U.S. patent application number 15/794869 was filed with the patent office on 2018-02-22 for uses of histone acetyltransferase activators.
The applicant listed for this patent is THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEWYORK. Invention is credited to Ottavio ARANCIO, Mauro FA, Yitshak FRANCIS.
Application Number | 20180050982 15/794869 |
Document ID | / |
Family ID | 47296520 |
Filed Date | 2018-02-22 |
United States Patent
Application |
20180050982 |
Kind Code |
A1 |
FRANCIS; Yitshak ; et
al. |
February 22, 2018 |
USES OF HISTONE ACETYLTRANSFERASE ACTIVATORS
Abstract
The invention provides methods for enhancing histone acylation,
learning, memory and/or cognition in subjects with compound (I) or
compositions comprising compound (I), or a pharmaceutically
acceptable salt thereof. ##STR00001##
Inventors: |
FRANCIS; Yitshak; (New York,
NY) ; ARANCIO; Ottavio; (New York, NY) ; FA;
Mauro; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEWYORK |
New York |
NY |
US |
|
|
Family ID: |
47296520 |
Appl. No.: |
15/794869 |
Filed: |
October 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14125214 |
Aug 12, 2014 |
9809532 |
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PCT/US12/41907 |
Jun 11, 2012 |
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15794869 |
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61495495 |
Jun 10, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 9/00 20180101; A61P 9/02 20180101; A61K 31/167 20130101; A61P
25/32 20180101; A61P 25/28 20180101; A61P 31/18 20180101; A61P
25/16 20180101; C07C 237/44 20130101; A61P 31/00 20180101; A61P
25/00 20180101; A61P 25/14 20180101 |
International
Class: |
C07C 237/44 20060101
C07C237/44; A61K 31/167 20060101 A61K031/167 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
R01.sup.-NS049442 awarded by the National Institute of Neurological
Disorders and Stroke (NINDS), and under AG034248 awarded by the
National Institute of Aging (NIA). The government has certain
rights in the invention.
Claims
1-25. (canceled)
26. A method for enhancing learning or memory in a subject, which
comprises administering to the subject a therapeutically effective
amount of compound (I): ##STR00005## or a pharmaceutically
acceptable salt thereof.
27. The method of claim 26, wherein the subject is not afflicted
with a neurodegenerative condition or disease.
28. The method of claim 27, wherein the neurodegenerative condition
or disease is selected from one or more of the group consisting of
adrenoleukodystrophy (ALD), alcoholism, Alexander's disease,
Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis
(Lou Gehrig's Disease or ALS), ataxia telangiectasia, batten
disease (Spielmeyer-Vogt-Sjogren-Batten disease), bovine spongiform
encephalopathy (BSE), canavan disease, cockayne syndrome,
corticobasal degeneration, Creutzfeldt-Jakob disease, familial
fatal insomnia, frontotemporal lobar degeneration, Huntington's
disease, HIV-associated dementia, Kennedy's disease, Krabbe's
disease, lewy body dementia, neuroborreliosis, Machado-Joseph
disease (spinocerebellar ataxia type 3), multiple system atrophy,
multiple sclerosis, narcolepsy, Niemann Pick disease, Parkinson's
disease, Pelizaeus-Merzbacher disease, Pick's disease, primary
lateral sclerosis, prion diseases, progressive supranuclear palsy,
Rett's syndrome, tau-positive frontotemporal dementia, tau-negative
frontotemporal dementia, Refsum's disease, sandhoff disease,
Schilder's disease, subacute combined degeneration of spinal cord
secondary to pernicious anaemia, spinocerebellar ataxia (multiple
types with varying characteristics), spinal muscular atrophy,
Steele-Richardson-Olszewski disease, Tabes dorsalis, and toxic
encephalopathy.
29. The method of claim 27, wherein the neurodegenerative condition
or disease is Alzheimer's.
30. The method of claim 26, wherein the subject is a mammal.
31. The method of claim 26, wherein the therapeutically effective
amount is at least about 1 mg/kg body weight.
32. The method of claim 26, wherein the therapeutically effective
amount is at least about 25 mg/kg body weight.
33. The method of claim 26, wherein the therapeutically effective
amount is at least about 100 mg/kg body weight.
34. A method for improving memory retention in a subject, which
comprises administering to the subject a therapeutically effective
amount of compound (I): ##STR00006## or a pharmaceutically
acceptable salt thereof.
35. The method of claim 34, wherein the subject is not afflicted
with a neurodegenerative condition or disease.
36. The method of claim 35, wherein the neurodegenerative condition
or disease is selected from the group consisting of
adrenoleukodystrophy (ALD), alcoholism, Alexander's disease,
Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis
(Lou Gehrig's Disease or ALS), ataxia telangiectasia, batten
disease (Spielmeyer-Vogt-Sjogren-Batten disease), bovine spongiform
encephalopathy (BSE), canavan disease, cockayne syndrome,
corticobasal degeneration, Creutzfeldt-Jakob disease, familial
fatal insomnia, frontotemporal lobar degeneration, Huntington's
disease, HIV-associated dementia, Kennedy's disease, Krabbe's
disease, lewy body dementia, neuroborreliosis, Machado-Joseph
disease (spinocerebellar ataxia type 3), multiple system atrophy,
multiple sclerosis, narcolepsy, Niemann Pick disease, Parkinson's
disease, Pelizaeus-Merzbacher disease, Pick's disease, primary
lateral sclerosis, prion diseases, progressive supranuclear palsy,
Rett's syndrome, tau-positive frontotemporal dementia, tau-negative
frontotemporal dementia, Refsum's disease, sandhoff disease,
Schilder's disease, subacute combined degeneration of spinal cord
secondary to pernicious anaemia, spinocerebellar ataxia (multiple
types with varying characteristics), spinal muscular atrophy,
Steele-Richardson-Olszewski disease, Tabes dorsalis, and toxic
encephalopathy.
37. The method of claim 35, wherein the neurodegenerative condition
or disease is Alzheimer's.
38. The method of claim 34, wherein the subject is a mammal.
39. The method of claim 34, wherein the therapeutically effective
amount is at least about 1 mg/kg body weight.
40. The method of claim 34, wherein the therapeutically effective
amount is at least about 25 mg/kg body weight.
41. The method of claim 34, wherein the therapeutically effective
amount is at least about 100 mg/kg body weight.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 61/495,495, filed on Jun. 10, 2011, the entirety of
which is incorporated herein by reference.
[0003] All patents, patent applications and publications cited
herein are hereby incorporated by reference in their entirety. The
disclosures of these publications in their entireties are hereby
incorporated by reference into this application in order to more
fully describe the state of the art as known to those skilled
therein as of the date of the invention described and claimed
herein.
[0004] This patent disclosure contains material that is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or the
patent disclosure as it appears in the U.S. Patent and Trademark
Office patent file or records, but otherwise reserves any and all
copyright rights.
BACKGROUND OF THE INVENTION
[0005] Cognitive neurodegenerative disorders are characterized by
synaptic dysfunction, cognitive abnormalities, and/or the presence
of inclusion bodies throughout the CNS containing, for example, but
not limited to native beta-amyloid fragments, native and
phosphorylated Tau, native and phosphorylated alpha-synuclein,
lipofuscin, cleaved TARDBP (TDB-43), in various percentages and in
relation to the specific disease. Alzheimer's disease (AD) is one
of the most prevalent neurodegenerative disorders characterized by
memory loss, and significant research toward discovering treatment
for this devastating disease has been undertaken.
[0006] Cognitive disorders that are not neurodegenerative, such as
normal memory loss, as well as neurocognitive enhancement of normal
individuals has become of increasing interest in the medical
community (Farah, et al., Nat. Rev. Neuroscience 2004, 5, 421-425).
Enhancement of learning and memory has been reported with
amphetamines and derivatives thereof as well as other
centrally-acting drugs. Certain nutritional supplements have also
been reported to improve mental functions such as cognition and
memory (Lanni, C., et al., Pharmacol. Res. 2004, 57, 196-213).
However, many of these suffer from limited efficacy and/or untoward
side effects due to their mechanisms of action.
[0007] Histone Acetyltransferases (HATs) are involved in histone
acetylation (leading to gene activation), chromosome
decondensation, DNA repair and non-histone substrate
modification.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention is directed to methods and
compositions for enhancing memory and learning in subjects.
[0009] In one aspect, the invention is directed to methods for
increasing histone acetylation in a subject.
[0010] In one aspect, the invention is directed to methods for
improving memory retention in a subject.
[0011] In one aspect, the invention is directed to methods for
treating memory loss or a learning disability in a subject
[0012] In some embodiments, the methods and compositions that are
useful for treating, suppressing and/or preventing afflictions
related to memory loss or learning disabilities in subjects.
[0013] In some embodiments, the methods comprise administering to
the subject a therapeutically effective amount of compound (I),
##STR00002##
or a pharmaceutically acceptable salt thereof, or a composition
comprising compound I, or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier.
[0014] In some embodiments, the methods and compositions are useful
for enhancing memory and/or learning in subjects. In some
embodiments, the methods and compositions are useful for treating,
suppressing and/or preventing afflictions related to memory loss or
learning disabilities in subjects.
[0015] In some embodiments, the subject is not afflicted with a
neurodegenerative disease. In some embodiments, the
neurodegenerative disease comprises Adrenoleukodystrophy (ALD),
Alcoholism, Alexander's disease, Alper's disease, Alzheimer's
disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease),
Ataxia telangiectasia, Batten disease (also known as
Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform
encephalopathy (BSE), Canavan disease, Cockayne syndrome,
Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial
fatal insomnia, Frontotemporal lobar degeneration, Huntington's
disease, HIV-associated dementia, Kennedy's disease, Krabbe's
disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph
disease (Spinocerebellar ataxia type 3), Multiple System Atrophy,
Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's
disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary
lateral sclerosis, Prion diseasesm Progressive Supranuclear Palsy,
Rett's syndrome, Tau-positive FrontoTemporal dementia, Tau-negative
FrontoTemporal dementia, Refsum's disease, Sandhoff disease,
Schilder's disease, Subacute combined degeneration of spinal cord
secondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten
disease (also known as Batten disease), Spinocerebellar ataxia
(multiple types with varying characteristics), Spinal muscular
atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, or
Toxic encephalopathy.
[0016] In some embodiments, the present invention provides a method
for enhancing memory in normal subjects. In some embodiments, the
present invention provides for a method of improving learning in
subjects. In some embodiments, the subject does not suffer from a
neurodegenerative condition or disease.
[0017] In some embodiments, compound (I) increases histone
acetylation. In some embodiments, histone acetylation comprises
acetylation of histones H2B, H3, H4, or a combination thereof. In
some embodiments, histone acetylation comprises acetylation of
histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12,
H4K16, or a combination thereof.
[0018] In some embodiments, the subject is a mammal. In some
embodiments, the subject is a mouse, rat, monkey, guinea pig, dog,
or human. In some embodiments, the subject is a mouse, rat, monkey
or human. In some embodiments, the subject is a mouse or a human.
In some embodiments, the subject is a human.
[0019] In some embodiments, the methods reduce pain, anxiety or
fear. In some embodiments, the methods reduce anxiety or fear. In
some embodiments, the methods reduce anxiety. In some embodiments,
the methods increase neurotransmission.
[0020] These and other embodiments of the invention are further
described in the following sections of the application, including
the Detailed Description, Examples, and Claims. Still other objects
and advantages of the invention will become apparent by those of
skill in the art from the disclosure herein, which are simply
illustrative and not restrictive. Thus, other embodiments will be
recognized by the ordinarily skilled artisan without departing from
the spirit and scope of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 is a photograph of a western blot showing acetylation
levels of H3 in the cortex and hippocampus. Mice were administered
with MOM via cannula (100 .mu.g/.mu.l per side) or mice were
administered YF2 (Compound 1) (50 mg/kg, i.p.).
[0022] FIG. 2 is a photograph of a western blot showing acetylation
levels of H3 in the hippocampus. Mice were administered with YF2
(Compound 1) (i.p. dissolved in saline) at 5 mg/kg, 10 mg/kg, or 20
mg/kg.
[0023] FIG. 3 is a graph showing the effect of OA2 (Compound 1) on
HDAC1 activity.
[0024] FIG. 4 is a graph showing the effect of OA2 (Compound 1) on
HDAC3/NCOR2 activity.
[0025] FIG. 5 is a graph showing the effect of OA2 (Compound 1) on
HDAC5FL activity.
[0026] FIG. 6 is a graph showing the effect of OA2 (Compound 1) on
HDAC7 activity.
[0027] FIG. 7 is a graph showing the effect of OA2 (Compound 1) on
HDAC8 activity.
[0028] FIG. 8 is a graph showing the effect of OA2 (Compound 1) on
HDAC10 activity.
[0029] FIG. 9 is a graph showing the effect of OA2 (Compound 1) on
HDAC11 activity.
[0030] FIG. 10 is a graph showing the effect of OA2 (Compound 1) on
Sirtuin1 activity.
[0031] FIG. 11 is a graph showing the effect of OA2 (Compound 1) on
Sirtuin2 activity.
[0032] FIG. 12 is a graph showing the effect of OA2 (Compound 1) on
HDAC6 activity.
[0033] FIG. 13 is a graph showing the effect of the HDAC inhibitor,
SAHA, on HDAC1 activity.
[0034] FIG. 14 is a graph showing the effect of the HDAC inhibitor,
SAHA, on HDAC3/NCOR2 activity.
[0035] FIG. 15 is a graph showing the effect of the HDAC inhibitor,
SAHA, on HDAC6 activity.
[0036] FIG. 16 shows dose-response curves for human CBP, p300, PCAF
and GCN5 activation by different concentrations of Compound 1.
Calculated Compound 1 EC.sub.50 values for CBP, p300, PCAF and GCN5
are: 0.82 .mu.M, 0.79 .mu.M, 18.11 .mu.M and 65.19 .mu.M are
respectively.
[0037] FIG. 17 is a graph showing pharmacokinetic properties of
Compound 1. The amount of Compound 1 in the brain is higher than
that in the plasma. Compound 1 is rapidly absorbed in the brain
(see Table 14). The elimination half-lives of Compound 1 in the
brain and plasma are .about.40 min i.p. and 60 min. p.o. The
distribution of Compound 1 to the brain is fast. Compound 1 does
not induce any adverse effects up to 300 mg/kg (i.p.) in acute
toxicity experiments. Chronic administration (45 days, i.p., daily,
20 mg/kg) of Compound 1 did not have adverse effects.
[0038] FIG. 18 is a bar graph demonstrating contextual fear
conditioning responses after administration of Compound 1 (1, 5,
and 20 mg/kg) to wild-type mice.
[0039] FIG. 19 is a bar graph demonstrating the assessment of
sensory threshold in mice treated with vehicle or YF2 (Compound 1)
(5 mg/kg).
[0040] FIG. 20 is a graph showing the kinetics of the HAT agonist,
Compound 1, in the blood. Compound 1 was administered (20 mg/kg.
i.p.) to mice and then blood was sampled from tails at different
time points.
[0041] FIG. 21 is photograph of a western blot that shows histone 3
acetylation levels of mice hippocampus.
[0042] FIG. 22 shows that an acute administration of YF2 (Compound
1) increased specific acetylation of H3, H4, and H2B in hippocampal
lysates. n=3 and p<0.05 per group.
[0043] FIG. 23 shows beneficial effect of YF2 (Compound 1) on
Ab42-induced reduction in BDNF levels. YF2 rescued A.beta.-induced
decrease in hippocampal BDNF levels (n=3 per each group,
p<0.05). A.beta. was infused through cannulas.
DETAILED DESCRIPTION OF THE INVENTION
[0044] Memory is known to be modulated by epigenetics through
regulation of gene expression. Epigenetics is defined as the
mechanism that changes gene expression by `marking` DNA or its
associated proteins, through processes such as DNA methylation and
histone (H) modification, without changing the DNA sequence itself
(Rakyan, V. K., et al., Biochem J., 2001. 356(Pt 1): p. 1-10;
herein incorporated by reference in its entirety). Modification of
histones by, for example, the addition or removal of acetyl or
methyl functional groups causes the chromatin structure to open or
close, so that the information contained within the DNA is made
more or less accessible to transcription factors. Hence,
deregulation of one of the epigenetic mechanisms might lead to
memory disruption. For instance, reduction of histone acetylation
causes the chromatin structure to close, so that the information
contained within the DNA might be less accessible to transcription
factors and memory formation (Rakyan, V. K., et al., Biochem J.,
2001. 356(Pt 1):1-10; herein incorporated by reference in its
entirety).
[0045] The main strategy that is currently used to up-regulate
histone acetylation involves inhibition of histone deacetylases
(HDACs), enzymes that remove an acetyl group from histones.
However, the pleiotropic effect of nonspecific HDAC inhibition may
hamper the therapeutic potential of HDAC inhibitors (J. Virol.
2001. 75(4): 1909-17; J. Virol. 2003. 77(21): 11425-35; Knutson, S.
K., Biochemistry. 2008, Vanderbilt: Nashville.167; PLoS One, 2009.
4(8): p. e6612; each herein incorporated by reference in its
entirety).
[0046] HATs share a highly conserved motif containing an acetyl-CoA
binding site. Specific HAT activators are potential tools for
pharmacological research and might find therapeutic applications.
HAT activators have been reported; however these compounds are
poorly soluble and poorly membrane permeant, and thus not
considered acceptable drug candidates for the treatment of diseases
and other afflictions. For example,
N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide is very
poorly solubile in water and precipitated as soon as it was put in
H.sub.2O (J. Phys Chem B, 2007. 111(17): 4527-34).
[0047] In one aspect, the invention is directed to methods and
compositions for enhancing memory and learning in subjects.
[0048] In one aspect, the invention is directed to methods for
increasing histone acetylation in a subject.
[0049] In one aspect, the invention is directed to methods for
improving memory retention in a subject.
[0050] In one aspect, the invention is directed to methods for
treating memory loss or a learning disability in a subject
[0051] In some embodiments, the methods and compositions that are
useful for treating, suppressing and/or preventing afflictions
related to memory loss or learning disabilities in subjects.
