag cggcatcaag gtgcggctgc agctctaccg gacggccaag 3301 atgggctggg gggtccgtgc cttgcagacc attccccagg gcacgttcat ctgcgagtac 3361 gtcggggagc tgatctccga cgccgaggca gacgtgagag aggatgattc ttacctcttc 3421 gacttagaca acaaggatgg tgaggtctac tgcattgatg cccgttacta tggcaacatc 3481 agccgcttca ttaaccacct gtgcgacccc aacatcatcc ctgttcgggt cttcatgctg 3541 catcaagacc tacggttccc tcgcatcgcc ttcttcagct ccagggacat ccggaccggg 3601 gaggagctgg ggtttgacta cggcgaccgg ttctgggata tcaagagcaa gtatttcacc 3661 tgccagtgtg gctctgagaa gtgcaagcat tcagctgagg ccatcgccct ggagcagagc 3721 cgcctggccc gtctggaccc ccacccggag ctgctccctg acctcagctc cctgcccccc 3781 atcaacacct gaggactgtt aaaatccagg ccggggcact gcccctcaga cattccccca 3841 tcggaggacc ccagtaaggc ctggaaggtt tacagcccct ctcccagagc tggtttctca 3901 ctgggagtga gtgacttcag ggctggccta ccccactgag cctggcctca gttcgctcat 3961 tgaagttggg cctctgccaa ctgattttct gtgctctcaa taaatgttgg gtttggtaat 4021
SEQ ID NO: 12
Rattus norvegicus histone- lysine N-methyltransferase, H3 lysine-9 specific 3, amino acid sequence
NP 997628.1
1 mrglprgrgl mrargrgraa ptggrgrgrg gahrgrgrpr sllslpraqa swapqlpagl
61 tgppvpclps qgeapaemga lllekeprga aervhgslgd tshseetlpk anpdsleptg 121 psspasvtvt vgdegadtpv gatpligeep enlegdggri llghatksfp sspskggacp 181 srakmsmtga gksppsvqsl amrllsmpga qgaatagpep ppattagqeg qpkvhrarkt 241 mskpsngqpp vpekrppevq hfrmsddmhl gkvtsdvakr rkltsgslse dlgsaggsge 301 vilekgeprp leewet vgd dfslyydays vdervdsdsk sevealaeql seeeeeeeee 361 eeeeeeeeee eeeeeedees gnqsdrsgss grrkakkkwr kdspwvkpsr krrkreppra 421 keprgvngvg ssgpseymev plgslelpse gtlspnhagv sndtsslete rgfeelplcs 481 crmeapkidr iseraghkcm atesvdgell gcnaailkre tmrpssrval mvlceahrar 541 mvkhhccpgc gyfctagtf1 echpdfrvah rfhkacvsql ngmvfcphcg edaseaqevt 601 iprgdggtpp vgtvapappp lahdapgrad tsqpsarmrg hgeprrppcd pladtidssg 661 psltlpnggc lsavglppgp grealekalv iqeserrkkl rfhprqlyls vkqgelqkvi 721 lmlldnldpn fqsdqqskrt plhaaaqkgs veichvllqa ganinavdkq qrtplmea v 781 nnhlevarym vqlggcvysk eedgstclhh aakignlemv slllstgqvd vnaqdsggwt 841 piiwaaehkh idvirmlltr gadvtltdne eniclhwasf tgsaaiaevl lnaqcdlhav 901 nyhgdtplhi aaresyhdcv 11flsrganp elrnkegdta wdltpersdv wfalqlnrkl 961 rlgvgnravr tekiicrdva rgyenvpipc vngvdgepcp edykyisenc etstmnidrn 1021 ithlqhctcv ddcsssnclc gqlsircwyd kdgrllqefn kiepplifec nqacscwrsc 1081 knr vqsgik vrlqlyrtak mgwgvralqt ipqgtficey vgelisdaea dvreddsylf 1141 dldnkdgevy cidaryygni srfinhlcdp niipvrvfml hqdlrfpria ffssrdirtg 1201 eelgfdygdr fwdikskyft cqcgsekckh saeaialeqs rlarldphpe llpdlsslpp 1261 int
SEQ ID NO 13:
Rattus norvegicus strain BN/SsNHsdMCW chromosome 20, RGSC_v3.4, G9a genomic sequence
NC 005119.2
1 cagcctccag tccctgagaa gcggccccct gaagtccagc atttccgcat gagtgacgac 61 atgcatctgg gaaaggtgac ttcaggtcag ttgactccac tccccccaca cacactccag 121 gaggcttggt gtattctgtc ccaggagtta gagttcagtc ctcacttttc tgtctttcca 181 gatgtggcca aacggaggaa gttgacctcg ggcagcctgg tgagctggag aggaagaccc 241 gtccgggccg ggaagggggt tggtgggtca acgccaggag cctcagactt tgtgatcttt 301 tctctagtca gaggacttgg gctctgctgg gggctcagga gaagtgatcc tggagaaggg 361 agagcccagg cctctggagg agtgggagac ggtggtgggc gatgacttca gcctgtacta 421 tgacgcgtac tctgtggatg agcgggtgga ctctgacagc aaggtgtgct ggtgtgctgt 481 tctcacgtct ccctttctaa tctgttcttc ccctttaata cgaaccctct agttcagtgc 541 cccaccatgt ctgcaaccat ttcttttttt ttttttgggt tctttttttt ttttccggag 601 ctggggaccg aacccagggc cttgcgcttc ctaggcaagc gctctaccac tgagctaaat 661 ccccaacccc