U.S. patent application number 14/801262 was filed with the patent office on 2015-10-29 for compounds and related methods for treatment of neurodegenerative diseases.
This patent application is currently assigned to UNIVERSITY OF SOUTH FLORIDA. The applicant listed for this patent is Bill Baker, Laurent Calcul, Umesh Jinwal. Invention is credited to Bill Baker, Laurent Calcul, Umesh Jinwal.
Application Number | 20150306068 14/801262 |
Document ID | / |
Family ID | 51841737 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150306068 |
Kind Code |
A1 |
Jinwal; Umesh ; et
al. |
October 29, 2015 |
COMPOUNDS AND RELATED METHODS FOR TREATMENT OF NEURODEGENERATIVE
DISEASES
Abstract
Aspects of the invention pertain to chemical compounds,
therapeutic compositions, and methods for treating
neurodegenerative diseases, in particular, neurodegenerative
diseases associated with abnormal accumulation of protein tau.
Inventors: |
Jinwal; Umesh; (Tampa,
FL) ; Baker; Bill; (Tampa, FL) ; Calcul;
Laurent; (Tampa, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Jinwal; Umesh
Baker; Bill
Calcul; Laurent |
Tampa
Tampa
Tampa |
FL
FL
FL |
US
US
US |
|
|
Assignee: |
UNIVERSITY OF SOUTH FLORIDA
Tampa
FL
|
Family ID: |
51841737 |
Appl. No.: |
14/801262 |
Filed: |
July 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14271177 |
May 6, 2014 |
9114130 |
|
|
14801262 |
|
|
|
|
61819701 |
May 6, 2013 |
|
|
|
Current U.S.
Class: |
514/183 ;
514/510 |
Current CPC
Class: |
A61K 31/222 20130101;
A61K 31/395 20130101 |
International
Class: |
A61K 31/395 20060101
A61K031/395; A61K 31/222 20060101 A61K031/222 |
Claims
1. A method of reducing intracellular tau phosphorylation levels,
wherein the method comprises administering, to cells comprising
phosphorylated protein tau, an effective amount of compound B:
##STR00010##
2. The method according to claim 1, wherein the cells are in a
subject in need of treatment for a neurodegenerative disease.
3. The method of claim 2, wherein the neurodegenerative disease is
a tauopathy.
4. The method of claim 3, wherein the subject is a human.
5. The method of claim 4, wherein the neurodegenerative disease is
selected from Alzheimer's disease, Parkinson's disease,
frontotemporal dementia, frontotemporal dementia with Parkinsonism,
frontotemporal lobe dementia, pallidopontonigral degeneration,
progressive supranuclear palsy, multiple system tauopathy, multiple
system tauopathy with presenile dementia, Wilhelmsen-Lynch disease,
Pick's disease, or Pick's disease-like dementia.
6. The method of claim 4, wherein the neurodegenerative disease is
Alzheimer's disease.
7. A pharmaceutical composition comprising a therapeutically
effective amount of a compound selected from compound A
##STR00011## compound B ##STR00012## and combinations thereof; and
a pharmaceutically acceptable carrier or adjuvant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/271,177, filed May 6, 2014, and now
allowed, which claims the benefit of U.S. Provisional Application
Ser. No. 61/819,701, filed May 6, 2013, which are hereby
incorporated by reference in its entireties.
BACKGROUND
[0002] Intracellular aggregation of abnormal species of
phosphorylated tau (protein tau) is a major pathologic feature of a
family of neurodegenerative diseases collectively referred to as
the tauopathies. Tau normally functions to stabilize microtubules
in neurons; however, it pathologically aggregates more than 15
neurodegenerative diseases, including Alzheimer's disease (AD) and
Parkinson's disease. The most common tauopathy is Alzheimer's
disease, in which paired helical filaments (PHFs) of mis-folded
protein tau aggregates in neurofibrillary tangles, in dystrophic
neuritis of senile plaques, and in cell processes in the neuropil.