[0052] In some embodiments, the methods comprise administering to
the subject a therapeutically effective amount of compound (I),
##STR00003##
or a pharmaceutically acceptable salt thereof, or a composition
comprising compound (I), or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier.
[0053] In some embodiments, the subject is not afflicted with a
neurodegenerative disease. In one embodiment, the neurodegenerative
disease comprises Adrenoleukodystrophy (ALD), Alcoholism,
Alexander's disease, Alper's disease, Alzheimer's disease,
Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia
telangiectasia, Batten disease (also known as
Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform
encephalopathy (BSE), Canavan disease, Cockayne syndrome,
Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial
fatal insomnia, Frontotemporal lobar degeneration, Huntington's
disease, HIV-associated dementia, Kennedy's disease, Krabbe's
disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph
disease (Spinocerebellar ataxia type 3), Multiple System Atrophy,
Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's
disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary
lateral sclerosis, Prion diseasesm Progressive Supranuclear Palsy,
Rett's syndrome, Tau-positive FrontoTemporal dementia, Tau-negative
FrontoTemporal dementia, Refsum's disease, Sandhoff disease,
Schilder's disease, Subacute combined degeneration of spinal cord
secondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten
disease (also known as Batten disease), Spinocerebellar ataxia
(multiple types with varying characteristics), Spinal muscular
atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, or
Toxic encephalopathy.
[0054] In some embodiments, the present invention provides a method
for enhancing memory in normal subjects. In some embodiments, the
present invention provides for a method of improving learning in
subjects. In some embodiments, the subject suffers from age-related
memory impairment. In some embodiments, the subject does not suffer
from a neurodegenerative condition. In some embodiments, the
subject does not suffer from Alzheimer's Disease.
[0055] In some embodiments, the invention provides for memory
enhancement in normal subjects. In some embodiments, the invention
provides for memory enhancement and/or learning improvement in
cognitively deficient subjects.
[0056] In some embodiments, the invention provides for memory
enhancement in aging subjects. In some embodiments, the subject is
greater than about 40 years old. In some embodiments, the subject
is greater than about 45 years old, greater than about 50 years
old, greater than about 55 years old, greater than about 60 years
old, or greater than about 65 years old.
[0057] In some embodiments, the subject is a mammal. In some
embodiments, the subject is a mouse, rat, monkey, guinea pig, dog,
or human. In some embodiments, the subject is a mouse, rat, monkey
or human. In some embodiments, the subject is a mouse or a human.
In some embodiments, the subject is a human.
[0058] In some embodiments, the methods reduce pain, anxiety or
fear. In some embodiments, the methods reduce anxiety or fear. In
some embodiments, the methods reduce anxiety. In some embodiments,
the methods increase neurotransmission.
[0059] In some embodiments, the invention provides for methods of
treatment using compound I, which has histone acetyltransferase
activity, HAT activation potency, high selectivity, reasonable
pharmacokinetics and good permeability across the
blood-brain-barrier (BBB).
[0060] In some embodiments, the methods increase gene expression in
a subject resulting in enhanced memory and cognition.
[0061] Abbreviations and Definitions
[0062] The term "compound (I)" or "compound 1" as used herein means
the compound designated as formula I or 1. It is also referred to
herein as "YF2" or "OA2".
[0063] The term "composition(s) of the invention" as used herein
means compositions comprising compound (I) or pharmaceutically
acceptable salts thereof. The compositions of the invention may
further comprise other agents such as, for example, excipients,
stabilants, lubricants, solvents, and the like.
[0064] The term "method(s) of the invention" as used herein means
methods comprising treatment with the compound (I) and/or
compositions of the invention.
[0065] A "pharmaceutical composition" refers to a mixture of
compound (I) described herein, or pharmaceutically acceptable salts
thereof, with other chemical components, such as physiologically
acceptable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0066] The term "pharmaceutically acceptable salt" is intended to
include salts derived from inorganic or organic acids including,
for example hydrochloric, hydrobromic, sulfuric, nitric,
perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric,
succinic, tartaric, glycolic, salicylic, citric, methanesulfonic,
benzenesulfonic, benzoic, malonic, trifluroacetic, trichloroacetic,
naphthalene-2 sulfonic and other acids; and salts derived from
inorganic or organic bases including, for example sodium,
potassium, calcium, ammonium or tetrafluoroborate. Exemplary
pharmaceutically acceptable salts are found, for example, in Berge,
et al. (J. Pharm. Sci. 1977, 66(1), 1 and Gould, P., Int. J.
Pharmaceutics 1986, 33, 201-217; each herein incorporated by
reference in its entirety). Pharmaceutically acceptable salts are
also intended to encompass hemi-salts, wherein the ratio of
compound:acid is respectively 2:1. Exemplary hemi-salts are those
salts derived from acids comprising two carboxylic acid groups,
such as malic acid, fumaric acid, maleic acid, succinic acid,
tartaric acid, glutaric acid, oxalic acid, adipic acid and citric
acid. Other exemplary hemi-salts are those salts derived from
diprotic mineral acids such as sulfuric acid. Exemplary preferred
hemi-salts include, but are not limited to, hemimaleate,
hemifumarate, and hemisuccinate.
[0067] The term "acid" contemplates all pharmaceutically acceptable
inorganic or organic acids. Inorganic acids include mineral acids
such as hydrohalic acids, such as hydrobromic and hydrochloric
acids, sulfuric acids, phosphoric acids and nitric acids. Organic
acids include all pharmaceutically acceptable aliphatic, alicyclic
and aromatic carboxylic acids, dicarboxylic acids, tricarboxylic
acids, and fatty acids. Preferred acids are straight chain or
branched, saturated or unsaturated C1-C20 aliphatic carboxylic
acids, which are optionally substituted by halogen or by hydroxyl
groups, or C6-C12 aromatic carboxylic acids. Examples of such acids
are carbonic acid, formic acid, fumaric acid, acetic acid,
propionic acid, isopropionic acid, valeric acid, alpha-hydroxy
acids, such as glycolic acid and lactic acid, chloroacetic acid,
benzoic acid, methane sulfonic acid, and salicylic acid. Examples
of dicarboxylic acids include oxalic acid, malic acid, succinic
acid, tataric acid and maleic acid. An example of a tricarboxylic
acid is citric acid. Fatty acids include all pharmaceutically
acceptable saturated or unsaturated aliphatic or aromatic
carboxylic acids having 4 to 24 carbon atoms. Examples include
butyric acid, isobutyric acid, sec-butyric acid, lauric acid,
palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic
acid, and phenylsteric acid. Other acids include gluconic acid,
glycoheptonic acid and lactobionic acid.
[0068] As used herein the term "about" is used herein to mean
approximately, roughly, around, or in the region of. When the term
"about" is used in conjunction with a numerical range, it modifies
that range by extending the boundaries above and below the
numerical values set forth. In general, the term "about" is used
herein to modify a numerical value above and below the stated value
by a variance of 20 percent up or down (higher or lower).
[0069] An "effective amount", "sufficient amount" or
"therapeutically effective amount" as used herein is an amount of a
compound that is sufficient to effect beneficial or desired
results, including clinical results. As such, the effective amount
may be sufficient, for example, to reduce or ameliorate the
severity and/or duration of the memory loss or cognition, or one or
more symptoms thereof, prevent the advancement of conditions
related to memory loss or cognition, improve cognition, learning or
memory in subjects not afflicted with a neurodegenerative disorder,
or enhance or otherwise improve the prophylactic or therapeutic
effect(s) of another therapy. An effective amount also includes the
amount of the compound that avoids or substantially attenuates
undesirable side effects.
[0070] As used herein and as well understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. Beneficial or desired clinical
results may include, but are not limited to, alleviation or
amelioration of one or more symptoms or conditions, diminution of
extent of disease, a stabilized (i.e., not worsening) state of
disease, preventing spread of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
[0071] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which a compound is administered. Non-limiting
examples of such pharmaceutical carriers include liquids, such as
water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. The pharmaceutical carriers may also be
saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal
silica, urea, and the like. In addition, auxiliary, stabilizing,
thickening, lubricating and coloring agents may be used. Other
examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E.W. Martin (hereby
incorporated by reference in its entirety).
[0072] The terms "animal," "subject" and "patient" as used herein
include all members of the animal kingdom including, but not
limited to, mammals (e.g., mice, rats, cats, monkeys, dogs, horses,
swine, etc.) and humans.
[0073] Acetylation and Methylation of DNA and Histones
[0074] Histone deacetylase (HDAC) and histone acetyltransferase
(HAT) are enzymes that influence transcription by selectively
deacetylating or acetylating the .epsilon.-amino groups of lysine
located near the amino termini of core histone proteins. Chromatin
acetylation correlates with transcriptional activity (euchromatin),
whereas deacetylation correlates with gene silencing.
Interestingly, it was shown that increased acetylation of H3 in
area CA1 of the hippocampus (an area in the brain that plays an
important role in long-term memory) occurs following associative
memory. Additionally, by inhibiting HDAC, they were able to
manipulate changes in the chromatin and enhance the formation of
long-term memory.
[0075] The DNA is firstly wrapped around an octamer complex of
histones (H) to form nucleosomal units, giving the appearance of
beads on a string (Nature, 2001. 409(6822): 860-921; herein
incorporated by reference in its entirety). In turn, these
nucleosomal units, fold into a higher-order chromatin fiber (Cell,
1999. 98(3): 285-94; herein incorporated by reference in its
entirety). Each histone-octamer complex contains two copies of
histones H3 and H4 bordered by two copies of histones 2A and 2B.
Histone H1 and its avian variant H5 are linker histones that bind
the nucleosome and both the entry and exit sites of the DNA, thus
locking the DNA into place and allowing the formation of higher
order structure. Every histone has a globular domain, which
mediates histone-histone interactions, and an N-terminal `tail`
extension. The histone cores and in particular their tails, are
targets for a considerable number of covalent modifications, such
as acetylation, ubiquitination, sumoylation, phosphorylation,
citrullination, ADP-ribosylation, and methylation (Angew Chem Int
Ed Engl, 2005. 44(21): 3186-216; herein incorporated by reference
in its entirety). Histone modifications associated with active gene
transcription, such as H3 Lys4 methylation and H3 Lys56
acetylation, were found to lead to gene expression. On the other
hand, histone modifications associated with the inactivation of
gene transcription, such as H3 Lys27 methylation and H2A Lys119
ubiquitination were found to cause gene silencing. Of particular
interest for this application are histone 2B, 3 and 4 because they
have been shown to be involved in memory processes (Nature, 2007.
447(7141): 178-82; Neuron, 2004. 42(6): 947-59; each herein
incorporated by reference in its entirety). Studies of
aging-associated memory dysfunction are discussed in Science 2010,
328, 701; herein incorporated by reference in its entirety.
[0076] HATs and HDACs. Histone modifications and their combinations
have been proposed to be involved in gene regulation by modifying
the chromatin accessibility and by acting as docking sites for
transcription factors and modifying enzymes (Bioessays, 2005.
27(2): 164-75; Nature, 2000. 403(6765): 41-5; herein incorporated
by reference in its entirety). One of the most studied histone
modifications is the acetylation of the evolutionary-conserved
lysine residues on the histone N-termini by histone
acetyltransferase (HAT). In contrast, histone deacetylation,
catalyzed by histone deacetylase (HDAC), was found to package the
DNA into a more condensed form, limiting the access of
transcription factors and thus acting as a gene silencer (Trends
Biochem Sci, 2000. 25(3): 121-6; herein incorporated by reference
in its entirety). The HATs involved in the regulation of gene
expression include at least three groups of enzymes (J. Biochem,
2005. 138(6): 647-62; herein incorporated by reference in its
entirety). The general control non-derepressible 5 (Gcn5) is the
founding member of the Gcn5 N-acetyltransferases (GNATs). The GNAT
family members include Gcn5, PCAF, Elp3, HAT1m Hpa2 and Nut1. The
MYST family is named after the founding members of the family:
Morf, Ybf2, Sas2 and Tip60. In addition, other proteins including
CBP/p300, Taf1 and a number of nuclear receptor co-activators have
been shown to possess intrinsic HAT activity. However, these
proteins do not contain a consensus domain and therefore represent
an `orphan class` of HAT enzymes.
[0077] HDACs form repressor complexes with transcription activators
and with other HDACs (Biochem J, 2003. 370(Pt 3): 737-49; herein
incorporated by reference in its entirety). Mammalian HDACs can be
divided into the classical and the silent information regulator 2
(Sir2)-related protein (sirtruin) families (Oncogene, 2007. 26(37):
5310-8; herein incorporated by reference in its entirety). In
humans, members of the classical family have another subdivision,
which include class I, II and IV, that share sequence similarity
and require Zn+ for deacetylase activity. Class I HDACs (HDAC1-3,
HDAC8) are related to the yeast gene repressor Rpd3p, and are
subunits of at least two distinct co-repressor complexes, the Sin3
complex and the NuRD complex. Class II HDACs (HDAC4-7, 9 and 10)
are similar to the yeast Hdalp HDAC, they act as gene repressors
and have been implicated in various roles in cell differentiation
and development. Class IV comprises HDAC11, which has some features
of both class I and II HDACs. The sirtruin family includes class
III HDACs (SIRT1-7), which are similar to yeast Sir2. Class III
HDACs are biochemically and structurally distinct from the
classical family and require NAD.sup.+ as a cofactor. HDACs appear
to be involved in gene silencing and heterochromatin formation at
centromeres and telomeres (for a review see (J Mol Biol, 2004.
338(1):17-31; herein incorporated by reference in its
entirety).
[0078] Alterations in epigenetic modifications including
acetylation and methylation of DNA and histones may contribute to
gene expression changes in cancer and neurological diseases.
Addition of acetyl group on histones by Histone Acetyltransferases
(HATs) enhances gene expression, while its removal by Histone
Deacytylases (HDAC) reduces gene expression. Reduction in histone
acetylation has been found in a variety of ailments such as tumors,
mood disorders, and neurodegenerative diseases. Examples of HATs
include, but are not limited to GCN5, GCN5L, PCAF, HAT1, ELP3,
HPA2, ESA1, SAS2, SAS3, TIP60, HBO1, MOZ, MORF, MOF, SRC1, SRC3,
TIF2, GRIP1, ATF-2 [see Lee and Workman (2007) Nat Rev Mol Cell
Biol., 8(4):284-95, Marmorstein (2001) J Molec Biol. 311: 433-444;
and Kimura et al., (2005) J Biochem. 138(6): 647-662; each of which
are hereby incorporated by reference in their entireties]. In some
embodiments, the HAT activator compound is directed to GCN5, GCN5L,
HAT1, PCAF, or a combination thereof. In some embodiments, the HAT
activator compound is directed to proteins that possess intrinsic
HAT activity, such as nuclear receptor co-activators (for example,
CBP/p300 and Taf1). In some embodiments, the acetylation of H2, H3,
and/or H4 histones is increased.
[0079] Increasing histone acetylation has been shown to improve
outcome in a wide variety of diseases as diverse as asthma,
infectious disease and psychiatric diseases. Although clinical
trials of several HDAC inhibitors are currently underway, the
alternative strategy where by histone acetylation is increased by
HAT activation has not been extensively explored. For example,
compounds in U.S. Patent Publication No. US2009076155 and PCT
Publication No. WO2004053140 (each herein incorporated by reference
in its entirety) have poor solubility and membrane permeability.
Furthermore, the compounds disclosed in the patent applications do
not disclose any data for the treatment of any diseases. Regulation
of HAT is also discussed, for example, in U.S. Patent Publication
No. US20040091967 and U.S. Pat. No. 7,750,047 (each herein
incorporated by reference in its entirety).
[0080] No HAT activator is currently in drug trials, however
several HDAC inhibitors are currently in clinical trials. Some of
these HDAC inhibitors (HDACi) have shown therapeutic efficacy in
preclinical trials. Without being bound by theory, it is believed
that HAT activators may be useful drug candidates with a role
similar to HDACi. However, previously available HAT activators had
little solubility and membrane permeability, making them unsuitable
as drugs.
[0081] Some HDACi are or were being developed for neurological
diseases, such as an HDACi from Merck (Whitehouse Station, N.J.)
that is being used for the treatment of neurodegenerative diseases;
and HDACi from TopoTarget (Rockaway, N.J.) that was being used for
the treatment of Huntington's disease, now discontinued;
isovaleramide NPS-1776 (NPS Pharmaceutical, Bedminster, N.J.) that
was being used for bipolar disorder, epilepsy, and migraines, now
discontinued; and a histone acetyltransferase inhibitor for cancer
from TopoTarget A/S (Kobenhavn, Denmark), which was discontinued in
the preclinical stage. Histone Acylation is discussed in Science
2010, 328, 753 and Nature 2009, 459, 55; each herein incorporated
by reference in its entirety.
[0082] Here, a HAT activator with improved solubility and membrane
permeability is described and its potency in-vitro as well as in an
animal model are shown. Compound (I) and other HAT activator
compounds are also described in PCT/US10/59925, incorporated herein
by reference in its entirety. In vitro and behavioral data show
that HAT activator compound (I) can acetylate histone H3 in brain
and ameliorate memory deficits in a mouse model of Alzheimer's
disease. For example, compound (I) can be used as adjuvant therapy
in several cancers, psychiatric and neurodegenerative diseases and
may improve efficacy and safety of treatment for these disorders.
Furthermore, the compound (I) exhibits good solubility and
permeability of the Blood-Brain-Barrier (See Abel and Zukin (2008)
Current Opinion in Pharmacology 8:57-64; and Lee and Workman (2007)
Nat Rev Mol Cell Biol 8:284-295; each herein incorporated by
reference in its entirety).
[0083] HAT1 is also known as KAT1 (K(lysine) acetyltransferase 1).
The protein encoded by this gene is a type B histone
acetyltransferase (HAT) that is involved in the rapid acetylation
of newly synthesized cytoplasmic histones, which are in turn
imported into the nucleus for de novo deposition onto nascent DNA
chains. Histone acetylation, particularly of histone H4, plays an
important role in replication-dependent chromatin assembly.