tgcaaccatt tcttgtattc cttctctcat ctctgctcct ctccttccaa 721 gacggtctcc tccctgcttc tgacggcttg tttgctccct tcgtattgtt cgttcgcttt 781 tggccagttt agtttgtttg acagggtctt actagtagac caagcccgcc tcaaattctt 841 agaaatctgt ctgcctctgc ctctttagtg ttgggatgaa aggcatatac tgcatacacc 901 atgtgcgcgt gtgcgtgcgt gcatgtatgt gcatgcgcac acatgtgcac atgtgtgtgt 961 gagcatgtat tccgggcatg agtgcgccag tgtggtgtac gtgcatgtgt gtgtgcatgt 1021 gtgtgtgtgt gtgcgcgcac gtgtggcgtg cgtgcatgcg ggtactcatc tggggttaga 1081 ggacggcatc tctggcttgg ctctctttcg atgtagactc tggggatttt aatctacaat 1141 gtatggggtt cagttttcag aatgcttccc ccagtgtttg aggttgaacc tggagctcgc 1201 ttggcaagca cagtagtcag gtgtgtgttg ccaggcaggc tatgttggtg agcacctggc 1261 ttttttctgt gacctctgag gagtctgaag atcactcaga tctcacccca gagcggcagg 1321 cactttccca ctcagccatc cccccatccc tcgggttcct gtttcattta tttaacactc 1381 cgctgtggaa actttccact gatcagaaaa gggaagaacg gtgagcccca acacacagcc 1441 agccacccgc acacacgcgg acagagcgag tcgtagggct ttgcttgtgt ggaagcgact 1501 tctctttgca cttccaaaca ggcctctgct ttatttttgc tgactttggg gttaaactca 1561 aggcctctca tggcgagcac tgagctgcta cttaaataat ttgaggcctc atcatttacc 1621 gagtggcctt ggagtggcca caccttcact cactcctcat ggtccgcctg ccccggcctt 1681 ccagggggct gggattaaag gcaagcttta atggaggatg gggcttgaca cagagcctca 1741 ctttgtagcc ttggttggcc ttgagcctct gcagcacctg cttcttcctc ctatttgctt 1801 gtgagctgat gaagctgttc acagttctag aggtgagatt cccttcgagt gcctaatacg 1861 ccgcttgtct cggggtgcgg tcgtgtaacc caggctgtcc tcagacttga tatgtagcca 1921 gggatgaccc tgagcgtctg ggttgctttt tagcgtgtat gagtggtttt ccctgcgtgt 1981 atattgtacc aagtgtgtgc ctcgggccct tgggagtcag aagagccttg gtactctggg 2041 accggagtaa cggacaggta cgagccaccg tgtgggtgct gggagttgaa cccaggccct 2101 ttgcaagagc cacagtgttc ttaaccgctg agccgtctct ccagccccag ctcctgagct 2161 tctggtcctc ctccccgctc tgtctcccac atgtttgggc tgtaggcgtg tctggtgctt 2221 tcttctagtt tttgggaaga agacgtggaa gtgggtacag gtgtttcctt cctgttgcct 2281 gtcgctggaa cctgtgtggg ttgtgcggtc atttctttta ctggtaaatg ttgacacgct 2341 ccagaggcag aggtgtggga tctctgtgag ttctaggtag tggccaggac taccctcctg 2401 agtgaggcct tgtcaccacc tcctccacac ctcatgagct tttcatagag tggtttggtt 2461 gccgtgggta gtcgtgtctg tgtgtttcct aaacccagcc cttttcactg ctgtgtcacg 2521 ctctgcacct cggtccctat ccagcccttc cctgcccaga tccaccccct ggagtttccc 2581 tggtggggtg gcaatgactg gggacctcag gattcaccat gtcttgctct tgaaggagat 2641 ggctgccgtt tgctggtttc ccagcctaat ttttctcttt cagtctgaag tcgaagctct 2701 agctgaacag ttgagtgaag aggaggagga ggaagaggag gaagaagaag aggaagagga 2761 ggaagaggag gaggaggaag aggaggagga ggatgaggag tcgggcaacc agtcagacag 2821 ggtaagagga gggactggcc tacctagagg agtcggggac tgttctgggc ctggagcaga 2881 gctctcgaat cttcctcttc cacagagtgg ctctagcggc cgacgcaagg ccaagaagaa 2941 atggcggaaa gacagcccat gggtgaagcc atccaggaaa cggcggaagc gagagcctcc 3001 gagggccaag gagcccagag gtaagaaggc ccctctgctc ccgggtcccc tcctctggcc 3061 ccagagagca cacactcgga tgctcgagca tgttgactgt gtagaccgca tgtgcgcacg 3121 cgcccaggcc ccgtgagccc ccactctcac tctctctgtc tctgtgtcag gagtgaatgg 3181 tgtgggttcc tcagggccca gtgagtacat ggaggttcct ctggggtccc tggagctgcc 3241 cagcgagggg accctctccc ccaaccacgc tggtaattaa ttaccaactt ccgggatagg 3301 gagccatcgg ggtgcccacg ggggtggagg gaaatcctgc cagagccggg gtgtcctgtg 3361 ccaggctttg gggttctaga atgtgaaagg gtggcggggc gggaaggagg ggacccttcc 3421 agtgggtgag gggcaaggtc ccccatcgtc tctgtctctt cctgtctaag gggtctccaa 3481 tgacacgtct tcgctggaga cggagcgtgg gtttgaggag ctgcccctct gcagctgccg 3541 