Abnormal accumulation of protein tau is closely linked with
postsymptomatic progression in Alzheimer's disease. Abnormal
accumulation of protein tau in the cytoplasm of neuronal and glial
cells also represents major structural hallmarks in the pathology
of Pick's disease, corticobasal degeneration, and progressive
supranuclear palsy.
[0003] At present, researchers on the development of therapeutics
for tauopathies focus primarily on agents that prevent abnormal
phosphorylation or aggregation of tau proteins. However, it has
been discovered that while aggregation of hyperphosphorylated
protein tau is visible evidence of tauopathies, these
neurofibrillary tangles appear to be less toxic than soluble
intermediates of protein tau. High levels of tau intermediates,
particularly aberrant tau species failed to be cleared from cells,
cause cognitive dysfunction in AD and tauopathies. Therefore,
agents that degrade or destabilize tau intermediates, clear
aberrant tau species from cells, or otherwise reduce intracellular
tau levels, are promising therapeutics for AD and tauopathies.
[0004] Existing therapeutics for the treatment tauopathies (such as
AD) only demonstrate limited efficacy; as such, additional
therapeutics for the treatment of tauopathies are needed.
BRIEF SUMMARY
[0005] Aspects of the invention provide chemical compounds for
treating neurodegenerative diseases, in particular,
neurodegenerative diseases associated with abnormal accumulation of
protein tau.
[0006] One aspect of the invention provides a method of reducing
intracellular tau levels comprising administering, to cells
comprising protein tau, an effective amount of compound A:
##STR00001##
[0007] In another aspect, the invention provides a method of
reducing intracellular tau levels comprising administering, to
cells comprising protein tau, an effective amount of compound
B:
##STR00002##
[0008] In some embodiments of aspects of the invention, the cells
are in a subject, such as a human, in need of treatment for a
neurodegenerative disease. The neurodegenerative disease may be a
tauopathy. In some embodiments, the neurodegenerative disease is
selected from Alzheimer's disease, Parkinson's disease,
frontotemporal dementia, frontotemporal dementia with Parkinsonism,
frontotemporal lobe dementia, pallidopontonigral degeneration,
progressive supranuclear palsy, multiple system tauopathy, multiple
system tauopathy with presenile dementia, Wilhelmsen-Lynch disease,
Pick's disease, or Pick's disease-like dementia. In one embodiment,
the neurodegenerative disease is Alzheimer's disease.
[0009] In additional aspects, the invention provides pharmaceutical
compositions comprising any of compounds A, B, and combinations
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows a western blot analysis of phosphorylated tau
following Palmadorin M and Beauvericin treatment in HeLa cells
transfected with (A) wild type human tau and (B) mutant P301L human
tau.
BRIEF DESCRIPTION OF THE SEQUENCES
[0011] SEQ ID NO:1 is an amino acid sequence of a tau protein
isoform (tau 352) useful according to aspects of the invention.
[0012] SEQ ID NO:2 is an amino acid sequence of a tau protein
isoform (tau 441) useful according to aspects of the invention.
[0013] SEQ ID NO:3 is an amino acid sequence of a tau protein
isoform (tau 383) useful according to aspects of the invention.
[0014] SEQ ID NO:4 is an amino acid sequence of a tau protein
isoform (tau 758) useful according to aspects of the invention.
[0015] SEQ ID NO:5 is an amino acid sequence of a tau protein
isoform (tau 776) useful according to aspects of the invention.
[0016] SEQ ID NO:6 is an amino acid sequence of a tau protein
isoform (tau 412) useful according to aspects of the invention.
DETAILED DESCRIPTION
[0017] Aspects of the invention provide chemical compounds and
compositions for treating neurodegenerative diseases, in
particular, neurodegenerative diseases associated with abnormal
accumulation of protein tau.