[0084] SEQ ID NO: 1 is the human wild type amino acid sequence
corresponding to the HAT protein, the HAT1 enzyme (residues
1-419):
TABLE-US-00001 1 MAGFGAMEKF LVEYKSAVEK KLAEYKCNTN TAIELKLVRF
PEDLENDIRT FFPEYTHQLF 61 GDDETAFGYK GLKILLYYIA GSLSTMFRVE
YASKVDENFD CVEADDVEGK IRQIIPPGFC 121 TNTNDFLSLL EKEVDFKPFG
TLLHTYSVLS PTGGENFTFQ IYKADMTCRG FREYHERLQT 181 FLMWFIETAS
FIDVDDERWH YFLVFEKYNK DGATLFATVG YMTVYNYYVY PDKTRPRVSQ 241
MLILTPFQGQ GHGAQLLETV HRYYTEFPTV LDITAEDPSK SYVKLRDFVL VKLCQDLPCF
301 SREKLMQGFN EDMAIEAQQK FKINKQHARR VYEILRLLVT DMSDAEQYRS
YRLDIKRRLI 361 SPYKKKQRDL AKMRKCLRPE ELTNQMNQIE ISMQHEQLEE
SFQELVEDYR RVIERLAQE
[0085] SEQ ID NO: 2 is the human wild type nucleotide sequence
corresponding to HAT protein, the HAT1 enzyme (residues 1-1682),
wherein the underscored ATG denotes the beginning of the open
reading frame:
TABLE-US-00002 1 ctgtgcggtc acttccggcc cgggagcgcg cgggttgatt
cgtccttcct cagccgcggg 61 tgatcgtagc tcggaaatgg cgggatttgg
tgctatggag aaatttttgg tagaatataa 121 gagtgcagtg gagaagaaac
tggcagagta caaatgtaac accaacacag caattgaact 181 aaaattagtt
cgttttcctg aagatcttga aaatgacatt agaactttct ttcctgagta 241
tacccatcaa ctctttgggg atgatgaaac tgcttttggt tacaagggtc taaagatcct
301 gttatactat attgctggta gcctgtcaac aatgttccgt gttgaatatg
catctaaagt 361 tgatgagaac tttgactgtg tagaggcaga tgatgttgag
ggcaaaatta gacaaatcat 421 tccacctgga ttttgcacaa acacgaatga
tttcctttct ttactggaaa aggaagttga 481 tttcaagcca ttcggaacct
tacttcatac ctactcagtt ctcagtccaa caggaggaga 541 aaactttacc
tttcagatat ataaggctga catgacatgt agaggctttc gagaatatca 601
tgaaaggctt cagacctttt tgatgtggtt tattgaaact gctagcttta ttgacgtgga
661 tgatgaaaga tggcactact ttctagtatt tgagaagtat aataaggatg
gagctacgct 721 ctttgcgacc gtaggctaca tgacagtcta taattactat
gtgtacccag acaaaacccg 781 gccacgtgta agtcagatgc tgattttgac
tccatttcaa ggtcaaggcc atggtgctca 841 acttcttgaa acagttcata
gatactacac tgaatttcct acagttcttg atattacagc 901 ggaagatcca
tccaaaagct atgtgaaatt acgagacttt gtgcttgtga agctttgtca 961
agatttgccc tgtttttccc gggaaaaatt aatgcaagga ttcaatgaag atatggcgat
1021 agaggcacaa cagaagttca aaataaataa gcaacacgct agaagggttt
atgaaattct 1081 tcgactactg gtaactgaca tgagtgatgc cgaacaatac
agaagctaca gactggatat 1141 taaaagaaga ctaattagcc catataagaa
aaagcagaga gatcttgcta agatgagaaa 1201 atgtctcaga ccagaagaac
tgacaaacca gatgaaccaa atagaaataa gcatgcaaca 1261 tgaacagctg
gaagagagtt ttcaggaact agtggaagat taccggcgtg ttattgaacg 1321
acttgctcaa gagtaaagat tatactgctc tgtacaggaa gcttgcaaat tttctgtaca
1381 atgtgctgtg aaaaatctga tgactttaat tttaaaatct tgtgacattt
tgcttatact 1441 aaaagttatc tatctttagt tgaatatttt cttttggaga
gattgtatat tttaaaatac 1501 tgtttagagt ttatgagcat atattgcatt
taaagaaaga taaagcttct gaaatactac 1561 tgcaattgct tcccttctta
aacagtataa taaatgctta gttgtgatat gttaatgtgt 1621 gatgatatga
ttcttaaata cttacaataa acctcattct taaatactta aaaaaaaaaa 1681 aa
[0086] The polypeptide sequence of a HAT protein, human PCAF, is
depicted in SEQ ID NO: 3. The nucleotide sequence of human PCAF is
shown in SEQ ID NO: 4. Sequence information related to PCAF is
accessible in public databases by GenBank Accession numbers
NM_003884 (for mRNA) and NP_003875 (for protein). PCAF is also
known as KAT2B (K(lysine) acetyltransferase 2B). CBP and p300 are
large nuclear proteins that bind to many sequence-specific factors
involved in cell growth and/or differentiation, including c-jun and
the adenoviral oncoprotein E1A. The protein encoded by this gene
associates with p300/CBP. It has in vitro and in vivo binding
activity with CBP and p300, and competes with E1A for binding sites
in p300/CBP. It has histone acetyl transferase activity with core
histones and nucleosome core particles, indicating that this
protein plays a direct role in transcriptional regulation.
[0087] SEQ ID NO: 3 is the human wild type amino acid sequence
corresponding to the HAT protein, the PCAF enzyme (residues
1-832):
TABLE-US-00003 1 MSEAGGAGPG GCGAGAGAGA GPGALPPQPA ALPPAPPQGS
PCAAAAGGSG ACGPATAVAA 61 AGTAEGPGGG GSARIAVKKA QLRSAPRAKK
LEKLGVYSAC KAEESCKCNG WKNPNPSPTP 121 PRADLQQIIV SLTESCRSCS
HALAAHVSHL ENVSEEEMNR LLGIVLDVEY LFTCVHKEED 181 ADTKQVYFYL
FKLLRKSILQ RGKPVVEGSL EKKPPFEKPS IEQGVNNFVQ YKFSHLPAKE 241
RQTIVELAKM FLNRINYWHL EAPSQRRLRS PNDDISGYKE NYTRWLCYCN VPQFCDSLPR
301 YETTQVFGRT LLRSVFTVMR RQLLEQARQE KDKLPLEKRT LILTHFPKFL
SMLEEEVYSQ 361 NSPIWDQDFL SASSRTSQLG IQTVINPPPV AGTISYNSTS
SSLEQPNAGS SSPACKASSG 421 LEANPGEKRK MTDSHVLEEA KKPRVMGDIP
MELINEVMST ITDPAAMLGP ETNFLSAHSA 481 RDEAARLEER RGVIEFHVVG
NSLNQKPNKK ILMWLVGLQN VFSHQLPRMP KEYITRLVFD 541 PKHKTLALIK
DGRVIGGICF RMFPSQGFTE IVFCAVTSNE QVKGYGTHLM NHLKEYHIKH 601
DILNFLTYAD EYAIGYFKKQ GFSKEIKIPK TKYVGYIKDY EGATLMGCEL NPRIPYTEFS
661 VIIKKQKEII KKLIERKQAQ IRKVYPGLSC FKDGVRQIPI ESIPGIRETG
WKPSGKEKSK 721 EPRDPDQLYS TLKSILQQVK SHQSAWPFME PVKRTEAPGY
YEVIRFPMDL KTMSERLKNR 781 YYVSKKLFMA DLQRVFTNCK EYNPPESEYY
KCANILEKFF FSKIKEAGLI DK
[0088] SEQ ID NO: 4 is the human wild type nucleotide sequence
corresponding to HAT protein, the PCAF enzyme (residues 1-4824),
wherein the underscored ATG denotes the beginning of the open
reading frame:
TABLE-US-00004 1 gcggaaaaga ggccgtgggg ggcctcccag cgctggcaga
caccgtgagg ctggcagccg 61 ccggcacgca cacctagtcc gcagtcccga
ggaacatgtc cgcagccagg gcgcggagca 121 gagtcccggg caggagaacc
aagggagggc gtgtgctgtg gcggcggcgg cagcggcagc 181 ggagccgcta
gtcccctccc tcctggggga gcagctgccg ccgctgccgc cgccgccacc 241
accatcagcg cgcggggccc ggccagagcg agccgggcga gcggcgcgct agggggaggg
301 cgggggcggg gaggggggtg ggcgaagggg gcgggagggc gtggggggag
ggtctcgctc 361 tcccgactac cagagcccga gagggagacc ctggcggcgg
cggcggcgcc tgacactcgg 421 cgcctcctgc cgtgctccgg ggcggcatgt
ccgaggctgg cggggccggg ccgggcggct 481 gcggggcagg agccggggca
ggggccgggc ccggggcgct gcccccgcag cctgcggcgc 541 ttccgcccgc
gcccccgcag ggctccccct gcgccgctgc cgccgggggc tcgggcgcct 601
gcggtccggc gacggcagtg gctgcagcgg gcacggccga aggaccggga ggcggtggct
661 cggcccgaat cgccgtgaag aaagcgcaac tacgctccgc tccgcgggcc
aagaaactgg 721 agaaactcgg agtgtactcc gcctgcaagg ccgaggagtc
ttgtaaatgt aatggctgga 781 aaaaccctaa cccctcaccc actcccccca
gagccgacct gcagcaaata attgtcagtc 841 taacagaatc ctgtcggagt
tgtagccatg ccctagctgc tcatgtttcc cacctggaga 901 atgtgtcaga
ggaagaaatg aacagactcc tgggaatagt attggatgtg gaatatctct 961
ttacctgtgt ccacaaggaa gaagatgcag ataccaaaca agtttatttc tatctattta
1021 agctcttgag aaagtctatt ttacaaagag gaaaacctgt ggttgaaggc
tctttggaaa 1081 agaaaccccc atttgaaaaa cctagcattg aacagggtgt
gaataacttt gtgcagtaca 1141 aatttagtca cctgccagca aaagaaaggc
aaacaatagt tgagttggca aaaatgttcc 1201 taaaccgcat caactattgg
catctggagg caccatctca acgaagactg cgatctccca 1261 atgatgatat
ttctggatac aaagagaact acacaaggtg gctgtgttac tgcaacgtgc 1321
cacagttctg cgacagtcta cctcggtacg aaaccacaca ggtgtttggg agaacattgc
1381 ttcgctcggt cttcactgtt atgaggcgac aactcctgga acaagcaaga
caggaaaaag 1441 ataaactgcc tcttgaaaaa cgaactctaa tcctcactca
tttcccaaaa tttctgtcca 1501 tgctagaaga agaagtatat agtcaaaact
ctcccatctg ggatcaggat tttctctcag 1561 cctcttccag aaccagccag
ctaggcatcc aaacagttat caatccacct cctgtggctg 1621 ggacaatttc
atacaattca acctcatctt cccttgagca gccaaacgca gggagcagca 1681
gtcctgcctg caaagcctct tctggacttg aggcaaaccc aggagaaaag aggaaaatga
1741 ctgattctca tgttctggag gaggccaaga aaccccgagt tatgggggat
attccgatgg 1801 aattaatcaa cgaggttatg tctaccatca cggaccctgc
agcaatgctt ggaccagaga 1861 ccaattttct gtcagcacac tcggccaggg
atgaggcggc aaggttggaa gagcgcaggg 1921 gtgtaattga atttcacgtg
gttggcaatt ccctcaacca gaaaccaaac aagaagatcc 1981 tgatgtggct
ggttggccta cagaacgttt tctcccacca gctgccccga atgccaaaag 2041
aatacatcac acggctcgtc tttgacccga aacacaaaac ccttgcttta attaaagatg
2101 gccgtgttat tggtggtatc tgtttccgta tgttcccatc tcaaggattc
acagagattg 2161 tcttctgtgc tgtaacctca aatgagcaag tcaagggcta
tggaacacac ctgatgaatc 2221 atttgaaaga atatcacata aagcatgaca
tcctgaactt cctcacatat gcagatgaat 2281 atgcaattgg atactttaag
aaacagggtt tctccaaaga aattaaaata cctaaaacca 2341 aatatgttgg
ctatatcaag gattatgaag gagccacttt aatgggatgt gagctaaatc 2401
cacggatccc gtacacagaa ttttctgtca tcattaaaaa gcagaaggag ataattaaaa
2461 aactgattga aagaaaacag gcacaaattc gaaaagttta ccctggactt
tcatgtttta 2521 aagatggagt tcgacagatt cctatagaaa gcattcctgg
aattagagag acaggctgga 2581 aaccgagtgg aaaagagaaa agtaaagagc
ccagagaccc tgaccagctt tacagcacgc 2641 tcaagagcat cctccagcag
gtgaagagcc atcaaagcgc ttggcccttc atggaacctg 2701 tgaagagaac
agaagctcca ggatattatg aagttataag gttccccatg gatctgaaaa 2761
ccatgagtga acgcctcaag aataggtact acgtgtctaa gaaattattc atggcagact
2821 tacagcgagt ctttaccaat tgcaaagagt acaacccccc tgagagtgaa
tactacaaat 2881 gtgccaatat cctggagaaa ttcttcttca gtaaaattaa
ggaagctgga ttaattgaca 2941 agtgattttt tttcccctct gcttcttaga
aactcaccaa gcagtgtgcc taaagcaagg 3001 tggtttagtt ttttacaaag
aattggacat gatgtattga agagacttgt aaatgtaata 3061 attagcactt
ttgaaaaaac aaaaaacctc cttttagctt ttcagatatg tatttaaatt 3121
gaagtcatag gacattttta ttttatggaa tagattttaa tctatttact actattaagg
3181 taaattttct atggcatgtc cattagctat ttcatgatag atgattaggg
gtttcctcaa 3241 aacctgtgtg tgaggaaatt gcacacagta gcaaaatttg
gggaaatcca taacattttc 3301 agaccatgaa tgaatgtttc catttttttc
taatggaatg tgagagttta cttttatttt 3361 attctgaagg actttaagga
agggatacat gattttaaaa aagcctgtaa gaggtgaaat 3421 atgtgatgtt
tgaagtctct ttatagactt tttatatata ttttttaaaa cactcatcta 3481
gatgaggtgc tttgagcagt tctgaaaaat gcagttccag gaaagcaact gctttggttc
3541 ctaaggaaga aattctaaat aatgcaaact tttaaaataa gcatctaggt
ttttgataat 3601 tctgtctact tacaacaaac ttgttagtac ataaccacta
ttttaataat tattttctct 3661 acacaaatgt gtaatatcat atttgacttt
gcttatgcag gccataagtt ccaaaagata 3721 atttccctgc ccacaaaggc
ataaacttga aaacacatga gattgaatca acatgcttta 3781 ataggaaaag
atgtatggtc tatatatgta tcaatctggt gaatcctcgt tctaataaag 3841
gttctttttc ttttctatga tacacacagc cacgctgata atatgcaaat gaacattttc
3901 ctttatgtct ctccagataa tgtttattgt ctgaggtaaa ttaaattccc
accagggttt 3961 gctgtcagta ttttaacacc cacattagta tatgcgtcca
gggtcataac cccctaaaat 4021 ccatcatgca accttattaa tctgtcttgg
gattccagtt tagtgcttgg atttatttcc 4081 tgattacact acatagaaaa
gtgagacatc tgccattccc aactctggga aaaccaacta 4141 atatacaacc
atataaatga aggccatctt gatggtctca acactaattt ttatgatgca 4201
aatttataca ctgatttttg taaaggacaa agttttaaaa gcgtatttaa cttgatgttt
4261 tctatcagca taaataaaat ggtcatgaat agtcattaaa aacagttgcc
agtgataatc 4321 tgcatgaagg aaaaagaacc ctgcaaatgg ctattgagtt
ggaagtattg tttttgatat 4381 gtaagagata ttcagaatgc tcacactgaa
aatgcctcaa ctttttaaag tgtaagaaac 4441 caccatgagt ggtgtctaga
tttctaatga agaatcatga tacagtttgg attaagtatc 4501 ttggactggt
tttaaacagt gctttgtacc ggatctgctg aagcatctgt ccagctggta 4561
tcctgtgaaa gtttgttatt ttctgagtag acattcttat agagtattgt ctttaaaatc
4621 agattgtctc ttctatattg aaagcatttt tatgttttct aatttaaaaa
ttaatatttt 4681 cttatagata ttgtgcaata aagctgaagt agaatgtgtg
gtttttgcaa atgctttaac 4741 agctgataaa aattttacat ttgtaaaatt
aatatattgt actggtacaa aatagtttta 4801 aattatattt taaaaagctt
ccaa
[0089] The polypeptide sequence of a HAT protein, human GCN5L, is
depicted in SEQ ID NO: 5. The nucleotide sequence of human GCN5L is
shown in SEQ ID NO: 6. Sequence information related to GCN5L is
accessible in public databases by GenBank Accession numbers
NM_021078 (for mRNA) and NP_066564.2 (for protein). GCN5L is also
known as KAT2A (K(lysine) acetyltransferase 2A). KAT2A, or GCN5, is
a histone acetyltransferase (HAT) that functions primarily as a
transcriptional activator. It also functions as a repressor of
NF-kappa-B by promoting ubiquitination of the NF-kappa-B subunit
RELA in a HAT-independent manner (Mao et al., Genes Dev. 2009 Apr.
1; 23(7):849-61; each herein incorporated by reference in its
entirety).