catggaggcg cccaagattg accgcatcag cgagagagca ggacacaagt gtatggccac 3601 cgaaagtgtg gacggagagg tgagtctggg tactgttggc ttaggaggcc cagggtgaac 3661 tagcctgggc ccgtgcctca ccctcatacc cccaccttcc tctaccgcta gctgctgggt 3721 tgcaatgccg ccatcttgaa gcgggagacc atgcggccgt ctagccgtgt ggcgctgatg 3781 gtactgtgtg aggcccatcg agcccgcatg gtcaagcacc attgctgccc aggctgtggc 3841 tacttctgca cggcggtgag tggccactgg ggtagatggg cggcctgagc agcaagagag 3901 agcttggagt gactgctgtc tccctttccc agggcacctt cctggagtgt caccccgact 3961 tccgtgtagc tcaccgcttc cataaggcct gcgtgtccca gctcaatggg atggttttct 4021 gtccccactg tggggaggac gcctcagagg cccaggaggt gaccatccct cggggcgatg 4081 ggggaacacc cccggttggc actgtggcgc ctgccccgcc acccctggca cacgacgccc 4141 cagggcgagc ggatacctcc cagcccaggt actggcctcc ccggcccgcc ccttccccat 4201 cttaattccc ttgcccccca gacctcagac ccttttccca cagtgcccga atgcgaggac 4261 acggagagcc aaggcgccca ccctgtgatc ccctggccga caccattgac agctcggggc 4321 cttctctgac tctgcctaat ggcggctgcc tctcagctgt gggtctgccc ccagggccag 4381 gccgggaagc cctggaaaaa gccttggtca tccaggagtc cgagaggtga gttggtgctg 4441 ttttcagggt acgactggtc cggaaccgca ggtgctgcta tcggtactta aatctgagca 4501 gcctaccttc tccccctccg tccccgggca ggaggaagaa gctgcggttc cacccgcggc 4561 agctctacct gtcggtgaag cagggggagc tgcagaaggt gatcctcatg ctgcgtgagt 4621 gtccctccca cccggggaac tgaggccttg tatagcgagg aaggtcacgt ggccctctcg 4681 ctgatggcag tggtggtgtc ctgtgggtga tgggcattgt tccagtgcgc agctcacaag 4741 ctttcgcaga accgcagaac cgaggctgag tgctcctcta gtcctgaggg gctggcgcag 4801 ctgcgtaccg ggcggtcctg ggacgcctcc ctagatgtag ggctgtcccg tccccagccc 4861 tcttactgct ttgtctcctc tcggcctgca gtcgacaacc tggaccccaa cttccagagc 4921 gaccagcaga gcaagcgcac gcccctgcac gcagccgccc agaaggggtc cgtggagatc 4981 tgtcacgtgc tgctacaggt cggtcgtggg gacccttccc cgcctcgctg tccagccccc 5041 tccgcagcat gccctgtccc tggtgccagc cttcctgctc ctcctcacag gcaggagcca 5101 acatcaacgc cgtggataag cagcagcgca ccccgctgat ggaggccgtg gtgaacaacc 5161 acctggaggt ggctcgctac atggtgcagc tgggcggctg tgtgtacagc aaggtgtggg 5221 gcggagccgt gcggggcggg gcccggcggt gggagggagg acggatgcct gggcaccaag 5281 tgccagccga agggctcact ggctcctgtc tccctcagga agaggatggc tctacctgtc 5341 tccatcatgc ggccaaaatt gggaacttgg aaatggtcag cctgctgctg agcacagggc 5401 aggtggacgt caatgcccag gtcggtggcc tggccgctgc tcctgtacat aggccccttg 5461 gccgtccctt gggcttcagt cctcacttct tcctcagctg ctctgaccct ctcccaccgc 5521 tcacattgct tccctcgttt ctttggttca tggaacttga gtccctcctc ttaacctctg 5581 agctcatcca gcgaggaccc cgctgagcct gggtggggag ggtgtgcatc tgtggaatgc 5641 cccatgctct ggctttggct gttcccagag gtgcccctca gcttccgttt tctggccagc 5701 aagtctgggc cctgattcca ttcctccttc cgtccctcag gacagtgggg gctggacacc 5761 catcatctgg gcagccgagc acaagcacat cgacgtgatc cgtatgctgc tgacccgggg 5821 cgccgacgtc acccttaccg acaacgtgag gggcactcaa gggagctggg gggactggtg 5881 gggagcaggg ggaccccgat cctgtcacct gcaacccgat ctctgcccct cacctccaca 5941 ggaggaaaac atctgcctgc actgggcttc cttcacgggt agtgccgcta ttgccgaggt 6001 cctcctgaat gcccagtgcg acctccatgc tgtcaactac cacggggaca cgcccctgca 6061 catagccgcc agggagagct accatgactg cgttctgtaa gccccagctt ccggacccac 6121 ctccccacac cccactcccg gcacccacac cccactgtcc agtctctgac tcaggttttc 6181 tatccctctc agggttgttc ctgtcccgtg gagccaaccc tgagcttcgg aacaaagaag 6241 gggacacggc gtgggatctg acccccagag cgctccgatg tgtggtttgc actgcagctc 6301 aatcgcaagc ttaggctcgg ggtagggaac cgggctgtcc gcaccgagaa gatcatctgc 6361 cggtgagtgc agcccagatg ccacagctcc tccacgtctt cagtctcagg ccccttgcca 