[0018] In one aspect, the invention provides compounds A and B, or
salts thereof:
##STR00003##
[0019] In one embodiment, the invention provides an isolated or
substantially pure compound A, or a salt thereof,
##STR00004##
[0020] In one embodiment, the invention provides an isolated or
substantially pure compound B, or a salt thereof,
##STR00005##
[0021] In certain embodiments, the invention pertains to the uses
of compound A (palmadorin M) and/or compound B (beauvericin), or
salts thereof, for treating neurodegenerative diseases, in
particular, neurodegenerative diseases associated with abnormal
accumulation of protein tau.
[0022] In some embodiments, the compounds of the present invention
are at least 75% pure, preferably at least 90% pure, more
preferably are more than 95% pure, and most preferably are more
than 99% pure (substantially pure). The compounds of the present
invention can also be synthesized.
[0023] The compounds and compositions of the present invention,
through administration to a subject, are useful for treating or
ameliorating neurodegenerative diseases or conditions, in
particular, neurodegenerative diseases or conditions associated
with abnormally high levels of protein tau and/or abnormal
accumulation of protein tau in neurons. In some embodiments, the
compounds and compositions are useful to treat or ameliorate
Alzheimer's disease or Parkinson's disease.
[0024] In one aspect, the invention provides methods for treating
or ameliorating a neurodegenerative disease or condition,
particularly a disease or condition associated with abnormally high
levels of protein tau and/or abnormal accumulation of protein tau
in neurons, wherein the method comprises administering, to a
subject in need of such treatment, an effective amount of a
composition comprising a compound selected from compound A and
compound B, or salts thereof.
[0025] Another aspect of the invention provides a method of
reducing intracellular tau levels comprising administering, to
cells comprising protein tau, an effective amount of compound
A:
##STR00006##
[0026] In another aspect, the invention provides a method of
reducing intracellular tau levels comprising administering, to
cells comprising protein tau, an effective amount of compound
B:
##STR00007##
[0027] In yet another aspect, the invention provides a method of
reducing intracellular tau levels comprising administering, to
cells comprising protein tau, an effective amount of compounds
selected from compound A (Palmadorin M) and compound B
(Beauvericin), and combinations thereof.
[0028] The term "subject," as used herein, describes an organism,
including mammals, to which treatment with the compositions and
compounds according to the subject invention can be administered.
Mammalian species that can benefit from the disclosed methods of
treatment include, but are not limited to, apes, chimpanzees,
orangutans, humans, monkeys; and other animals such as dogs, cats,
horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea
pigs, and hamsters. Typically, the subject is a human.
[0029] The term "tau protein" or any grammatical variation thereof
(e.g., protein tau and tau etc.), as used herein, refers generally
to any protein of the microtubule-associated tau protein family.
Members of the tau family share the common features of a
characteristic N-terminal segment, sequences of approximately 50
amino acids inserted in the N-terminal segment, a characteristic
tandem repeat region consisting of 3 or 4 tandem repeats of 31-32
amino acids, and a C-terminal tail. Tau proteins of the present
invention may be in a form of soluble tau intermediates,
functional, aberrant, abnormally-truncated, mis-folded or
mis-processed tau, and phosphorylated tau.
[0030] In some embodiments, tau protein of the invention is of
mammalian origin, and more preferably, of human origin.
Specifically, tau proteins of the subject invention include
microtubule-associated protein translated from the human
chromosomal sequence of GenBank Accession No. AH005895 and
naturally-occurring mammalian variants or isoforms thereof. Six
human brain tau isoforms are currently known, including tau352
(GenBank Accession No. NP.sub.--058525) (SEQ ID NO:1), tau441
(GenBank Accession No. NP.sub.--005901) (SEQ ID NO:2), tau383
(GenBank Accession No. NP.sub.--058518) (SEQ ID NO:3), tau758
(GenBank Accession No. NP.sub.--058519) (SEQ ID NO:4), tau776
(GenBank Accession No. NP.sub.--001116538) (SEQ ID NO:5), and
tau412 (GenBank Accession No. NP.sub.--001116539) (SEQ ID
NO:6).