[0090] SEQ ID NO: 5 is the human wild type amino acid sequence
corresponding to the HAT protein, the GCN5L enzyme (residues
1-837):
TABLE-US-00005 1 MAEPSQAPTP APAAQPRPLQ SPAPAPTPTP APSPASAPIP
TPTPAPAPAP AAAPAGSTGT 61 GGPGVGSGGA GSGGDPARPG LSQQQRASQR
KAQVRGLPRA KKLEKLGVFS ACKANETCKC 121 NGWKNPKPPT APRMDLQQPA
ANLSELCRSC EHPLADHVSH LENVSEDEIN RLLGMVVDVE 181 NLFMSVHKEE
DTDTKQVYFY LFKLLRKCIL QMTRPVVEGS LGSPPFEKPN IEQGVLNFVQ 241
YKFSHLAPRE RQTMFELSKM FLLCLNYWKL ETPAQFRQRS QAEDVATYKV NYTRWLCYCH
301 VPQSCDSLPR YETTHVFGRS LLRSIFTVTR RQLLEKFRVE KDKLVPEKRT
LILTHFPKFL 361 SMLEEEIYGA NSPIWESGFT MPPSEGTQLV PRPASVSAAV
VPSTPIFSPS MGGGSNSSLS 421 LDSAGAEPMP GEKRTLPENL TLEDAKRLRV
MGDIPMELVN EVMLTITDPA AMLGPETSLL 481 SANAARDETA RLEERRGIIE
FHVIGNSLTP KANRRVLLWL VGLQNVFSHQ LPRMPKEYIA 541 RLVFDPKHKT
LALIKDGRVI GGICFRMFPT QGFTEIVFCA VTSNEQVKGY GTHLMNHLKE 601
YHIKHNILYF LTYADEYAIG YFKKQGFSKD IKVPKSRYLG YIKDYEGATL MECELNPRIP
661 YTELSHIIKK QKEIIKKLIE RKQAQIRKVY PGLSCFKEGV RQIPVESVPG
IRETGWKPLG 721 KEKGKELKDP DQLYTTLKNL LAQIKSHPSA WPFMEPVKKS
EAPDYYEVIR FPIDLKTMTE 781 RLRSRYYVTR KLFVADLQRV IANCREYNPP
DSEYCRCASA LEKFFYFKLK EGGLIDK
[0091] SEQ ID NO: 6 is the human wild type nucleotide sequence
corresponding to HAT protein, the GCN5L enzyme (residues 1-3127),
wherein the underscored ATG denotes the beginning of the open
reading frame:
TABLE-US-00006 1 ggttgcccat gcggccctag ggctgggagc gcggcgccgc
tctccgctgc gggggaggcc 61 atggcggaac cttcccaggc cccgaccccg
gccccggctg cgcagccccg gccccttcag 121 tccccagccc ctgccccaac
tccgactcct gcacccagcc cggcttcagc cccgattccg 181 actcccaccc
cggcaccagc ccctgcccca gctgcagccc cagccggcag cacagggact 241
ggggggcccg gggtaggaag tgggggggcc gggagcgggg gggatccggc tcgacctggc
301 ctgagccagc agcagcgcgc cagtcagagg aaggcgcaag tccgggggct
gccgcgcgcc 361 aagaagcttg agaagctagg ggtcttctcg gcttgcaagg
ccaatgaaac ctgtaagtgt 421 aatggctgga aaaaccccaa gccccccact
gcaccccgca tggatctgca gcagccagct 481 gccaacctga gtgagctgtg
ccgcagttgt gagcacccct tggctgacca cgtatcccac 541 ttggagaatg
tgtcagagga tgagataaac cgactgctgg ggatggtggt ggatgtggag 601
aatctcttca tgtctgttca caaggaagag gacacagaca ccaagcaggt ctatttctac
661 ctcttcaagc tactgcggaa atgcatcctg cagatgaccc ggcctgtggt
ggaggggtcc 721 ctgggcagcc ctccatttga gaaacctaat attgagcagg
gtgtgctgaa ctttgtgcag 781 tacaagttta gtcacctggc tccccgggag
cggcagacga tgttcgagct ctcaaagatg 841 ttcttgctct gccttaacta
ctggaagctt gagacacctg cccagtttcg gcagaggtct 901 caggctgagg
acgtggctac ctacaaggtc aattacacca gatggctctg ttactgccac 961
gtgccccaga gctgtgatag cctcccccgc tacgaaacca ctcatgtctt tgggcgaagc
1021 cttctccggt ccattttcac cgttacccgc cggcagctgc tggaaaagtt
ccgagtggag 1081 aaggacaaat tggtgcccga gaagaggacc ctcatcctca
ctcacttccc caaattcctg 1141 tccatgctgg aggaggagat ctatggggca
aactctccaa tctgggagtc aggcttcacc 1201 atgccaccct cagaggggac
acagctggtt ccccggccag cttcagtcag tgcagcggtt 1261 gttcccagca
cccccatctt cagccccagc atgggtgggg gcagcaacag ctccctgagt 1321
ctggattctg caggggccga gcctatgcca ggcgagaaga ggacgctccc agagaacctg
1381 accctggagg atgccaagcg gctccgtgtg atgggtgaca tccccatgga
gctggtcaat 1441 gaggtcatgc tgaccatcac tgaccctgct gccatgctgg
ggcctgagac gagcctgctt 1501 tcggccaatg cggcccggga tgagacagcc
cgcctggagg agcgccgcgg catcatcgag 1561 ttccatgtca tcggcaactc
actgacgccc aaggccaacc ggcgggtgtt gctgtggctc 1621 gtggggctgc
agaatgtctt ttcccaccag ctgccgcgca tgcctaagga gtatatcgcc 1681
cgcctcgtct ttgacccgaa gcacaagact ctggccttga tcaaggatgg gcgggtcatc
1741 ggtggcatct gcttccgcat gtttcccacc cagggcttca cggagattgt
cttctgtgct 1801 gtcacctcga atgagcaggt caagggttat gggacccacc
tgatgaacca cctgaaggag 1861 tatcacatca agcacaacat tctctacttc
ctcacctacg ccgacgagta cgccatcggc 1921 tacttcaaaa agcagggttt
ctccaaggac atcaaggtgc ccaagagccg ctacctgggc 1981 tacatcaagg
actacgaggg agcgacgctg atggagtgtg agctgaatcc ccgcatcccc 2041
tacacggagc tgtcccacat catcaagaag cagaaagaga tcatcaagaa gctgattgag
2101 cgcaaacagg cccagatccg caaggtctac ccggggctca gctgcttcaa
ggagggcgtg 2161 aggcagatcc ctgtggagag cgttcctggc attcgagaga
caggctggaa gccattgggg 2221 aaggagaagg ggaaggagct gaaggacccc
gaccagctct acacaaccct caaaaacctg 2281 ctggcccaaa tcaagtctca
ccccagtgcc tggcccttca tggagcctgt gaagaagtcg 2341 gaggcccctg
actactacga ggtcatccgc ttccccattg acctgaagac catgactgag 2401
cggctgcgaa gccgctacta cgtgacccgg aagctctttg tggccgacct gcagcgggtc
2461 atcgccaact gtcgcgagta caaccccccg gacagcgagt actgccgctg
tgccagcgcc 2521 ctggagaagt tcttctactt caagctcaag gagggaggcc
tcattgacaa gtaggcccat 2581 ctttgggccg cagccctgac ctggaatgtc
tccacctcgg attctgatct gatccttagg 2641 gggtgccctg gccccacgga
cccgactcag cttgagacac tccagccaag ggtcctccgg 2701 acccgatcct
gcagctcttt ctggaccttc aggcaccccc aagcgtgcag ctctgtccca 2761
gccttcactg tgtgtgagag gtctcctggg ttggggccca gcccctctag agtagctggt
2821 ggccagggat gaaccttgcc cagccgtggt ggcccccagg cctggtcccc
aagagctttg 2881 gaggcttgga ttcctgggcc tggcccaggt ggctgtttcc
ctgaggacca gaactgctca 2941 ttttagcttg agtgatggct tcaggggttg
gaagttcagc ccaaactgaa gggggccatg 3001 ccttgtccag cactgttctg
tcagtctccc ccaggggtgg ggggtatggg gaccattcat 3061 tccctggcat
taatccctta gagggaataa taaagctttt tatttctctg tgaaaaaaaa 3121
aaaaaaa
[0092] Knowledge of the primary sequence of a molecule of interest,
such as a HAT polypeptide, and the similarity of that sequence with
other proteins of the same histone acetyltransferase family (such
as the GNAT family, the MYST family or the GCN5 family [see Lee and
Owrkman (2007) Nat Rev Mol Cell Biol., 8(4):284-95, Marmorstein
(2001) J Molec Biol. 311: 433-444; and Kimura et al., (2005) J.
Biochem. 138(6): 647-662; each herein incorporated by reference in
its entirety]), can provide information as to the inhibitors or
antagonists of the protein of interest. Identification and
screening antagonists can be further facilitated by determining
structural features of the protein, e.g., using X-ray
crystallography, neutron diffraction, nuclear magnetic resonance
spectrometry, and other techniques for structure determination.
These techniques may provide for rational approaches to the design
or identification of antagonists, in addition to protein
agonists.
[0093] A HAT Activator compound can be a compound that increases
the activity and/or expression of a HAT molecule (e.g., GCN5,
GCN5L, PCAF, or HAT1) in vivo and/or in vitro. HAT Activator
compounds can be compounds that exert their effect on the activity
of a HAT protein via the expression, via post-translational
modifications, or by other means. In some embodiments, a HAT
Activator compound can increase HAT protein or mRNA expression, or
acetyltransferase activity by at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 97%, at least about 99%, or 100%.
[0094] In some embodiments, the methods comprise administering to
the subject an effective amount of a composition comprising
compound (I). In some embodiments, the subject does not exhibit
abnormally elevated amyloid beta plaques, elevated Tau protein
levels, accumulations of alpha-synuclein, accumulations of
lipofuscin, or accumulation of cleaved TARDBP (TDB-43) levels, or
any combination thereof. In some embodiments, the subject is not
afflicted with Alzheimer's disease, Lewy body dementia, inclusion
body myositis, Huntington's Disease, Parkinson's Disease, or
cerebral amyloid angiopathy. In some embodiments, the subject is
not afflicted with cancer.
[0095] In some embodiments, the method of treatment comprises the
steps of: i) identifying a subject in need of such treatment; (ii)
providing compound (I), or a pharmaceutically acceptable salt
thereof; and (iii) administering said compound in a therapeutically
effective amount to a subject in need of such treatment.
[0096] In some embodiments, the method of treatment comprises the
steps of: i) identifying a subject in need of such treatment; (ii)
providing a composition comprising compound (I), or a
pharmaceutically acceptable salt thereof; and (iii) administering
said composition in a therapeutically effective amount to a subject
in need of such treatment.
[0097] In some embodiments, the methods comprise administering to
the subject an effective amount of compound (I), or a
pharmaceutically acceptable salt thereof, or a composition
comprising compound (I), or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carriers are well-known to those
skilled in the art, and include, for example, adjuvants, diluents,
excipients, fillers, lubricants and vehicles. Often, the
pharmaceutically acceptable carrier is chemically inert toward the
active compounds and is non-toxic under the conditions of use.
Examples of pharmaceutically acceptable carriers may include, for
example, water or saline solution, polymers such as polyethylene
glycol, carbohydrates and derivatives thereof, oils, fatty acids,
or alcohols.
[0098] In one aspect, the invention is directed to the use of
compound (I), or a pharmaceutically acceptable salt thereof, in
preparation of a medicament for enhancing learning or memory in a
subject.
[0099] In one aspect, the invention is directed to the use of
compound (I), or a pharmaceutically acceptable salt thereof, in
preparation of a medicament for increasing histone acetylation in a
subject.
[0100] In one aspect, the invention is directed to the use of
compound (I), or a pharmaceutically acceptable salt thereof, in
preparation of a medicament for improving memory retention in a
subject.
[0101] In one aspect, the invention is directed to the use of
compound (I), or a pharmaceutically acceptable salt thereof, in
preparation of a medicament for treating memory loss or a learning
disability in a subject.
[0102] In some embodiments, compound (I) is formulated into
pharmaceutical compositions for administration to subjects in a
biologically compatible form suitable for administration in vivo.
According to some embodiments, the present invention provides a
pharmaceutical composition comprising compound (I) in admixture
with a pharmaceutically acceptable diluent and/or carrier. The
pharmaceutically-acceptable carrier is "acceptable" in the sense of
being compatible with the other ingredients of the composition and
not deleterious to the recipient thereof. The
pharmaceutically-acceptable carriers employed herein may be
selected from various organic or inorganic materials that are used
as materials for pharmaceutical formulations and which are
incorporated as absorption delaying agents, analgesics,
antibacterials, antifungals, buffers, binders, coatings,
disintegrants, diluents, dispersants, emulsifiers, excipients,
extenders, glidants, solubilizers, solvents, stabilizers,
suspending agents, tonicity agents, vehicles and
viscosity-increasing agents. Pharmaceutical additives, such as
antioxidants, aromatics, colorants, flavor-improving agents,
preservatives, and sweeteners, may also be added. Examples of
acceptable pharmaceutical carriers include carboxymethyl cellulose,
crystalline cellulose, glycerin, gum arabic, lactose, magnesium
stearate, methyl cellulose, powders, saline, sodium alginate,
sucrose, starch, talc and water, among others. In some embodiments,
the term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans.
[0103] Surfactants such as, for example, detergents, are also
suitable for use in the formulations. Specific examples of
surfactants include polyvinylpyrrolidone, polyvinyl alcohols,
copolymers of vinyl acetate and of vinylpyrrolidone, polyethylene
glycols, benzyl alcohol, mannitol, glycerol, sorbitol or
polyoxyethylenated esters of sorbitan; lecithin or sodium
carboxymethylcellulose; or acrylic derivatives, such as
methacrylates and others, anionic surfactants, such as alkaline
stearates, in particular sodium, potassium or ammonium stearate;
calcium stearate or triethanolamine stearate; alkyl sulfates, in
particular sodium lauryl sulfate and sodium cetyl sulfate; sodium
dodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or
fatty acids, in particular those derived from coconut oil, cationic
surfactants, such as water-soluble quaternary ammonium salts of
formula N.sup.+R'R''R'''R''''Y.sup.-, in which the R radicals are
identical or different optionally hydroxylated hydrocarbon radicals
and Y.sup.- is an anion of a strong acid, such as halide, sulfate
and sulfonate anions; cetyltrimethylammonium bromide is one of the
cationic surfactants which can be used, amine salts of formula
N.sup.+R'R''R''', in which the R radicals are identical or
different optionally hydroxylated hydrocarbon radicals;
octadecylamine hydrochloride is one of the cationic surfactants
which can be used, non-ionic surfactants, such as optionally
polyoxyethylenated esters of sorbitan, in particular Polysorbate
80, or polyoxyethylenated alkyl ethers; polyethylene glycol
stearate, polyoxyethylenated derivatives of castor oil,
polyglycerol esters, polyoxyethylenated fatty alcohols,
polyoxyethylenated fatty acids or copolymers of ethylene oxide and
of propylene oxide, amphoteric surfactants, such as substituted
lauryl compounds of betaine,
[0104] When administered to a subject, compound (I) and
pharmaceutically acceptable carriers can be sterile. Suitable
pharmaceutical carriers may also include excipients such as starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol,
polyethylene glycol 300, water, ethanol, polysorbate 20, and the
like. The present compositions, if desired, may also contain minor
amounts of wetting or emulsifying agents, or pH buffering
agents.
[0105] The pharmaceutical formulations of the present invention are
prepared by methods well-known in the pharmaceutical arts.
Optionally, one or more accessory ingredients (e.g., buffers,
flavoring agents, surface active agents, and the like) also are
added. The choice of carrier is determined by the solubility and
chemical nature of the compounds, chosen route of administration
and standard pharmaceutical practice.
[0106] Additionally, the compound and/or compositions of the
present invention are administered to a human or animal subject by
known procedures including oral administration, intraperitoneal,
parenteral (e.g., intravenous), intradermal, subcutaneous,
intranasal, transdermal, topical, transmucosal, rectal, sublingual
or buccal administration. In some embodiments, compound (I) or a
composition comprising compound (I) is administered orally. In some
embodiments, compound (I) or a composition comprising compound (I)
is administered intraperitoneally.
[0107] For oral administration, a formulation of compound (I) or
compositions thereof may be presented in dosage forms such as
capsules, tablets, powders, granules, or as a suspension or
solution. Capsule formulations may be gelatin, soft-gel or solid.
Tablets and capsule formulations may further contain one or more
adjuvants, binders, diluents, disintegrants, excipients, fillers,
or lubricants, each of which are known in the art. Examples of such
include carbohydrates such as lactose or sucrose, dibasic calcium
phosphate anhydrous, corn starch, mannitol, xylitol, cellulose or
derivatives thereof, microcrystalline cellulose, gelatin,
stearates, silicon dioxide, talc, sodium starch glycolate, acacia,
flavoring agents, preservatives, buffering agents, disintegrants,
and colorants. Orally administered compositions may contain one or
more optional agents such as, for example, sweetening agents such
as fructose, aspartame or saccharin; flavoring agents such as
peppermint, oil of wintergreen, or cherry; coloring agents; and
preservative agents, to provide a pharmaceutically palatable
preparation.
[0108] The compositions may further comprise one or more sterile
diluents, such as water, saline solutions, fixed oils, polyalkylene
glycols, polyoxyalkylene glycols, glycerine, or other solvents;
antibacterial agents such as benzyl alcohol or methyl parabens,
antioxidants such as ascorbic acid, citric acid or sodium
bisulfite, chelating agents such as EDTA, buffers such as acetate,
citrate, phosphate and the like, tonicity adjusters such as sodium
chloride or dextrose, pH adjusters such as weak acids or bases,
etc.
[0109] In some embodiments, the composition is in unit dose form
such as a tablet, capsule or single-dose vial. Suitable unit doses,
i.e., therapeutically effective amounts, may be determined during
clinical trials designed appropriately for each of the conditions
for which administration of a chosen compound is indicated and
will, of course, vary depending on the desired clinical
endpoint.