6421 gacgcaggac gtcccagtag ctcctacaat catcttgcct ctaagggagg tggaaataag 6481 gggccctcag tgggcaaggg aacctgcaac caagcctgac aaactgagtg cagtccctgg 6541 agagccatgt ttttctctgg cttacgaaca cacagcatgg cacagtcgca cacctgaaaa 6601 aagctaaagg cagggtgtgg tggccacccc tgtaatctca gcacgcagga ggcagacggt 6661 tggttgtttt gaacctgagg ccagcttggt ttgtatgtat tagtgagttc caggccaagg 6721 agggccacat ggtgagacct tatctttctt aaaataaatg aaaaataaat aaatatgtaa 6781 gaaaaataaa gaaaaggaca tgttttccaa gccttagagg tagctgaggc aggaggattg 6841 tgagtttgag gcagataccc aaataagagt ttcagacagg ctggagagat ggctcagagg 6901 ctaagaacac tgactgctct tccagaggtc ctgagttcaa ttcccagcaa ccacatggtg 6961 actcacagcc atctgaaagg ggatcttccg ccctcttctg gcctgcaggt gtacatgcag 7021 atagagcgct cataaacata agataaataa agctttttaa aaaaaaaaaa aaaaagaatt 7081 tcagacagga gctgggtgtc acctgtgacc ccctcctatt ctgaaggctg gaggaagacc 7141 tgcctcttga gcccacgggt tggagatcag tatttgttat tattgagact cagaccttaa 7201 aaaggaaaat caaaccaaac caaaaaaccc cgcaaggcta gtgtgaggca acccaactac 7261 tgaatgtgca ttctgagttc aagccaagaa cccaccagga gagagctgcc tcctgaaggt 7321 tgccctctga ccttaattcc acactgtatc ctgtgtgagc gcgaaaggtc ctcaagccag 7381 gcgtggtggt ttagaccttt aatccactac tctggaggga ggcgaaggca gatctctggg 7441 ctctagggct acacagtgag atgctgtctc aagaagggag cggttggagg ccaccaggaa 7501 tgttcactgt ctttgaggag aggttttttt tttttttttt ttttttaaga cagggtttct 7561 ctgtgtagcc ccggctgtcc tggaagtcac tctgtagacc aggctggcct tgaactcaca 7621 gagatccctg tgcctctgcc tccctagtgc tgggattaaa aggagtgcat cactctgcca 7681 gaaaggactg tgacctctaa tgttagcact ggggaggagt agcagatgga tctgagttct 7741 aggatagcca gagctacaga atgagagtgt ctcaaagaaa ggaaggaaga ggcaacacgt 7801 taaaaagtta aaaaaggggg tcggggattt agctcagtgg tagagcactt gcctagcaag 7861 ggcaaggccc tgggttcggt cctcagctcc gggggggggg gggggggtaa aaaagggatc 7921 tggagagatg gctcagcggt taagagcact ggctgctctt ccagaggtcc tgagttcaat 7981 tcccagcacc cacagggtgg ctcacagcca tctgtaactt cagatccatg acccagagga 8041 tccatgcctc cttctgacct ctgcgggcag caggcgtgca tgtggggtgc atacagaagt 8101 gcaggcaaag tattcttaac acgtaaaatg agaaaatgtt ttttcaggtg agcaggaata 8161 ctagtgcgtc ctgtgctagc tgggtgcttg cagtctgtgt cagtgctaca ctaaggaagc 8221 gggaaggcca tggtctcact tttaaggcca gccaagcgag gcattatacc aaggctcagc 8281 ctccaactaa aatttaaaag tccagagtcc agcagggtga tgacatatac ctctaatcct 8341 agccacttgg gagctagggc aggagagtca agagtccaag tcattcttgg ctatctcgct 8401 atttggggcc agcaggctat atgatgctgt atctcacaga ggaatataag gggccggggc 8461 cggggccggg gctggagtag agtgtttatt ggcctcacat gcacaatgtt gttgggcctc 8521 accaagaaac aaagataacc tgtgcgatgt tgaggacata attatttcta agctgagatt 8581 gtttccctgg tgtcttaact tccctttttt tgttttcaag aatgtctcac cgcatgcact 8641 gggattaaag gtgccaccac cccagcaccc ttatacggag gaatccttga gcttcccctc 8701 aggaatgctc cggcccctac ctaactttac tgcccttctg tagggatgta gcccgaggct 8761 atgagaatgt gcccattccc tgtgtcaatg gcgtggatgg ggagccatgc ccagaggact 8821 ataagtacat ctccgagaac tgtgaaacgt ccaccatgaa catcgaccga aacatcactc 8881 acctgcaggt gagacacagg ccccaccggg ctcgagggag caggggctga aggagcctga 8941 tgtgggagtc cctccacact ggggcacatg aagactcccc ctctcgctgg gaccgtcacc 9001 agccagcgtc ctgaaacgaa cccatgctcc aggggagagc cctctcactc acaccactcc 9061 ttcccgccca gcactgcacg tgtgttgacg actgctccag ctccaactgc ctgtgtggcc 9121 agctaagcat ccgatgctgg tatgataagg tgcgtgtccc tcacctggcc actcccacca 9181 aggagccagg accctgctct gttggggaag agatacaggg tgtagcagga agtcctggtg 9241 ccctgccagg aatgcaatgg gcctctctgc tcacgtatat gcaaatggac