[0110] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EM.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition is
preferably sterile and should be fluid to the extent that easy
syringability exists. It is preferably stable under the conditions
of manufacture and storage and is preferably preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, a pharmaceutically acceptable polyol like
glycerol, propylene glycol, liquid polyetheylene glycol, and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, and thimerosal. In many cases, it can be useful to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
[0111] Sterile injectable solutions can be prepared by
incorporating compound (I) or a composition thereof in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated herein, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated herein. In the case of sterile powders for the
preparation of sterile injectable solutions, examples of useful
preparation methods are vacuum drying and freeze-drying which
yields a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof.
[0112] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
[0113] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0114] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0115] The parenteral preparation can be enclosed in ampoules,
disposable syringes or multiple dose vials made of glass or
plastic.
[0116] In accordance with the methods of the present invention, in
some embodiments the compounds and/or compositions of the invention
are administered to the subject in a therapeutically effective
amount to enhance or increase memory or cognition in the subject.
This amount is readily determined by the skilled artisan, based
upon known procedures, including analysis of titration curves
established in vivo and methods and assays disclosed herein.
[0117] The dosage administered can be a therapeutically effective
amount of the composition sufficient to result in memory or
cognitive enhancement, increasing learning, or reducing memory loss
in a subject.
[0118] In some embodiments, the methods comprise administration of
a therapeutically effective dosage of compound (I). In some
embodiments, the therapeutically effective dosage is at least about
0.05 mg/kg body weight, at least about 0.1 mg/kg body weight, at
least about 0.25 mg/kg body weight, at least about 0.3 mg/kg body
weight, at least about 0.5 mg/kg body weight, at least about 0.75
mg/kg body weight, at least about 1 mg/kg body weight, at least
about 2 mg/kg body weight, at least about 3 mg/kg body weight, at
least about 4 mg/kg body weight, at least about 5 mg/kg body
weight, at least about 6 mg/kg body weight, at least about 7 mg/kg
body weight, at least about 8 mg/kg body weight, at least about 9
mg/kg body weight, at least about 10 mg/kg body weight, at least
about 15 mg/kg body weight, at least about 20 mg/kg body weight, at
least about 25 mg/kg body weight, at least about 30 mg/kg body
weight, at least about 40 mg/kg body weight, at least about 50
mg/kg body weight, at least about 75 mg/kg body weight, at least
about 100 mg/kg body weight, at least about 200 mg/kg body weight,
at least about 250 mg/kg body weight, at least about 300 mg/kg body
weight, at least about 350 mg/kg body weight, at least about 400
mg/kg body weight, at least about 450 mg/kg body weight, at least
about 500 mg/kg body weight, at least about 550 mg/kg body weight,
at least about 600 mg/kg body weight, at least about 650 mg/kg body
weight, at least about 700 mg/kg body weight, at least about 750
mg/kg body weight, at least about 800 mg/kg body weight, at least
about 900 mg/kg body weight, or at least about 1000 mg/kg body
weight. It will be recognized that any of the dosages listed herein
may constitute an upper or lower dosage range, and may be combined
with any other dosage to constitute a dosage range comprising an
upper and lower limit.
[0119] In some embodiments, the methods comprise a single dosage or
administration (e.g., as a single injection or deposition).
Alternatively, the methods comprise administration once daily,
twice daily, three times daily or four times daily to a subject in
need thereof for a period of from about 2 to about 28 days, or from
about 7 to about 10 days, or from about 7 to about 15 days, or
longer. In some embodiments, the methods comprise chronic
administration. In some embodiments, the methods comprise
administration over the course of several years or decades.
[0120] The dosage administered can vary depending upon known
factors such as the pharmacodynamic characteristics of the active
ingredient and its mode and route of administration; time of
administration of active ingredient; age, sex, health and weight of
the recipient; nature and extent of symptoms; kind of concurrent
treatment, frequency of treatment and the effect desired; and rate
of excretion. These are all readily determined and may be used by
the skilled artisan to adjust or titrate dosages and/or dosing
regimens.
[0121] The precise dose to be employed in the compositions will
also depend on the route of administration, and should be decided
according to the judgment of the practitioner and each patient's
circumstances. In some embodiments of the invention, suitable dose
ranges for oral administration of compound (I) are generally about
5 mg/day to about 1000 mg/day. In some embodiments, the oral dose
of compound (I) is about 5 mg/day to about 800 mg/day. In some
embodiments, the oral dose of compound (I) is about 5 mg/day to
about 500 mg/day. In some embodiments, the oral dose of compound
(I) is about 5 mg/day to about 250 mg/day. In some embodiments, the
oral dose of compound (I) is about 5 mg/day to about 100 mg/day. In
some embodiments, the oral dose of compound (I) is about 5 mg/day
to about 50 mg/day. In some embodiments, the oral dose of compound
(I) is about 5 mg/day. In some embodiments, the oral dose of
compound (I) is about 10 mg/day. In some embodiments, the oral dose
of compound (I) is about 20 mg/day. In some embodiments, the oral
dose of compound (I) is about 50 mg/day. In some embodiments, the
oral dose of compound (I) is about 100 mg/day. In some embodiments,
the oral dose of compound (I) is about 250 mg/day. In some
embodiments, the oral dose of compound (I) is about 500 mg/day. In
some embodiments, the oral dose of compound (I) is about 750
mg/day. In some embodiments, the oral dose of compound (I) is about
1000 mg/day.
[0122] In some embodiments of the invention, suitable dose ranges
for i.p. administration of compound (I) are generally about 5
mg/day to about 1000 mg/day. In some embodiments, the i.p. dose of
compound (I) is about 5 mg/day to about 800 mg/day. In some
embodiments, the i.p. dose of compound (I) is about 5 mg/day to
about 500 mg/day. In some embodiments, the i.p. dose of compound
(I) is about 5 mg/day to about 250 mg/day. In some embodiments, the
i.p. dose of compound (I) is about 5 mg/day to about 100 mg/day. In
some embodiments, the i.p. dose of compound (I) is about 5 mg/day
to about 50 mg/day. In some embodiments, the i.p. dose of compound
(I) is about 5 mg/day. In some embodiments, the i.p. dose of
compound (I) is about 10 mg/day. In some embodiments, the i.p. dose
of compound (I) is about 20 mg/day. In some embodiments, the i.p.
dose of compound (I) is about 50 mg/day. In some embodiments, the
i.p. dose of compound (I) is about 100 mg/day. In some embodiments,
the i.p. dose of compound (I) is about 250 mg/day. In some
embodiments, the i.p. dose of compound (I) is about 500 mg/day. In
some embodiments, the i.p. dose of compound (I) is about 750
mg/day. In some embodiments, the i.p. dose of compound (I) is about
1000 mg/day.
[0123] Any of the therapeutic applications described herein can be
applied to any subject in need of such therapy, including, for
example, a mammal such as a mouse, rat, dog, a cat, a cow, a horse,
a rabbit, a monkey, a pig, a sheep, a goat, or a human.
[0124] It will recognized that one or more features of any
embodiments disclosed herein may be combined and/or rearranged
within the scope of the invention to produce further embodiments
that are also within the scope of the invention.
[0125] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be within the scope of the
present invention.
[0126] The invention is further described by the following
non-limiting Examples.
EXAMPLES
[0127] Examples are provided below to facilitate a more complete
understanding of the invention. The following examples illustrate
the exemplary modes of making and practicing the invention.
However, the scope of the invention is not limited to specific
embodiments disclosed in these Examples, which are for purposes of
illustration only, since alternative methods can be utilized to
obtain similar results.
Example 1
Synthesis of Compound 1
##STR00004##
[0129] HAT Activator compound, compound 1 (I) was prepared
according to Scheme 1. A solution of commercially available ethyl
6-ethoxy-2-hydroxybenzoate (2.10 g, 10.0 mmol) in EtOH and NaOH 1N
(10 mL, 1:1) was heated to reflux for 2 h. The solution was
acidified by adding HCl 1N and the resulting precipitate was
diluted with CH.sub.2Cl.sub.2 (50 mL) and washed with HCl 1N
(3.times.50 mL). The organic layer was dried under
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure. A
white solid was obtained as the desired product 3(1.65 g, 91%).
[0130] EDC (2.19 mL, 12.35 mmol) was added dropwise to a solution
of 3 (1.5 g, 8.23 mmol) and 3-chloro-4-(trifluoromethyl)aniline
(1.61 g, 8.23 mmol) in CH.sub.2Cl.sub.2 (15 mL) at 0.degree. C. The
reaction was stirred at room temperature overnight. The solvent was
evaporated under reduced pressure and the final product 4 (2.42 g,
82%) was isolated by precipitation from MeOH as a white needle-like
solid.
[0131] To a solution of 4 (170 mg, 0.47 mmol),
2-(dimethylamino)ethanol (54 mg, 0.6 mmol), and PPh.sub.3 (157 mg,
0.6 mmol) in THF (5 mL) DIAD (121 mg, 0.6 mmol) was added dropwise.
The reaction was stirred for 24 h at room temperature. The solvent
was removed under reduced pressure, and the residue was dissolved
in AcOEt (30 mL). The organic layer was washed with water
(3.times.30 mL), dried under Na.sub.2SO.sub.4, filtered, and
concentrated to give a yellow oil, which was purified by flash
chromatography (AcOEt:MeOH 9:1) affording compound 1 (135 mg, 70%)
as a colorless oil. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 8.65
(s, 1H), 7.98 (d, 1H, J=7.8 Hz), 7.79 (d, 1H, J=1.5 Hz), 7.46 (d,
1H, J=8.7 Hz), 7.29 (t, 1H, J=8.4 Hz), 6.60 (dd, 2H, J.sub.1=1.8,
J.sub.2=8.4 Hz), 4.20 (t, 2H, J=5.1 Hz), 4.10 (q, 2H, J=6.9 Hz),
2.65 (t, 2H, J=5.4 Hz), 2.25 (s, 6H), 1.40 (t, 3H, J=7.2 Hz); Ms
ESI (m/z) 431 (M+1).sup.+. Compound 1 was treated with HCl 2M
solution in ethyl ether and the white salt of 1 was collected by
filtration.
Example 2
HAT Activator Compound Characteristics
[0132] The preparation of compound 1 was without a column and 2
phases were visible: clear and oily. Compound 1 (50 mg/kg, i.p.)
was subsequently administered to mice. The des-ethoxy analog of
compound 1, MOM, was also administered via cannula (100 .mu.g/.mu.L
per side). Two and four hrs after its administration, the mice were
sacrificed and hippocampi were extracted. Interestingly, while MOM
did not cross the BBB, YF2 (compound 1) was able to cross the BBB,
penetrate the cells and increase AcH3 (lane 1 vs. lanes 9, 10)
(FIG. 1). Given that the compound was not 100% clean and needed to
be further purified/verified, more compound 1 was synthesized and
purified. Purity was verified through Nuclear Magnetic Resonance
(NMR) spectroscopy. Mice were administered with compound 1 (i.p.
dissolved in saline) at 5, 10, 20 mg/Kg. Hippocampus extraction was
made at 3 different time points (0.5, 1 and 2 hrs post treatment).
A western blotting for AcH3 was then performed. Except for the 1
hr-10 mg/kg administration of YF2, YF2 dramatically increased AcH3
levels (FIG. 2), indicating that YF2 (compound 1) crosses the BBB
and the cell membrane.
Example 3
Compound 1 Increases Histone Acetylation by HAT Activation, Not
HDAC Inhibition
[0133] HDAC inhibition causes an increase in histone acetylation.
The inventors examined whether histone acetylation occurred via
HDAC inhibition. The summary of the results is depicted in Table 1.
The mean IC.sub.50 values of the compounds (compound 1 and SAHA)
are summarized in Table 1.
TABLE-US-00007 TABLE 1 Inhibitory Effects of the Compounds on HDAC
Activities IC.sub.50 (nM) or % Inhibition HDACs 1 SAHA HDAC1
>200 .mu.M 31 HDAC3/NCOR2 >200 .mu.M 38 HDAC5FL >200 .mu.M
>10 .mu.M HDAC6 >200 .mu.M 30 HADC7 >200 .mu.M >10
.mu.M HDAC8 >200 .mu.M 2,236 HDAC10 >200 .mu.M 65 HDAC11
>200 .mu.M >10 .mu.M Sirtuin 1 >200 .mu.M >10 .mu.M
Sirtuin 2 >200 .mu.M >10 .mu.M
[0134] The experiments were done blind, and the studies show that
compound 1 has no HDAC inhibition properties. Compound 1 does not
inhibit HDACs.
[0135] Materials and Methods
[0136] Materials:
[0137] HDAC Assay Buffer (BPS catalog number 50031)
[0138] HDAC Assay Developer (BPS catalog number 50030)
[0139] HDAC Substrate 1 (BPS number 50032)
[0140] HDAC Substrate 3 (BPS number 50037)
[0141] HDAC Class 2a Substrate 1 (BPS number 50040)
[0142] SAHA (Cayman Chemical, Ann Arbor, Mich., Catalog number
10009929)
TABLE-US-00008 TABLE 2 Compounds used in the studies Dis- Inter-
Compound Compound Stock solving Test Range mediate I.D. Supplied
Conc. Solvent (nM) Dilution 1* Solution 10 mM DMSO 3-200,000 10%
DMSO in HDAC Assay Buffer SAHA Powder 10 mM DMSO 0.3-10,000 10%
DMSO in HDAC Assay Buffer *Compound 1 is cloudy at 2 mM in 10% DMSO
(The highest test point). **SAHA, and HDACi, is a positive control
for HDACs.
[0143] Experimental Conditions
TABLE-US-00009 TABLE 3 Enzymes and Substrates Enzyme Used Assay
Catalog # (ng)/Reaction Substrate HDAC1 50051 1.5 10 .mu.M of 50037
HADC3/NCOR2 50003 1.33 10 .mu.M of 50037 HDAC5FL 50045 1.25 2 .mu.M
of 50040 HDAC6 50006 10 10 .mu.M of 50037 HDAC7 50007 0.3 2 .mu.M
of 50040 HDAC8 50008 20 2 .mu.M of 50040 HDAC10 50010 1,300 10
.mu.M of 50037 HADC11 50011 400 2 .mu.M of 50040 Sirtuin 1 50012
400 10 .mu.M of 50032 Sirtuin 2 50013 5,600 10 .mu.M of 50032
[0144] Assay Conditions. A series of dilution of the test compounds
were prepared with 10% DMSO in assay buffer and 5 .mu.l of the
dilution was added to a 50 .mu.l reaction so that the final
concentration of DMSO is 1% in all of reactions. All of the
enzymatic reactions were conducted in duplicate at 37.degree. C.
for 30 minutes except of HDAC11 at room temperature for 3 hours.
The 50 .mu.l reaction mixture contains HDAC assay buffer, 5 .mu.g
BSA, an HDAC substrate, an HDAC enzyme and a test compound. After
enzymatic reactions, 50 .mu.l of HDAC Developer was added to each
well and the plate was incubated at room temperature for an
additional 20 minutes. Fluorescence intensity was measured at an
excitation of 360 nm and an emission of 460 nm using a Tecan
Infinite M1000 microplate reader.
[0145] Data Analysis. HDAC activity assays were performed in
duplicates at each concentration. The fluorescent intensity data
were analyzed using the computer software, Graphpad Prism. In the
absence of the compound, the fluorescent intensity (F.sub.t) in
each data set was defined as 100% activity. In the absence of HDAC,
the fluorescent intensity (F.sub.b) in each data set was defined as
0% activity. Compound 1 has fluorescence at assay condition;
therefore the fluorescent intensity at different concentration of
compound 1 was defined as background (Fb). The percent activity in
the presence of each compound was calculated according to the
following equation: % activity=(F-F.sub.b)/(F.sub.t-F.sub.b), where
F=the fluorescent intensity in the presence of the compound.
[0146] The values of % activity versus a series of compound
concentrations were then plotted using non-linear regression
analysis of Sigmoidal dose-response curve generated with the
equation Y=B+(T-B)/1+10.sup.((Log EC50-X).times.Hill Slope), where
Y=percent activity, B=minimum percent activity, T=maximum percent
activity, X=logarithm of compound and Hill Slope=slope factor or
Hill coefficient. The IC.sub.50 value was determined by the
concentration causing a half-maximal percent activity.
[0147] Results of Effect of Compound 1 on HDAC Inhibition
TABLE-US-00010 TABLE 4 HDAC1 Assay - Data for the Effect of
Compound 1 on HDAC1 Activity HDAC Activity Background (Fluorescence
(Fluorescence Compound 1 count) count) % Activity (Log [nM])
Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD 17721 17257
796 803 101.39 98.61 0.5 17287 17200 796 798 98.80 98.28 1.0 17083
17178 788 786 97.64 98.21 1.5 16949 17020 830 784 96.72 97.14 2.0
16879 16826 796 779 96.42 96.10 2.5 16792 17072 827 775 95.81 97.49
3.0 16943 16784 829 802 96.63 95.68 3.5 16387 17135 866 827 93.12
97.60 4.0 16140 16336 920 868 91.35 92.53 4.5 16432 16128 1117 1035
92.01 90.19 5.3 24780 24451 14884 13403 63.73 61.76
[0148] FIG. 3 corresponds to the results shown in Table 4.
TABLE-US-00011 TABLE 5 HDAC3/NCOR2 Assay - Data for the Effect of
Compound 1 on HDAC3/NCOR2 Activity HDAC Activity Background
(Fluorescence (Fluorescence Compound 1 count) count) % Activity
(Log [nM]) Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD
10787 10452 805 828 101.71 98.29 0.5 10928 9694 813 976 102.35
89.76 1.0 10423 10379 812 818 98.01 97.56 1.5 10752 10231 813 803
101.44 96.12 2.0 10827 10078 809 798 102.25 94.61 2.5 10718 10173
818 803 101.07 95.51 3.0 10587 10073 831 811 99.62 94.38 3.5 10362
10080 854 824 97.14 94.27 4.0 11530 10216 927 898 108.31 94.90 4.5
9872 10001 1467 1091 87.66 88.97 5.3 20905 22163 13408 10875 89.40
102.23
[0149] FIG. 4 corresponds to the results shown in Table 5.
TABLE-US-00012 TABLE 6 HDAC5FL Assay - Data for the Effect of
Compound 1 on HDAC5FL Activity HDAC Activity Background
(Fluorescence (Fluorescence Compound 1 count) count) % Activity
(Log [nM]) Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD
4492 4892 345 348 95.40 104.60 0.5 4686 4386 355 343 99.80 92.90
1.0 4802 4581 341 347 102.59 97.50 1.5 4835 4874 359 342 103.20
104.10 2.0 5071 4991 344 356 108.64 106.80 2.5 5068 5006 344 354
108.60 107.17 3.0 4944 4685 342 354 105.76 99.80 3.5 4773 4686 353
389 101.30 99.30 4.0 4987 4983 449 407 104.91 104.82 4.5 4570 4514
451 398 95.40 94.11 5.3 9875 10983 7907 5878 68.63 94.13
[0150] FIG. 5 corresponds to the results shown in Table 6.
TABLE-US-00013 TABLE 7 HDAC7 Assay - Data for the Effect of
Compound 1 on HDAC7 Activity HDAC Activity Background (Fluorescence
(Fluorescence Compound 1 count) count) % Activity (Log [nM])
Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD 7528 7176
382 377 102.52 97.48 0.5 7578 7200 394 383 103.11 97.69 1.0 6756
6763 385 386 91.37 91.47 1.5 7471 7705 389 381 101.63 104.98 2.0
7679 7196 390 380 104.61 97.68 2.5 7071 7068 385 398 95.80 95.75
3.0 7083 7269 384 392 96.02 98.69 3.5 7453 6898 397 462 100.73
92.77 4.0 6801 7568 416 534 90.73 101.73 4.5 7238 7518 554 565
95.78 99.80 5.3 9692 9912 3002 2871 96.89 100.04
[0151] FIG. 6 corresponds to the results shown in Table 7.
TABLE-US-00014 TABLE 8 HDAC8 Assay - Data for the Effect of
Compound 1 on HDAC8 Activity HDAC Activity Background (Fluorescence
(Fluorescence Compound 1 count) count) % Activity (Log [nM])
Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD 3492 3483
346 346 100.14 99.86 0.5 3541 3581 339 342 101.88 103.15 1.0 3519
3391 349 342 101.02 96.94 1.5 3539 3456 336 331 102.04 99.40 2.0
3757 3425 338 340 108.80 98.23 2.5 3451 3428 335 341 99.09 98.36
3.0 3398 2995 337 347 97.28 84.45 3.5 3808 3407 346 366 109.88
97.12 4.0 3361 3365 433 374 94.14 94.27 4.5 3045 3090 375 364 85.17
86.60 5.3 6631 8117 4962 4635 58.33 105.63
[0152] FIG. 7 corresponds to the results shown in Table 8.
TABLE-US-00015 TABLE 9 HDAC10 Assay - Data for the Effect of
Compound 1 on HDAC10 Activity HDAC Activity Background
(Fluorescence (Fluorescence Compound 1 count) count) % Activity
(Log [nM]) Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD
11695 12141 497 507 98.05 101.95 0.5 10894 12032 492 501 91.08
101.05 1.0 12341 12402 497 492 103.77 104.31 1.5 12564 12368 525
500 105.57 103.85 2.0 12262 12573 500 497 103.04 105.77 2.5 12472
12556 500 493 104.90 105.64 3.0 11935 12471 530 521 99.94 104.64
3.5 11622 12684 501 607 96.95 106.25 4.0 11588 12318 597 547 96.50
102.89 4.5 11448 12305 623 495 95.38 102.89 5.3 25769 22285 12210
12714 116.56 86.05
[0153] FIG. 8 corresponds to the results shown in Table 9.
TABLE-US-00016 TABLE 10 HDAC11 Assay - Data for the Effect of
Compound 1 on HDAC11 Activity HDAC Activity Background
(Fluorescence (Fluorescence Compound 1 count) count) % Activity
(Log [nM]) Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD
2840 2860 426 406 99.59 100.41 0.5 2761 2530 411 423 96.30 86.81
1.0 2828 2898 425 415 98.93 101.81 1.5 2765 2851 411 406 96.82
100.35 2.0 2812 2864 408 409 98.75 100.88 2.5 2672 2655 412 408
92.93 92.24 3.0 2829 2806 417 424 98.95 98.01 3.5 2719 2712 427 463
93.43 93.14 4.0 2835 2860 467 524 96.12 97.14 4.5 3289 3064 699 617
108.09 98.85 5.3 6249 5842 2911 3158 132.07 115.35
[0154] FIG. 9 corresponds to the results shown in Table 10.
TABLE-US-00017 TABLE 11 Sirtuin 1 Assay - Data for the Effect of
Compound 1 on Sirtuin 1 Activity HDAC Activity Background
(Fluorescence (Fluorescence Compound 1 count) count) % Activity
(Log [nM]) Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD
5823 5974 412 410 97.91 100.64 0.5 5627 5940 414 420 94.26 99.92
1.0 5240 5913 422 413 87.25 99.42 1.5 5980 5273 418 457 100.27
87.48 2.0 5827 5527 411 411 97.98 92.56 2.5 6028 5987 413 416
101.56 100.81 3.0 6454 5681 422 452 108.86 94.87 3.5 5782 5964 422
426 96.93 100.23 4.0 5786 5408 442 441 96.69 89.85 4.5 5976 5697
502 524 98.83 93.79 5.3 7483 7591 2022 1997 99.02 100.98
[0155] FIG. 10 corresponds to the results shown in Table 11.
TABLE-US-00018 TABLE 12 Sirtuin 2 Assay - Data for the Effect of
Compound 1 on Sirtuin 2 Activity HDAC Activity Background
(Fluorescence (Fluorescence Compound 1 count) count) % Activity
(Log [nM]) Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD
3910 3919 413 419 99.87 100.13 0.5 3835 3981 420 413 97.71 101.89
1.0 3780 3821 406 422 96.21 97.38 1.5 3858 3954 408 410 98.59
101.33 2.0 3712 3912 420 413 94.20 99.91 2.5 3729 3788 409 420
94.74 96.43 3.0 3714 3861 405 409 94.53 98.73 3.5 3806 3856 422 417
96.80 98.23 4.0 3844 3883 425 426 97.71 98.83 4.5 3717 3811 485 480
92.45 95.14 5.3 5686 5717 2225 2245 98.64 99.53
[0156] FIG. 11 corresponds to the results shown in Table 12.
TABLE-US-00019 TABLE 13 HDAC6 Assay - Data for the Effect of
Compound 1 on HDAC6 Activity HDAC Activity Background (Fluorescence
(Fluorescence Compound 1 count) count) % Activity (Log [nM])
Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD 5844 5616
773 733 102.29 97.71 0.5 5998 6074 832 737 104.75 106.28 1.0 6006
5728 747 704 106.10 100.51 1.5 5541 6126 746 706 96.75 108.50 2.0
5733 5981 748 731 100.33 105.31 2.5 5678 5677 763 709 99.30 99.28
3.0 5717 5446 758 716 100.06 94.62 3.5 5575 5616 781 735 96.79
97.61 4.0 5516 5789 828 786 94.62 100.10 4.5 4994 5418 1081 1030
79.13 87.65 5.3 8327 9938 4925 4721 70.40 102.77
[0157] FIG. 12 corresponds to the results shown in Table 13.
[0158] Results of Effect of SAHA on HDAC Inhibition
[0159] SAHA is an HDAC inhibitor (HDACi). It serves as a positive
control for HDACs. FIGS. 13-15 show the inhibitory effect of SAHA
on the HDACs HDAC1, HDAC3/NCOR2, and HDAC6. SAHA also inhibited
HDAC5FL, HDAC7, HDAC8, HDAC10, Sirtuin 1, and Sirtuin 2 (see Table
1).
[0160] In In vitro assays, compound 1 has activity versus CBP,
PCAF, and GCN5. The EC.sub.50's of compound 1 for CBP, PCAF, and
GCN5 are 2.75 .mu.M, 29.04 .mu.M and 49.31 .mu.M, respectively.
Additionally, compound 1 did not interfere with p300 and HDAC
activity (HDAC 1, 3, 5, 6, 7, 8, 10, 11, and sirt1-2). Compound 1
also increases p300 activity as shown in FIG. 16.
Example 4
Pharmacokinetic Experiments with Compound 1
[0161] Compound 1 pharmacokinetic (PK) and blood-brain barrier
(BBB) penetration capability was assayed. After i.p. and i.v.
administration at 20 mg/kg to BALB/c mice, plasma and brain
concentrations were determined by LC-MS/MS. The data in Table 14
indicates that compound 1 is rapidly absorbed in the brain
(T.sub.max at 15min).
TABLE-US-00020 TABLE 14 Pharmacokinetic parameters of Compound 1.
IP Administration IV Administration Parameters Plasma Brain Ratio*
Plasma Brain Ratio* T.sub.max (h) 0.25 0.25 -- 0.125 0.125 --
C.sub.max (ng/mL or ng/g) 843 4878 5.8 2132 27892 13.1 AUC.sub.0-t
(ng h/mL or ng h/g) 806 6493 8.1 1967 22222 11.3 AUC.sub.0-.infin.
(ng h/mL or ng h/g) 813 6564 8.1 2020 22581 11.2 t1/2 (h) 0.60 0.63
-- 0.70 0.63 -- MRT (h) 0.85 1.03 -- 0.84 0.74 -- F (%) 41.0 29.2
-- -- -- -- *Ratio = brain/plasma YF2 EC.sub.50's for CBP, PCAF and
GCN5 are: 2.75 .mu.M, 29.04 .mu.M and 49.31 .mu.M, respectively
[0162] The amount of compound 1 in the brain was higher than that
in the plasma with an AUC.sub.0-t ratio of 8.2 and 10.8 for i.p.
and i.v. administration, respectively. The elimination half-lives
of compound 1 in the brain and plasma were .about.40 min. The
T.sub.max values in the brain and plasma were similar, indicating
that the distribution of compound 1 to the brain is also fast.
Additionally, in acute toxicity experiments compound 1 did not
induce any adverse effects up to 300 mg/kg (i.p.).
[0163] Pharmacokinetic properties of compound 1 dosed orally are
shown in FIG. 17 and Table 15, and indicate that the amount of
compound 1 in the brain is higher than that in the plasma.
TABLE-US-00021 TABLE 15 Oral pharmacokinetic parameters of Compound
1. Oral Administration Parameters Plasma Brain Ratio* T.sub.max (h)
0.5 1.0 -- C.sub.max (ng/mL or ng/g) 177 1008 5.7 AUC.sub.0-t (ng
h/mL or ng h/g) 328 2149 6.6 AUC.sub.0-.infin. (ng h/mL or ng h/g)
330 2159 6.5 t1/2 (h) 1.06 0.99 -- MRT (h) 1.50 1.68 --
Example 5
Contextual Fear Conditioning Experiments
[0164] Contextual fear conditioning was performed to assess whether
compound 1 is capable of enhancing memory. This type of cognitive
test is much faster than other behavioral tasks that require
multiple days of training and testing (J Clin Invest, 2004.
114(11): 1624-34; herein incorporated by reference in its
entirety). The conditioning chamber was in a sound-attenuating box.
A clear Plexiglas window allowed the experimenter to film the mouse
performance with a camera placed on a tripod and connected to the
Freezeframe software (MED Ass. Inc.). To provide background white
noise (72 dB), a single computer fan was installed in one of the
sides of the sound-attenuating chamber. The conditioning chamber
had a 36-bar insulated shock grid floor. The floor was removable,
and after each experimental subject, it was cleaned with 75%
ethanol and then with water. Only one animal at a time was present
in the experimentation room.
[0165] Training consisted of a 2.5 min period of acclimatizing to
the context, followed by pairing of a tone (2800 Hz, 85 dB, 30 s)
with a coterminating foot shock (0.4 mA, 1 s) for the weak training
protocol, or pairing of a tone (2800 Hz, 85 dB, 30 s) with a
coterminating foot shock (0.8 mA, 2 s) for the strong training
protocol. The mice remained in the chamber for an additional 30 sec
after the end of the last pairing, after which they were returned
to their home cages. Contextual fear conditioning was assayed 24 hr
after training by replacing the animals in the conditioning context
for a 5 min period, during which the incidence of freezing
(immobile except for respiration) was recorded.
[0166] The stronger training protocol leads to learning saturation,
whereby freezing/memory reaches it max (.about.25-30% in WT
animals) even if the foot shock is increased. On the other hand,
the weaker training protocol leads to much less freezing
(.about.15%), which allows the more freezing in case there is an
increase in memory. When the weaker protocol was used, compound 1
worked as a memory enhancer.
[0167] Freezing behavior, defined as the absence of all movement
except for that necessitated by breathing, was scored using the
Freezeview software.
[0168] To evaluate contextual fear learning, freezing was measured
for 5 min (consecutively) in the chamber in which the mice was
trained 24 hr after training. To evaluate cued fear learning,
following contextual testing, the mice were placed in a novel
context (triangular cage with smooth flat floor) for 2 min (pre-CS
test), after which they were exposed to the CS for 3 min (CS test),
and freezing was measured. Sensory perception of the shock was
determined through threshold assessment. A sequence of single foot
shocks was delivered to animals placed on the same electrified grid
used for fear conditioning. Initially, a 0.1 mV shock was delivered
for 1 sec, and the animal behavior was evaluated for flinching,
jumping, and vocalization. At 30 sec intervals the shock intensity
was increased by 0.1 mV to 0.7 mV and then returned to 0 mV in 0.1
mV increments at 30 sec intervals. Threshold to vocalization,
flinching, and then jumping was quantified for each animal by
averaging the shock intensity at which each animal manifests a
behavioral response to the foot shock.
[0169] Vehicle WT and compound 1 treated mice showed similar
freezing responses before the delivery of the foot shock (baseline)
(FIG. 18). However, 5 and 20 mg/kg compound 1 treated mice froze
nearly twice as often as did WT vehicle mice 24 h after training
protocol (a single pairing of a tone with a 0.35 mA foot shock).
Finally, no difference was observed among different groups of mice
in different sets of experiments in which we assessed sensory
threshold in the presence of vehicle or compound 1 (YF2) alone
(FIG. 19).
[0170] Based on the results obtained during the fear conditioning
tests, it was decided to determine the kinetics of compound 1 in
blood to verify the best time point for treatment. To this purpose,
compound 1 (20 mg/kg. i.p.) was administered and then sampled blood
from tails at different time points. The kinetics of compound 1
shows a peak around 30 minutes post-injection (FIG. 20).
[0171] Compound 1, a Histone Acetyltransferase (HAT) Activator of
the invention, is a good drug candidate to enhance memory and
cognition in subjects without neurodegenerative diseases. When
compound 1 (YF2)was administered to mice (i.p.), the western blot
showed that it not only crosses the BBB, but also increases histone
3 acetylation levels of the hippocampus (FIG. 21).
[0172] Compound 1 was then tested to ascertain increases in histone
acetylation in mouse hippocampus. The compound was i.p.
administered at 20 mg/Kg, mice were sacrificed 30 min later, and
hippocampi were removed and quickly frozen for WB analysis. As
shown in FIG. 22, compound 1 (YF2) increased acetylation of histone
lysines that were shown to be involved in memory formation (H3K4,
H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, and H2B) (Neuron, 2004,
42(6): 947-59; Science 328(5979): 753-6; each herein incorporated
by reference in its entirety).
Example 6
Compound 1 Rescues AO-Induced Reduction in BDNF Levels
[0173] Compound 1 increases levels of BDNF, a key protein necessary
for activity-dependent plasticity and memory. CBP was shown to
facilitate the transcription of key proteins necessary for
activity-dependent plasticity and memory (Korzus, E., M. G.
Rosenfeld, and M. Mayford, CBP histone acetyltransferase activity
is a critical component of memory consolidation. Neuron, 2004.
42(6): p. 961-72; herein incorporated by reference in its
entirety), such as brain-derived neurotrophic factor (BDNF), which
is known to facilitate synaptic plasticity and memory formation
(Cowansage, K. K., J. E. LeDoux, and M. H. Monfils, Brain-derived
neurotrophic factor: a dynamic gatekeeper of neural plasticity.
Current molecular pharmacology, 2010. 3(1): p. 12-29; Caccamo, A.,
et al., CBP gene transfer increases BDNF levels and ameliorates
learning and memory deficits in a mouse model of Alzheimer's
disease. Proc Natl Acad Sci USA, 2010. 107(52): p. 22687-92; each
herein incorporated by reference in its entirety). Interestingly,
BDNF was proposed to play a role in AD pathogenesis, with reduced
BDNF levels detected in brains of AD patients and AD animal models
(Hock, C., et al., Region-specific neurotrophin imbalances in
Alzheimer disease: decreased levels of brain-derived neurotrophic
factor and increased levels of nerve growth factor in hippocampus
and cortical areas. Archives of neurology, 2000. 57(6): p. 846-51;
Connor, B., et al., Brain-derived neurotrophic factor is reduced in
Alzheimer's disease. Brain research. Molecular brain research,
1997. 49(1-2): p. 71-81; Garzon, D. J. and M. Fahnestock,
Oligomeric amyloid decreases basal levels of brain-derived
neurotrophic factor (BDNF) mRNA via specific downregulation of BDNF
transcripts IV and V in differentiated human neuroblastoma cells.
The Journal of Neuroscience: The Official Journal of the Society
for Neuroscience, 2007. 27(10): p. 2628-35; each herein
incorporated by reference in its entirety). Thus, preliminary
studies on Compound 1 efficacy were extended to to BDNF. BDNF
levels in the hippocampi of A.beta.-infused mice were measured
compared to vehicle infused animals. Consistent with the decrease
in BDNF levels in brains of AD patients and animal models of AD, a
reduction of BDNF levels following A.beta. infusion was found (FIG.