gctggtgccc 9301 gcctgcacac ttcctggtct ctgacctcag cctgcagact ctgcaccgtc ctctcctgct 9361 ctgggctggg gtcagagtgg agtttcctgt ggagtcctct gggtcctggg cagctgaaat 9421 gggccccaca gagaaaccac tgtcccctcc ctccgcaccc caggacgggc ggctgctcca 9481 ggagtttaac aagatcgagc cccccctgat ctttgagtgt aaccaggcat gctcctgctg 9541 gagaagctgc aagaaccgcg tggtgcagag cggcatcaag tgaggcgcgc gccccggcac 9601 cctgcctcgc ctccgcctcc ccttccctgg ggccctccca ggcgtcccac actcagtgtg 9661 actccctctc cacctccagg gtgcggctgc agctctaccg gacggccaag atgggctggg 9721 gggtccgtgc cttgcagacc attccccagg gcacgttcat ctgcgagtaa gttcacgcta 9781 aggccgtggc tgacacctgc tgacacccag gccccactct gacctgcctg ctgctcctcc 9841 tgcactgagg acagagatgg ggcccgctct gcctgaaccc ctgatctccc tgtcaccatg 9901 cacgtctcca cccacccagt gcgtgtattc cagacatcat gtcccccctt tttattgcac 9961 actgcctaga tgtggcctgt ggcagacgac ccgggtttga gttgtgcact gttgtgtgtg 10021 agctgtgcac tgttgtatgt ccccggccac tttatttaac ctctttcagc cttaatcgtc 10081 tttgtcgagc agagaatagc ttcttcctgt gaatttcctc cccgtggcca ttagactccc 10141 ctaggtctcc tgtggccttc agagcagaga aaccctctca cctgctccct ccaaggtttc 10201 tttctctgct ccctgaattt ttcagacacc catgtctgtc tttgcgttgt gcagtttctc 10261 tgccagccac acggggcatc ctcacggctg atgtagctgt gcctgggccc accacctgtg 10321 tgattctgtg ttctgacacc tccccatgtc taaacctgga actttccacc cagcctgcca 10381 ccagggatgc taatccccag aacacgggcc tacgccctcc cccaacatct cctgccatgc 10441 ctttgcaggt cttgcaggct ctttcctccc aacccccacc tcctgcctac ctgctgttcc 10501 agctgcacag ggggaccccc agtggctgga ctgtgtcctg ctccctcccc ctgggtggga 10561 ggtgggaccc ggagccccgc tcacacgcag aaccagtttc tgtccaatag gtacgtcggg 10621 gagctgatct ccgacgccga ggcagacgtg agagaggatg attcttacct cttcgactta 10681 gacaacaagg tgggaaggca gcacctttgt cctcactacc aaaggctgct ttctggagcc 10741 acctgggaca tctgggagtg gagacctggg tggagggact gggtgtcgct gaaggaaaac 10801 tagctggtca agtgggtaga ggtgggcatt aagagcactg tgtgccccac tccccaggat 10861 ggtgaggtct actgcattga tgcccgttac tatggcaaca tcagccgctt cattaaccac 10921 ctgtgcgacc ccaacatcat ccctgttcgg gtcttcatgc tgcatcaaga cctacggttc 10981 cctcgcatcg ccttcttcag ctccagggac atccggaccg gggaggagct ggggtgagca 11041 gtctcagcag tcctgggcag ccattggtga tcttgtgtcc cctgggaggg cttgtcaaac 11101 agtgcaaggc aggtgtcggg gataacacgt tcatgatcac agcactcagg tggctgaggc 11161 aagaggatca cagagagtcc aggctacaga gtgagaccct gtgtcaaaga aaacgacaaa 11221 aagaggagtc cagtctgccc tgttatcact tggccacaaa attcaaggcc acccaggggc 11281 atagagagag accctatctt aaaaacaaac aacaaggaag ggggtggggg ctaggatggt 11341 tgaatacttg gagagtgtac tagaccctgg gctgcatccc acctcccaca gagcctctgg 11401 agattctggt cacacggctg gtaggccaga agcctgcatt tatgacagtt ctctgggagt 11461 gtgagtcaac agacccaaca agggcttgga gaccatctgg aggagaggtt gggcgggagg 11521 aagtggctcc ctcctagaga gggggctccc gtggctcttt gagatgggct ttgagtgtct 11581 ggatccgtgc tccaagaggt cctgtgtttc ctctgccagg tttgactacg gcgaccggtt 11641 ctgggatatc aagagcaagt atttcacctg ccagtgtggc tctgagaagt gcaagcattc 11701 agctgaggcc atcgccctgg agcagagccg cctggcccgt ctggaccccc acccggagct 11761 gctccctgac ctcagctccc tgccccccat caacacctga ggactgttaa aatccaggcc 11821 ggggcactgc ccctcagaca ttcccccatc ggaggacccc agtaaggcct ggaaggttta 11881 cagcccctct cccagagctg gtttctcact gggagtgagt gacttcaggg ctggcctacc 11941 ccactgagcc tggcctcagt tcgctcattg aagttgggcc tctgccaact gattttctgt 12001 gctctcaata aatgttgggt ttggtaataa
Interaction domainof histone methyl transferase G9a
1) Homo Sapiens (AS 1-300)
SEQ ID NO: 14