23). This effect was rescued by Compound 1 (20 mg/kg, i.p., 90 min
before harvesting the hippocampi, FIG. 23). Interestingly, Compound
1 increased BDNF levels in vehicle-infused mice (FIG. 23),
consistent with the observation that basal levels of BDNF are
increased following stimulation of the gene transcription machinery
relevant to memory formation (Arancio, O. and M. V. Chao,
Neurotrophins, synaptic plasticity and dementia. Current Opinion in
Neurobiology, 2007. 17(3): p. 325-30; herein incorporated by
reference in its entirety).
[0174] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific substances and procedures described
herein. Such equivalents are considered to be within the scope of
this invention, and are covered by the following claims.
[0175] Although the invention has been described and illustrated in
the foregoing illustrative embodiments, it is understood that the
present disclosure has been made only by way of example, and that
numerous changes in the details of implementation of the invention
can be made without departing from the spirit and scope of the
invention, which is limited only by the claims that follow.
Features of the disclosed embodiments can be combined and
rearranged in various ways to obtain additional embodiments within
the scope and spirit of the invention.
Sequence CWU 1
1
61419PRTHomo sapiens 1Met Ala Gly Phe Gly Ala Met Glu Lys Phe Leu
Val Glu Tyr Lys Ser 1 5 10 15 Ala Val Glu Lys Lys Leu Ala Glu Tyr
Lys Cys Asn Thr Asn Thr Ala 20 25 30 Ile Glu Leu Lys Leu Val Arg
Phe Pro Glu Asp Leu Glu Asn Asp Ile 35 40 45 Arg Thr Phe Phe Pro
Glu Tyr Thr His Gln Leu Phe Gly Asp Asp Glu 50 55 60 Thr Ala Phe
Gly Tyr Lys Gly Leu Lys Ile Leu Leu Tyr Tyr Ile Ala 65 70 75 80 Gly
Ser Leu Ser Thr Met Phe Arg Val Glu Tyr Ala Ser Lys Val Asp 85 90
95 Glu Asn Phe Asp Cys Val Glu Ala Asp Asp Val Glu Gly Lys Ile Arg
100 105 110 Gln Ile Ile Pro Pro Gly Phe Cys Thr Asn Thr Asn Asp Phe
Leu Ser 115 120 125 Leu Leu Glu Lys Glu Val Asp Phe Lys Pro Phe Gly
Thr Leu Leu His 130 135 140 Thr Tyr Ser Val Leu Ser Pro Thr Gly Gly
Glu Asn Phe Thr Phe Gln 145 150 155 160 Ile Tyr Lys Ala Asp Met Thr
Cys Arg Gly Phe Arg Glu Tyr His Glu 165 170 175 Arg Leu Gln Thr Phe
Leu Met Trp Phe Ile Glu Thr Ala Ser Phe Ile 180 185 190 Asp Val Asp
Asp Glu Arg Trp His Tyr Phe Leu Val Phe Glu Lys Tyr 195 200 205 Asn
Lys Asp Gly Ala Thr Leu Phe Ala Thr Val Gly Tyr Met Thr Val 210 215
220 Tyr Asn Tyr Tyr Val Tyr Pro Asp Lys Thr Arg Pro Arg Val Ser Gln
225 230 235 240 Met Leu Ile Leu Thr Pro Phe Gln Gly Gln Gly His Gly
Ala Gln Leu 245 250 255 Leu Glu Thr Val His Arg Tyr Tyr Thr Glu Phe
Pro Thr Val Leu Asp 260 265 270 Ile Thr Ala Glu Asp Pro Ser Lys Ser
Tyr Val Lys Leu Arg Asp Phe 275 280 285 Val Leu Val Lys Leu Cys Gln
Asp Leu Pro Cys Phe Ser Arg Glu Lys 290 295 300 Leu Met Gln Gly Phe
Asn Glu Asp Met Ala Ile Glu Ala Gln Gln Lys 305 310 315 320 Phe Lys
Ile Asn Lys Gln His Ala Arg Arg Val Tyr Glu Ile Leu Arg 325 330 335
Leu Leu Val Thr Asp Met Ser Asp Ala Glu Gln Tyr Arg Ser Tyr Arg 340
345 350 Leu Asp Ile Lys Arg Arg Leu Ile Ser Pro Tyr Lys Lys Lys Gln
Arg 355 360 365 Asp Leu Ala Lys Met Arg Lys Cys Leu Arg Pro Glu Glu
Leu Thr Asn 370 375 380 Gln Met Asn Gln Ile Glu Ile Ser Met Gln His
Glu Gln Leu Glu Glu 385 390 395 400 Ser Phe Gln Glu Leu Val Glu Asp
Tyr Arg Arg Val Ile Glu Arg Leu 405 410 415 Ala Gln Glu
21682DNAHomo sapiens 2ctgtgcggtc acttccggcc cgggagcgcg cgggttgatt
cgtccttcct cagccgcggg 60tgatcgtagc tcggaaatgg cgggatttgg tgctatggag
aaatttttgg tagaatataa 120gagtgcagtg gagaagaaac tggcagagta
caaatgtaac accaacacag caattgaact 180aaaattagtt cgttttcctg
aagatcttga aaatgacatt agaactttct ttcctgagta 240tacccatcaa
ctctttgggg atgatgaaac tgcttttggt tacaagggtc taaagatcct
300gttatactat attgctggta gcctgtcaac aatgttccgt gttgaatatg
catctaaagt 360tgatgagaac tttgactgtg tagaggcaga tgatgttgag
ggcaaaatta gacaaatcat 420tccacctgga ttttgcacaa acacgaatga
tttcctttct ttactggaaa aggaagttga 480tttcaagcca ttcggaacct
tacttcatac ctactcagtt ctcagtccaa caggaggaga 540aaactttacc
tttcagatat ataaggctga catgacatgt agaggctttc gagaatatca
600tgaaaggctt cagacctttt tgatgtggtt tattgaaact gctagcttta
ttgacgtgga 660tgatgaaaga tggcactact ttctagtatt tgagaagtat
aataaggatg gagctacgct 720ctttgcgacc gtaggctaca tgacagtcta
taattactat gtgtacccag acaaaacccg 780gccacgtgta agtcagatgc
tgattttgac tccatttcaa ggtcaaggcc atggtgctca 840acttcttgaa
acagttcata gatactacac tgaatttcct acagttcttg atattacagc
900ggaagatcca tccaaaagct atgtgaaatt acgagacttt gtgcttgtga
agctttgtca 960agatttgccc tgtttttccc gggaaaaatt aatgcaagga
ttcaatgaag atatggcgat 1020agaggcacaa cagaagttca aaataaataa
gcaacacgct agaagggttt atgaaattct 1080tcgactactg gtaactgaca
tgagtgatgc cgaacaatac agaagctaca gactggatat 1140taaaagaaga
ctaattagcc catataagaa aaagcagaga gatcttgcta agatgagaaa
1200atgtctcaga ccagaagaac tgacaaacca gatgaaccaa atagaaataa
gcatgcaaca 1260tgaacagctg gaagagagtt ttcaggaact agtggaagat
taccggcgtg ttattgaacg 1320acttgctcaa gagtaaagat tatactgctc
tgtacaggaa gcttgcaaat tttctgtaca 1380atgtgctgtg aaaaatctga
tgactttaat tttaaaatct tgtgacattt tgcttatact 1440aaaagttatc
tatctttagt tgaatatttt cttttggaga gattgtatat tttaaaatac
1500tgtttagagt ttatgagcat atattgcatt taaagaaaga taaagcttct
gaaatactac 1560tgcaattgct tcccttctta aacagtataa taaatgctta
gttgtgatat gttaatgtgt 1620gatgatatga ttcttaaata cttacaataa
acctcattct taaatactta aaaaaaaaaa 1680aa 16823832PRTHomo sapiens
3Met Ser Glu Ala Gly Gly Ala Gly Pro Gly Gly Cys Gly Ala Gly Ala 1
5 10 15 Gly Ala Gly Ala Gly Pro Gly Ala Leu Pro Pro Gln Pro Ala Ala
Leu 20 25 30 Pro Pro Ala Pro Pro Gln Gly Ser Pro Cys Ala Ala Ala
Ala Gly Gly 35 40 45 Ser Gly Ala Cys Gly Pro Ala Thr Ala Val Ala
Ala Ala Gly Thr Ala 50 55 60 Glu Gly Pro Gly Gly Gly Gly Ser Ala
Arg Ile Ala Val Lys Lys Ala 65 70 75 80 Gln Leu Arg Ser Ala Pro Arg
Ala Lys Lys Leu Glu Lys Leu Gly Val 85 90 95 Tyr Ser Ala Cys Lys
Ala Glu Glu Ser Cys Lys Cys Asn Gly Trp Lys 100 105 110 Asn Pro Asn
Pro Ser Pro Thr Pro Pro Arg Ala Asp Leu Gln Gln Ile 115 120 125 Ile
Val Ser Leu Thr Glu Ser Cys Arg Ser Cys Ser His Ala Leu Ala 130 135
140 Ala His Val Ser His Leu Glu Asn Val Ser Glu Glu Glu Met Asn Arg
145 150 155 160 Leu Leu Gly Ile Val Leu Asp Val Glu Tyr Leu Phe Thr
Cys Val His 165 170 175 Lys Glu Glu Asp Ala Asp Thr Lys Gln Val Tyr
Phe Tyr Leu Phe Lys 180 185 190 Leu Leu Arg Lys Ser Ile Leu Gln Arg
Gly Lys Pro Val Val Glu Gly 195 200 205 Ser Leu Glu Lys Lys Pro Pro
Phe Glu Lys Pro Ser Ile Glu Gln Gly 210 215 220 Val Asn Asn Phe Val
Gln Tyr Lys Phe Ser His Leu Pro Ala Lys Glu 225 230 235 240 Arg Gln
Thr Ile Val Glu Leu Ala Lys Met Phe Leu Asn Arg Ile Asn 245 250 255
Tyr Trp His Leu Glu Ala Pro Ser Gln Arg Arg Leu Arg Ser Pro Asn 260
265 270 Asp Asp Ile Ser Gly Tyr Lys Glu Asn Tyr Thr Arg Trp Leu Cys
Tyr 275 280 285 Cys Asn Val Pro Gln Phe Cys Asp Ser Leu Pro Arg Tyr
Glu Thr Thr 290 295 300 Gln Val Phe Gly Arg Thr Leu Leu Arg Ser Val
Phe Thr Val Met Arg 305 310 315 320 Arg Gln Leu Leu Glu Gln Ala Arg
Gln Glu Lys Asp Lys Leu Pro Leu 325 330 335 Glu Lys Arg Thr Leu Ile
Leu Thr His Phe Pro Lys Phe Leu Ser Met 340 345 350 Leu Glu Glu Glu
Val Tyr Ser Gln Asn Ser Pro Ile Trp Asp Gln Asp 355 360 365 Phe Leu
Ser Ala Ser Ser Arg Thr Ser Gln Leu Gly Ile Gln Thr Val 370 375 380
Ile Asn Pro Pro Pro Val Ala Gly Thr Ile Ser Tyr Asn Ser Thr Ser 385
390 395 400 Ser Ser Leu Glu Gln Pro Asn Ala Gly Ser Ser Ser Pro Ala
Cys Lys 405 410 415 Ala Ser Ser Gly Leu Glu Ala Asn Pro Gly Glu Lys
Arg Lys Met Thr 420 425 430 Asp Ser His Val Leu Glu Glu Ala Lys Lys
Pro Arg Val Met Gly Asp 435 440 445 Ile Pro Met Glu Leu Ile Asn Glu
Val Met Ser Thr Ile Thr Asp Pro 450 455 460 Ala Ala Met Leu Gly Pro
Glu Thr Asn Phe Leu Ser Ala His Ser Ala 465 470 475 480 Arg Asp Glu
Ala Ala Arg Leu Glu Glu Arg Arg Gly Val Ile Glu Phe 485 490 495 His
Val Val Gly Asn Ser Leu Asn Gln Lys Pro Asn Lys Lys Ile Leu 500 505
510 Met Trp Leu Val Gly Leu Gln Asn Val Phe Ser His Gln Leu Pro Arg
515 520 525 Met Pro Lys Glu Tyr Ile Thr Arg Leu Val Phe Asp Pro Lys
His Lys 530 535 540 Thr Leu Ala Leu Ile Lys Asp Gly Arg Val Ile Gly
Gly Ile Cys Phe 545 550 555 560 Arg Met Phe Pro Ser Gln Gly Phe Thr
Glu Ile Val Phe Cys Ala Val 565 570 575 Thr Ser Asn Glu Gln Val Lys
Gly Tyr Gly Thr His Leu Met Asn His 580 585 590 Leu Lys Glu Tyr His
Ile Lys His Asp Ile Leu Asn Phe Leu Thr Tyr 595 600 605 Ala Asp Glu
Tyr Ala Ile Gly Tyr Phe Lys Lys Gln Gly Phe Ser Lys 610 615 620 Glu
Ile Lys Ile Pro Lys Thr Lys Tyr Val Gly Tyr Ile Lys Asp Tyr 625 630
635 640 Glu Gly Ala Thr Leu Met Gly Cys Glu Leu Asn Pro Arg Ile Pro
Tyr 645 650 655 Thr Glu Phe Ser Val Ile Ile Lys Lys Gln Lys Glu Ile
Ile Lys Lys 660 665 670 Leu Ile Glu Arg Lys Gln Ala Gln Ile Arg Lys
Val Tyr Pro Gly Leu 675 680 685 Ser Cys Phe Lys Asp Gly Val Arg Gln
Ile Pro Ile Glu Ser Ile Pro 690 695 700 Gly Ile Arg Glu Thr Gly Trp
Lys Pro Ser Gly Lys Glu Lys Ser Lys 705 710 715 720 Glu Pro Arg Asp
Pro Asp Gln Leu Tyr Ser Thr Leu Lys Ser Ile Leu 725 730 735 Gln Gln
Val Lys Ser His Gln Ser Ala Trp Pro Phe Met Glu Pro Val 740 745 750
Lys Arg Thr Glu Ala Pro Gly Tyr Tyr Glu Val Ile Arg Phe Pro Met 755
760 765 Asp Leu Lys Thr Met Ser Glu Arg Leu Lys Asn Arg Tyr Tyr Val
Ser 770 775 780 Lys Lys Leu Phe Met Ala Asp Leu Gln Arg Val Phe Thr
Asn Cys Lys 785 790 795 800 Glu Tyr Asn Pro Pro Glu Ser Glu Tyr Tyr
Lys Cys Ala Asn Ile Leu 805 810 815 Glu Lys Phe Phe Phe Ser Lys Ile
Lys Glu Ala Gly Leu Ile Asp Lys 820 825 830 44824DNAHomo sapiens
4gcggaaaaga ggccgtgggg ggcctcccag cgctggcaga caccgtgagg ctggcagccg
60ccggcacgca cacctagtcc gcagtcccga ggaacatgtc cgcagccagg gcgcggagca
120gagtcccggg caggagaacc aagggagggc gtgtgctgtg gcggcggcgg
cagcggcagc 180ggagccgcta gtcccctccc tcctggggga gcagctgccg
ccgctgccgc cgccgccacc 240accatcagcg cgcggggccc ggccagagcg
agccgggcga gcggcgcgct agggggaggg 300cgggggcggg gaggggggtg
ggcgaagggg gcgggagggc gtggggggag ggtctcgctc 360tcccgactac
cagagcccga gagggagacc ctggcggcgg cggcggcgcc tgacactcgg
420cgcctcctgc cgtgctccgg ggcggcatgt ccgaggctgg cggggccggg
ccgggcggct 480gcggggcagg agccggggca ggggccgggc ccggggcgct
gcccccgcag cctgcggcgc 540ttccgcccgc gcccccgcag ggctccccct
gcgccgctgc cgccgggggc tcgggcgcct 600gcggtccggc gacggcagtg
gctgcagcgg gcacggccga aggaccggga ggcggtggct 660cggcccgaat
cgccgtgaag aaagcgcaac tacgctccgc tccgcgggcc aagaaactgg
720agaaactcgg agtgtactcc gcctgcaagg ccgaggagtc ttgtaaatgt
aatggctgga 780aaaaccctaa cccctcaccc actcccccca gagccgacct
gcagcaaata attgtcagtc 840taacagaatc ctgtcggagt tgtagccatg
ccctagctgc tcatgtttcc cacctggaga 900atgtgtcaga ggaagaaatg
aacagactcc tgggaatagt attggatgtg gaatatctct 960ttacctgtgt
ccacaaggaa gaagatgcag ataccaaaca agtttatttc tatctattta
1020agctcttgag aaagtctatt ttacaaagag gaaaacctgt ggttgaaggc
tctttggaaa 1080agaaaccccc atttgaaaaa cctagcattg aacagggtgt
gaataacttt gtgcagtaca 1140aatttagtca cctgccagca aaagaaaggc
aaacaatagt tgagttggca aaaatgttcc 1200taaaccgcat caactattgg
catctggagg caccatctca acgaagactg cgatctccca 1260atgatgatat
ttctggatac aaagagaact acacaaggtg gctgtgttac tgcaacgtgc
1320cacagttctg cgacagtcta cctcggtacg aaaccacaca ggtgtttggg
agaacattgc 1380ttcgctcggt cttcactgtt atgaggcgac aactcctgga
acaagcaaga caggaaaaag 1440ataaactgcc tcttgaaaaa cgaactctaa
tcctcactca tttcccaaaa tttctgtcca 1500tgctagaaga agaagtatat
agtcaaaact ctcccatctg ggatcaggat tttctctcag 1560cctcttccag
aaccagccag ctaggcatcc aaacagttat caatccacct cctgtggctg
1620ggacaatttc atacaattca acctcatctt cccttgagca gccaaacgca
gggagcagca 1680gtcctgcctg caaagcctct tctggacttg aggcaaaccc
aggagaaaag aggaaaatga 1740ctgattctca tgttctggag gaggccaaga
aaccccgagt tatgggggat attccgatgg 1800aattaatcaa cgaggttatg
tctaccatca cggaccctgc agcaatgctt ggaccagaga 1860ccaattttct
gtcagcacac tcggccaggg atgaggcggc aaggttggaa gagcgcaggg
1920gtgtaattga atttcacgtg gttggcaatt ccctcaacca gaaaccaaac
aagaagatcc 1980tgatgtggct ggttggccta cagaacgttt tctcccacca
gctgccccga atgccaaaag 2040aatacatcac acggctcgtc tttgacccga
aacacaaaac ccttgcttta attaaagatg 2100gccgtgttat tggtggtatc
tgtttccgta tgttcccatc tcaaggattc acagagattg 2160tcttctgtgc
tgtaacctca aatgagcaag tcaagggcta tggaacacac ctgatgaatc
2220atttgaaaga atatcacata aagcatgaca tcctgaactt cctcacatat
gcagatgaat 2280atgcaattgg atactttaag aaacagggtt tctccaaaga
aattaaaata cctaaaacca 2340aatatgttgg ctatatcaag gattatgaag
gagccacttt aatgggatgt gagctaaatc 2400cacggatccc gtacacagaa
ttttctgtca tcattaaaaa gcagaaggag ataattaaaa 2460aactgattga
aagaaaacag gcacaaattc gaaaagttta ccctggactt tcatgtttta
2520aagatggagt tcgacagatt cctatagaaa gcattcctgg aattagagag
acaggctgga 2580aaccgagtgg aaaagagaaa agtaaagagc ccagagaccc
tgaccagctt tacagcacgc 2640tcaagagcat cctccagcag gtgaagagcc
atcaaagcgc ttggcccttc atggaacctg 2700tgaagagaac agaagctcca
ggatattatg aagttataag gttccccatg gatctgaaaa 2760ccatgagtga
acgcctcaag aataggtact acgtgtctaa gaaattattc atggcagact
2820tacagcgagt ctttaccaat tgcaaagagt acaacccccc tgagagtgaa
tactacaaat 2880gtgccaatat cctggagaaa ttcttcttca gtaaaattaa
ggaagctgga ttaattgaca 2940agtgattttt tttcccctct gcttcttaga
aactcaccaa gcagtgtgcc taaagcaagg 3000tggtttagtt ttttacaaag
aattggacat gatgtattga agagacttgt aaatgtaata 3060attagcactt
ttgaaaaaac aaaaaacctc cttttagctt ttcagatatg tatttaaatt
3120gaagtcatag gacattttta ttttatggaa tagattttaa tctatttact
actattaagg 3180taaattttct atggcatgtc cattagctat ttcatgatag
atgattaggg gtttcctcaa 3240aacctgtgtg tgaggaaatt gcacacagta
gcaaaatttg gggaaatcca taacattttc 3300agaccatgaa tgaatgtttc
catttttttc taatggaatg tgagagttta cttttatttt 3360attctgaagg
actttaagga agggatacat gattttaaaa aagcctgtaa gaggtgaaat
3420atgtgatgtt tgaagtctct ttatagactt tttatatata ttttttaaaa
cactcatcta 3480gatgaggtgc tttgagcagt tctgaaaaat gcagttccag
gaaagcaact gctttggttc 3540ctaaggaaga aattctaaat aatgcaaact
tttaaaataa gcatctaggt ttttgataat 3600tctgtctact tacaacaaac
ttgttagtac ataaccacta ttttaataat tattttctct 3660acacaaatgt
gtaatatcat atttgacttt gcttatgcag gccataagtt ccaaaagata
3720atttccctgc ccacaaaggc ataaacttga aaacacatga gattgaatca
acatgcttta 3780ataggaaaag atgtatggtc tatatatgta tcaatctggt
gaatcctcgt tctaataaag 3840gttctttttc ttttctatga tacacacagc
cacgctgata atatgcaaat gaacattttc 3900ctttatgtct ctccagataa
tgtttattgt ctgaggtaaa ttaaattccc accagggttt 3960gctgtcagta
ttttaacacc cacattagta tatgcgtcca gggtcataac cccctaaaat
4020ccatcatgca accttattaa tctgtcttgg gattccagtt tagtgcttgg
atttatttcc 4080tgattacact acatagaaaa gtgagacatc tgccattccc
aactctggga aaaccaacta 4140atatacaacc atataaatga aggccatctt
gatggtctca acactaattt ttatgatgca 4200aatttataca ctgatttttg
taaaggacaa agttttaaaa gcgtatttaa cttgatgttt 4260tctatcagca
taaataaaat ggtcatgaat agtcattaaa aacagttgcc agtgataatc
4320tgcatgaagg aaaaagaacc ctgcaaatgg ctattgagtt ggaagtattg
tttttgatat 4380gtaagagata ttcagaatgc tcacactgaa aatgcctcaa
ctttttaaag tgtaagaaac 4440caccatgagt ggtgtctaga tttctaatga
agaatcatga tacagtttgg attaagtatc 4500ttggactggt tttaaacagt
gctttgtacc ggatctgctg aagcatctgt ccagctggta 4560tcctgtgaaa
gtttgttatt ttctgagtag acattcttat agagtattgt ctttaaaatc
4620agattgtctc ttctatattg aaagcatttt tatgttttct aatttaaaaa
ttaatatttt 4680cttatagata ttgtgcaata aagctgaagt agaatgtgtg
gtttttgcaa atgctttaac 4740agctgataaa aattttacat ttgtaaaatt
aatatattgt actggtacaa aatagtttta 4800aattatattt taaaaagctt ccaa
48245837PRTHomo sapiens 5Met Ala Glu Pro Ser Gln Ala Pro Thr Pro
Ala Pro Ala Ala Gln Pro 1 5 10 15 Arg Pro Leu Gln Ser Pro Ala Pro
Ala Pro Thr Pro Thr Pro Ala Pro 20 25
30 Ser Pro Ala Ser Ala Pro Ile Pro Thr Pro Thr Pro Ala Pro Ala Pro
35 40 45 Ala Pro Ala Ala Ala Pro Ala Gly Ser Thr Gly Thr Gly Gly
Pro Gly 50 55 60 Val Gly Ser Gly Gly Ala Gly Ser Gly Gly Asp Pro
Ala Arg Pro Gly 65 70 75 80 Leu Ser Gln Gln Gln Arg Ala Ser Gln Arg
Lys Ala Gln Val Arg Gly 85 90 95 Leu Pro Arg Ala Lys Lys Leu Glu
Lys Leu Gly Val Phe Ser Ala Cys 100 105 110 Lys Ala Asn Glu Thr Cys
Lys Cys Asn Gly Trp Lys Asn Pro Lys Pro 115 120 125 Pro Thr Ala Pro
Arg Met Asp Leu Gln Gln Pro Ala Ala Asn Leu Ser 130 135 140 Glu Leu
Cys Arg Ser Cys Glu His Pro Leu Ala Asp His Val Ser His 145 150 155
160 Leu Glu Asn Val Ser Glu Asp Glu Ile Asn Arg Leu Leu Gly Met Val
165 170 175 Val Asp Val Glu Asn Leu Phe Met Ser Val His Lys Glu Glu
Asp Thr 180 185 190 Asp Thr Lys Gln Val Tyr Phe Tyr Leu Phe Lys Leu
Leu Arg Lys Cys 195 200 205 Ile Leu Gln Met Thr Arg Pro Val Val Glu
Gly Ser Leu Gly Ser Pro 210 215 220 Pro Phe Glu Lys Pro Asn Ile Glu
Gln Gly Val Leu Asn Phe Val Gln 225 230 235 240 Tyr Lys Phe Ser His
Leu Ala Pro Arg Glu Arg Gln Thr Met Phe Glu 245 250 255 Leu Ser Lys
Met Phe Leu Leu Cys Leu Asn Tyr Trp Lys Leu Glu Thr 260 265 270 Pro
Ala Gln Phe Arg Gln Arg Ser Gln Ala Glu Asp Val Ala Thr Tyr 275 280
285 Lys Val Asn Tyr Thr Arg Trp Leu Cys Tyr Cys His Val Pro Gln Ser
290 295 300 Cys Asp Ser Leu Pro Arg Tyr Glu Thr Thr His Val Phe Gly
Arg Ser 305 310 315 320 Leu Leu Arg Ser Ile Phe Thr Val Thr Arg Arg
Gln Leu Leu Glu Lys 325 330 335 Phe Arg Val Glu Lys Asp Lys Leu Val
Pro Glu Lys Arg Thr Leu Ile 340 345 350 Leu Thr His Phe Pro Lys Phe
Leu Ser Met Leu Glu Glu Glu Ile Tyr 355 360 365 Gly Ala Asn Ser Pro
Ile Trp Glu Ser Gly Phe Thr Met Pro Pro Ser 370 375 380 Glu Gly Thr
Gln Leu Val Pro Arg Pro Ala Ser Val Ser Ala Ala Val 385 390 395 400
Val Pro Ser Thr Pro Ile Phe Ser Pro Ser Met Gly Gly Gly Ser Asn 405
410 415 Ser Ser Leu Ser Leu Asp Ser Ala Gly Ala Glu Pro Met Pro Gly
Glu 420 425 430 Lys Arg Thr Leu Pro Glu Asn Leu Thr Leu Glu Asp Ala
Lys Arg Leu 435 440 445 Arg Val Met Gly Asp Ile Pro Met Glu Leu Val
Asn Glu Val Met Leu 450 455 460 Thr Ile Thr Asp Pro Ala Ala Met Leu
Gly Pro Glu Thr Ser Leu Leu 465 470 475 480 Ser Ala Asn Ala Ala Arg
Asp Glu Thr Ala Arg Leu Glu Glu Arg Arg 485 490 495 Gly Ile Ile Glu
Phe His Val Ile Gly Asn Ser Leu Thr Pro Lys Ala 500 505 510 Asn Arg
Arg Val Leu Leu Trp Leu Val Gly Leu Gln Asn Val Phe Ser 515 520 525
His Gln Leu Pro Arg Met Pro Lys Glu Tyr Ile Ala Arg Leu Val Phe 530
535 540 Asp Pro Lys His Lys Thr Leu Ala Leu Ile Lys Asp Gly Arg Val
Ile 545 550 555 560 Gly Gly Ile Cys Phe Arg Met Phe Pro Thr Gln Gly
Phe Thr Glu Ile 565 570 575 Val Phe Cys Ala Val Thr Ser Asn Glu Gln
Val Lys Gly Tyr Gly Thr 580 585 590 His Leu Met Asn His Leu Lys Glu
Tyr His Ile Lys His Asn Ile Leu 595 600 605 Tyr Phe Leu Thr Tyr Ala
Asp Glu Tyr Ala Ile Gly Tyr Phe Lys Lys 610 615 620 Gln Gly Phe Ser
Lys Asp Ile Lys Val Pro Lys Ser Arg Tyr Leu Gly 625 630 635 640 Tyr
Ile Lys Asp Tyr Glu Gly Ala Thr Leu Met Glu Cys Glu Leu Asn 645 650
655 Pro Arg Ile Pro Tyr Thr Glu Leu Ser His Ile Ile Lys Lys Gln Lys
660 665 670 Glu Ile Ile Lys Lys Leu Ile Glu Arg Lys Gln Ala Gln Ile
Arg Lys 675 680 685 Val Tyr Pro Gly Leu Ser Cys Phe Lys Glu Gly Val
Arg Gln Ile Pro 690 695 700 Val Glu Ser Val Pro Gly Ile Arg Glu Thr
Gly Trp Lys Pro Leu Gly 705 710 715 720 Lys Glu Lys Gly Lys Glu Leu
Lys Asp Pro Asp Gln Leu Tyr Thr Thr 725 730 735 Leu Lys Asn Leu Leu
Ala Gln Ile Lys Ser His Pro Ser Ala Trp Pro 740 745 750 Phe Met Glu
Pro Val Lys Lys Ser Glu Ala Pro Asp Tyr Tyr Glu Val 755 760 765 Ile
Arg Phe Pro Ile Asp Leu Lys Thr Met Thr Glu Arg Leu Arg Ser 770 775
780 Arg Tyr Tyr Val Thr Arg Lys Leu Phe Val Ala Asp Leu Gln Arg Val
785 790 795 800 Ile Ala Asn Cys Arg Glu Tyr Asn Pro Pro Asp Ser Glu
Tyr Cys Arg 805 810 815 Cys Ala Ser Ala Leu Glu Lys Phe Phe Tyr Phe
Lys Leu Lys Glu Gly 820 825 830 Gly Leu Ile Asp Lys 835
63127DNAHomo sapiens 6ggttgcccat gcggccctag ggctgggagc gcggcgccgc
tctccgctgc gggggaggcc 60atggcggaac cttcccaggc cccgaccccg gccccggctg
cgcagccccg gccccttcag 120tccccagccc ctgccccaac tccgactcct
gcacccagcc cggcttcagc cccgattccg 180actcccaccc cggcaccagc
ccctgcccca gctgcagccc cagccggcag cacagggact 240ggggggcccg
gggtaggaag tgggggggcc gggagcgggg gggatccggc tcgacctggc
300ctgagccagc agcagcgcgc cagtcagagg aaggcgcaag tccgggggct
gccgcgcgcc 360aagaagcttg agaagctagg ggtcttctcg gcttgcaagg
ccaatgaaac ctgtaagtgt 420aatggctgga aaaaccccaa gccccccact
gcaccccgca tggatctgca gcagccagct 480gccaacctga gtgagctgtg
ccgcagttgt gagcacccct tggctgacca cgtatcccac 540ttggagaatg
tgtcagagga tgagataaac cgactgctgg ggatggtggt ggatgtggag
600aatctcttca tgtctgttca caaggaagag gacacagaca ccaagcaggt
ctatttctac 660ctcttcaagc tactgcggaa atgcatcctg cagatgaccc
ggcctgtggt ggaggggtcc 720ctgggcagcc ctccatttga gaaacctaat
attgagcagg gtgtgctgaa ctttgtgcag 780tacaagttta gtcacctggc
tccccgggag cggcagacga tgttcgagct ctcaaagatg 840ttcttgctct
gccttaacta ctggaagctt gagacacctg cccagtttcg gcagaggtct
900caggctgagg acgtggctac ctacaaggtc aattacacca gatggctctg
ttactgccac 960gtgccccaga gctgtgatag cctcccccgc tacgaaacca
ctcatgtctt tgggcgaagc 1020cttctccggt ccattttcac cgttacccgc
cggcagctgc tggaaaagtt ccgagtggag 1080aaggacaaat tggtgcccga
gaagaggacc ctcatcctca ctcacttccc caaattcctg 1140tccatgctgg
aggaggagat ctatggggca aactctccaa tctgggagtc aggcttcacc
1200atgccaccct cagaggggac acagctggtt ccccggccag cttcagtcag
tgcagcggtt 1260gttcccagca cccccatctt cagccccagc atgggtgggg
gcagcaacag ctccctgagt 1320ctggattctg caggggccga gcctatgcca
ggcgagaaga ggacgctccc agagaacctg 1380accctggagg atgccaagcg
gctccgtgtg atgggtgaca tccccatgga gctggtcaat 1440gaggtcatgc
tgaccatcac tgaccctgct gccatgctgg ggcctgagac gagcctgctt
1500tcggccaatg cggcccggga tgagacagcc cgcctggagg agcgccgcgg
catcatcgag 1560ttccatgtca tcggcaactc actgacgccc aaggccaacc
ggcgggtgtt gctgtggctc 1620gtggggctgc agaatgtctt ttcccaccag
ctgccgcgca tgcctaagga gtatatcgcc 1680cgcctcgtct ttgacccgaa
gcacaagact ctggccttga tcaaggatgg gcgggtcatc 1740ggtggcatct
gcttccgcat gtttcccacc cagggcttca cggagattgt cttctgtgct
1800gtcacctcga atgagcaggt caagggttat gggacccacc tgatgaacca
cctgaaggag 1860tatcacatca agcacaacat tctctacttc ctcacctacg
ccgacgagta cgccatcggc 1920tacttcaaaa agcagggttt ctccaaggac
atcaaggtgc ccaagagccg ctacctgggc 1980tacatcaagg actacgaggg
agcgacgctg atggagtgtg agctgaatcc ccgcatcccc 2040tacacggagc
tgtcccacat catcaagaag cagaaagaga tcatcaagaa gctgattgag
2100cgcaaacagg cccagatccg caaggtctac ccggggctca gctgcttcaa
ggagggcgtg 2160aggcagatcc ctgtggagag cgttcctggc attcgagaga
caggctggaa gccattgggg 2220aaggagaagg ggaaggagct gaaggacccc
gaccagctct acacaaccct caaaaacctg 2280ctggcccaaa tcaagtctca
ccccagtgcc tggcccttca tggagcctgt gaagaagtcg 2340gaggcccctg
actactacga ggtcatccgc ttccccattg acctgaagac catgactgag
2400cggctgcgaa gccgctacta cgtgacccgg aagctctttg tggccgacct
gcagcgggtc 2460atcgccaact gtcgcgagta caaccccccg gacagcgagt
actgccgctg tgccagcgcc 2520ctggagaagt tcttctactt caagctcaag
gagggaggcc tcattgacaa gtaggcccat 2580ctttgggccg cagccctgac
ctggaatgtc tccacctcgg attctgatct gatccttagg 2640gggtgccctg
gccccacgga cccgactcag cttgagacac tccagccaag ggtcctccgg
2700acccgatcct gcagctcttt ctggaccttc aggcaccccc aagcgtgcag
ctctgtccca 2760gccttcactg tgtgtgagag gtctcctggg ttggggccca
gcccctctag agtagctggt 2820ggccagggat gaaccttgcc cagccgtggt
ggcccccagg cctggtcccc aagagctttg 2880gaggcttgga ttcctgggcc
tggcccaggt ggctgtttcc ctgaggacca gaactgctca 2940ttttagcttg
agtgatggct tcaggggttg gaagttcagc ccaaactgaa gggggccatg
3000ccttgtccag cactgttctg tcagtctccc ccaggggtgg ggggtatggg
gaccattcat 3060tccctggcat taatccctta gagggaataa taaagctttt
tatttctctg tgaaaaaaaa 3120aaaaaaa 3127
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