1 maaaagaaaa aaaegeapae mgallleket rgatervhgs lgdtprseet lpkatpdsle
61 pagpsspasv tvtvgdegad tpvgatplig desenlegdg dlrggrillg hatksfpssp
121 skggscpsra kmsmtgagks ppsvqslamr llsmpgaqga aaagsepppa ttspegqpkv
181 hrarktmskp gngqppvpek rppeiqhfrm sddvhslgkv tsdlakrrkl nsggglseel
241 gsarrsgevt ltkgdpgsle ewet vgddf slyydsysvd ervdsdskse vealteqlse
2) Homo Sapiens (mRNA 11-910)
SEQ ID NO: 15
11 atggcggcgg cggcgggagc tgcagcggcg gcggccgccg agggggaggc
61 ccccgctgag atgggggcgc tgctgctgga gaaggaaacc agaggagcca ccgagagagt
121 tcatggctct ttgggggaca cccctcgtag tgaagaaacc ctgcccaagg ccacccccga
181 ctccctggag cctgctggcc cctcatctcc agcctctgtc actgtcactg ttggtgatga
241 gggggctgac acccctgtag gggctacacc actcattggg gatgaatctg agaatcttga
301 gggagatggg gacctccgtg ggggccggat cctgctgggc catgccacaa agtcattccc
361 ctcttccccc agcaaggggg gttcctgtcc tagccgggcc aagatgtcaa tgacaggggc
421 gggaaaatca cctccatctg tccagagttt ggctatgagg ctactgagta tgccaggagc
481 ccagggagct gcagcagcag ggtctgaacc ccctccagcc accacgagcc cagagggaca
541 gcccaaggtc caccgagccc gcaaaaccat gtccaaacca ggaaatggac agcccccggt
601 ccctgagaag cggccccctg aaatacagca tttccgcatg agtgatgatg tccactcact
661 gggaaaggtg acctcagatc tggccaaaag gaggaagctg aactcaggag gtggcctgtc
721 agaggagtta ggttctgccc ggcgttcagg agaagtgacc ctgacgaaag gggaccccgg
781 gtccctggag gagtgggaga cggtggtggg tgatgacttc agtctctact atgattccta 841 ctctgtggat gagcgcgtgg actccgacag caagtctgaa gttgaagctc taactgaaca 901 actaagtgaa
3) Mus Musculus (AS 1-300)
SEQ ID NO: 16
1 mrglprgrgl mrargrgraa ptggrgrgrg gahrgrgrpr sllslpraqa swapqlpagl
61 tgppvpclps qgeapaemga lllekeprga aervhsslgd tpqseetlpk anpdslepag
121 psspasvtvt vgdegadtpv gaasligdep eslegdggri vlghatksfp sspskggacp
181 srakmsmtga gksppsvqsl amrllsmpga qgaatagpep spattaaqeg qpkvhrarkt
241 mskpsngqpp ipekrppevq hfrmsddmhl gkvtsdvakr rklnsgslse dlgsaggsgd
4) Mus Musculus (mRNA 1-900)
SEQ ID NO: 17
1 atgcggggtc tgccgagagg gagggggctg atgcgggccc gggggcgggg gcgtgcggcc 61 cccacgggcg gccgcggccg cggtcggggg ggcgcccacc gagggcgagg taggccccga 121 agcctgctct cgctgcccag ggcccaggcg tcttgggccc cccagctgcc tgccgggctg 181 accggccccc cggttccttg tctcccctcc cagggggagg cccccgctga gatgggggcg 241 ctgctgctgg agaaggagcc ccgaggagcc gccgagagag ttcatagctc tttgggggac 301 acccctcaga gtgaggagac ccttcccaag gccaaccccg actccttgga gcctgccggc 361 ccctcctctc cggcctctgt cactgtcacc gtcggcgatg agggggctga cacccctgtc 421 ggggccgcat cactcatcgg ggacgaaccc gagagcctgg agggagatgg gggtcgcatc 481 gtgctgggcc atgccacaaa gtcgttcccc tcttccccca gcaagggggg tgcctgtccc 541 agtcgggcca aaatgtcaat gacaggggca ggaaagtcgc ccccctcggt ccagagtttg 601 gccatgaggc tgttgagcat gcccggggcc cagggagctg caactgctgg gcctgaaccc 661 tctccggcaa caactgccgc ccaggagggg cagcccaaag tgcaccgagc ccggaaaacc 721 atgtccaaac ctagcaacgg acagcctcca atccctgaga agcggccccc tgaagtccag 781 catttccgca tgagtgatga catgcatctg gggaaggtga cttcagatgt ggccaaaagg 841 aggaagctga actctggtag cctgtccgag gacttgggct ctgccggggg ctcaggagat
5) Rattus norvegicus (AS 1-300)
SEQ ID NO: 18
1 mrglprgrgl mrargrgraa ptggrgrgrg gahrgrgrpr sllslpraqa swapqlpagl
61 tgppvpclps qgeapaemga lllekeprga aervhgslgd tshseetlpk anpdsleptg
121 psspasvtvt vgdegadtpv gatpligeep enlegdggri llghatksfp sspskggacp
181 srakmsmtga gksppsvqsl amrllsmpga qgaatagpep ppattagqeg qpkvhrarkt
241 mskpsngqpp vpekrppevq hfrmsddmhl gkvtsdvakr rkltsgslse dlgsaggsge
6) Rattus norvegicus (mRNA 1-900)
SEQ ID NO: 19
1 atgcggggtc tgccgagagg gagggggctg atgcgggccc gggggcgggg gcgtgcggcc 61 cccacgggcg gccgcggccg gggtcgcggg ggcgcccacc gtggacgagg taggccccga 121 agcctgctct cgctgcccag ggcccaggcg tcttgggccc cccagctgcc tgccgggctg 181 accggccctc cggtcccttg tctcccctcc cagggggagg cccccgctga gatgggggcg 241 ctgctgctgg agaaggagcc ccgaggagcc gccgagagag ttcatggctc tttgggggac 301 acctctcata gtgaggagac ccttcccaag gccaaccccg actccttgga gcctaccggc 361 ccctcctctc cggcctctgt cactgtcacc gtcggcgatg agggggctga cacccctgtc
421 ggggccacac cgctcatcgg ggaggaaccc gagaacctgg agggagatgg gggtcgaatc
481 ctgctgggcc atgccacaaa gtcattcccc tcttccccca gcaagggggg tgcctgtccc
541 agtcgggcca aaatgtcaat gacaggggca ggaaagtcgc ccccctcggt ccagagtttg
601 gccatgaggc tgttgagcat gcctggggcc cagggagctg caactgctgg gcctgagccc
661 cctccggcaa caactgccgg ccaggagggg cagcccaaag tacaccgagc tcggaaaacc
721 atgtccaagc ctagcaacgg acagcctcca gtccctgaga agcggccccc tgaagtccag
781 catttccgca tgagtgacga catgcatctg ggaaaggtga cttcagatgt ggccaaacgg
841 aggaagttga cctcgggcag cctgtcagag gacttgggct ctgctggggg ctcaggagaa

Claims

CLAIMS:
1. A method of identifying whether a compound has a therapeutic activity in the treatment of stress-related disorders, comprising the steps of
a) contacting a compound with a neural cell under conditions suitable for cellular uptake of the compound, and
b) determining a value indicative for the cellular level of histone methyltransferase
G9a in said neural cell, wherein reduction of the cellular level compared to a control level is indicative of a therapeutic activity of the compound in the treatment of stress-related disorders.
2. The method of claim 1, wherein the stress-related disorder is selected from the group consisting of depression and a disorder caused by the experience of a traumatic event.
3. The method of claim 1 or 2, wherein the stress-related disorder is depression.
4. The method of any of claims 1 to 3, wherein the stress-related disorder is anxiety disorder.
5. The method of any of claims 1 to 4, wherein the stress-related disorder is posttraumatic stress disorder.
6. The method of any of claims 1 to 5, wherein determining the value indicative for the cellular level of histone methyltransferase G9a is determined by measuring the expression level and/or the amount of histone methyltransferase G9a protein in said neural cell.
7. The method of any of claims 1 to 6, wherein said neural cell is an astrocyte.
8. The method of any one of claims 1 to 7, wherein the neural cell is derived from a mammal, the neural cell being optionally a human neural cell.
9. The method of any one of claims 1 to 8, wherein the value indicative for the cellular level of histone methyltransferase G9a is determined by using a total or nuclear extract of said neural cell.
10. The method of any one of claims 1 to 9, wherein reduction of the cellular level to less than 80%, optionally less than 70% of a control level, is indicative of a therapeutic activity in the treatment of stress-related disorders.
11. The method of any one of claims 1 to 10, further comprising a step of determining whether said compound is capable of decreasing the activity of a DNA methyltransferase, optionally o f DNA methyltransferase DNMT 1.
12. The method of claim 11 , wherein the activity of the DNA methyltransferase is determined by i) contacting a DNA substrate with a methyl group donor and a total or nuclear extract of said neural cell and ii) detecting DNA methylation at cytosine residues.
13. The method of claim 12, wherein the DNA substrate is poly(dI-dC)-Poly(dI-dC) or hemi/unmethylated DNA substrate.
14. The method of any one of claims 11 to 13, wherein a decrease of DNA
methyltransferase activity of at least about 30%, optionally 40% is indicative of a therapeutic activity in the treatment of stress-related disorders.
15. The method of any one of claims 11 to 14, further comprising a step of determining whether the decrease of the activity of the DNA methyltransferase can be restored by the addition of purified histone methyltransferase G9a protein.
16. The method of any one of claims 1 to 15, wherein said reduction of the cellular level comprises inhibiting histone methyltransferase G9a by directly binding to histone
methyltransferase G9a protein.
17. The method of any one of claims 1 to 16, wherein said reduction of the cellular level comprises decrease or inhibition of histone methyltransferase G9a protein expression.
18. The method of any one of claims 1 to 17, wherein said reduction of the cellular level comprises enhancing degradation of histone methyltransferase G9a protein in said neural cell.
19. A method of identifying a compound having a therapeutic activity in the treatment of stress-related disorders, comprising the steps of
a) contacting said compound with a neural cell under conditions suitable for cellular uptake of the chemical compound, and
b) determining a value indicative for a loss of interaction between the histone methyltransferase G9a and DNA methyltransferase DNMTl, wherein said loss of interaction is indicative of a therapeutic activity in the treatment of stress-related disorders.
20. The method of claim 19, wherein the stress-related disorder is selected from the group consisting of depression and a disorder caused by the experience of a traumatic event.
21. The method of claim 19 or 20, wherein the stress-related disorder is depression.
22. The method of any of claims 19 to 21, wherein the stress-related disorder is anxiety disorder.
23. The method of any of claims 19 to 22, wherein the stress-related disorder is post- traumatic stress disorder.
24. The method of any of claims 19 to 23, wherein the loss of interaction is detected by determining the loss of binding of histone methyltransferase G9a to DNA methyltransferase DNMTl .
25. The method of any of claims 19 to 24, wherein the loss of interaction is determined by using a method selected from the group consisting of co-immunoprecipitation, pulldown- assay, resonance energy transfer (FRET), protein complementation assay, enzyme complementation assay, yeast two hybrid, mammalian two hybrid, fluorescent polarization, and surface plasmon resonance.
26. A method of providing a compound having a therapeutic activity in the treatment of stress-related disorders comprising the steps of
a) conducting the method of identifying a compound having a therapeutic activity in the treatment of stress-related disorders according to claim 1 to 25, and
b) synthesizing the compound identified in step a).
27. An inhibitory polynucleotide molecule capable of interfering with the expression of the histone methyltransferase G9a protein comprising
a) an antisense nucleic acid specific for histone methyltransferase G9a,
b) a small interfering RNA specific for histone methyltransferase G9a , or c) a microRNA specific for histone methyltransferase G9a,
for use as a medicament.
28. The inhibitory polynucleotide according to claim 27, for use in the treatment of a stress-related disorder.
29. The inhibitory polynucleotide according to claim 27 or 28, for use in the treatment of depression.
30. The inhibitory polynucleotide according to claim 27 or 28, for use in the treatment of disorders caused by the experience of a traumatic event.
31. The inhibitory polynucleotide according to claim 30, wherein the disorder caused by the experience of a traumatic event is selected from the group consisting of psychological trauma, excessive alertness, exhaustion, sleeping disorders, poor concentration and/or memory, social withdrawal, loss of interest in normal activities, acute stress reaction, fear, depression, anxiety, panic and/or post-traumatic stress disorder (PTSD).
32. The inhibitory polynucleotide according to claim 27 or 28, for use in the treatment of anxiety disorders.
33. The inhibitory polynucleotide according to claim 27 or 28, for use in the treatment of post-traumatic stress disorder (PTSD).
34. A vector comprising the polynucleotide molecule according to any of claims 27 to 33.
35. A pharmaceutical composition comprising the inhibitory polynucleotide according to claims 27 to 33 and at least one pharmaceutically acceptable excipient.
36. The pharmaceutical composition according to claim 35, for use in the treatment of a stress-related disorder.
37. The pharmaceutical composition according to claim 35 or 36, for use in the treatment of depression.
38. The pharmaceutical composition according to claim 35 or 36 for use in the treatment of disorders caused by the experience of a traumatic event.
39. The pharmaceutical composition according to claim 38, wherein the disorder caused by the experience of a traumatic event is selected from the group consisting of psychological trauma, excessive alertness, exhaustion, sleeping disorders, poor concentration and/or memory, social withdrawal, loss of interest in normal activities, acute stress reaction, fear, depression, anxiety, panic and/or post-traumatic stress disorder (PTSD).
40. The pharmaceutical composition according to claim 35 or 36, for use in the treatment of anxiety disorders.
41. The pharmaceutical composition according to claim 35 or 36, for use in the treatment of post-traumatic stress disorder (PTSD).
PCT/EP2012/074995 2011-12-09 2012-12-10 Screening assays for compounds for use in the treatment of stress-related disorders WO2013083851A1 (en)

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