[0031] The term "treatment" or any grammatical variation thereof
(e.g., treat, treating and treatment etc.), as used herein,
includes but is not limited to, ameliorating or alleviating a
symptom of a disease or condition, reducing, suppressing,
inhibiting, lessening, or affecting the progression, severity,
and/or scope of a condition, chance of re-occurrence or returning
of a disease after a remission. For instance, the term "treatment"
includes (i) ameliorating a symptom associated with a
neurodegenerative disease in a patient diagnosed with the
neurodegenerative disease; and/or (ii) relieving (such as
attenuating the progress of) or remedying a neurodegenerative
disease in a patient diagnosed with the neurodegenerative
disease.
[0032] In some embodiments, the treatment methods of the present
invention reduce tau levels and/or improve tau clearance. Normal,
functional tau is less affected by clearance pathways in the cell
than aberrant tau. In one embodiment, the treatment methods of the
present invention modulate tau clearance by selectively targeting
abnormal tau.
[0033] In an embodiment, the therapeutic composition is
administered to a human subject who has symptoms of, or is
diagnosed with, a neurodegenerative disease. In some embodiments,
the therapeutic composition is administered to a human subject who
has symptoms of, or is diagnosed with, a neurodegenerative disease
associated with abnormal accumulation of protein tau. For instance,
the therapeutic composition is administered to a human subject who
has elevated levels of soluble protein tau and/or
hyperphosphorylated protein tau in the nervous system, such as in
the brain or cytoplasm of neuronal and glial cells. In addition,
the therapeutic composition is administered to a human subject who
exhibits pathologic features such as neurofibrillary tangles or
senile plaques in neuronal cells and/or cell processes. In a
specific embodiment, the therapeutic composition is administered to
a human subject who has symptoms of, or is diagnosed with,
Alzheimer's disease.
[0034] The identification of subjects who are in need of treatment
for a neurodegenerative disease is well within the knowledge and
ability of one skilled in the art. By way of example, a clinician
skilled in the art can readily identify, by the use of clinical
tests, neurologic and physical examination, and medical/family
history, those patients who are suffering from a neurodegenerative
disease as well as those who are predisposed to developing a
neurodegenerative disease and thus readily determine if an
individual is in need of treatment. For instance, neurofibrillary
tangles or senile plaques present in neuronal cells and/or cell
processes can be determined using electron microscopy (EM) or other
clinical techniques known in the art. In addition, spinal fluid or
cerebral fluid samples or tissues samples from hippocampal tissue
or frontal cortex tissue samples may be obtained from a subject and
levels of protein tau present in the samples can be determined
using routine techniques such as enzyme-linked immunosorbant assay
(ELISA), western blot, and immunological assays.
[0035] The compounds and compositions of the present invention can
be administered to the subject being treated by standard routes,
including oral, inhalation, or parenteral administration including
intravenous, subcutaneous, topical, transdermal, intradermal,
transmucosal, intraperitoneal, intramuscular, intracapsular,
intraorbital, intracardiac, transtracheal, subcutaneous,
subcuticular, intraarticular, subcapsular, subarachnoid,
intraspinal, epidural and intrasternal injection, infusion, and
electroporation, as well as co-administration as a component of any
medical device or object to be inserted (temporarily or
permanently) into a subject. In a one embodiment, the compounds and
compositions of the subject invention are administered orally.
[0036] The term "effective amount" or "therapeutically effective
amount," as used herein, refers to an amount that is capable of
treating or ameliorating a disease or condition or otherwise
capable of producing an intended therapeutic effect. For instance,
the effective amount of the compounds and compositions of the
present invention is an amount capable of reducing levels of
protein tau in a subject. In certain embodiments, the effective
amount enables a 5%, 25%, 50%, 75%, 90%, 95%, 99% and 100%
reduction of levels of protein tau (e.g. soluble protein tau
intermediates and/or aberrant protein tau) in a subject.
[0037] The amount of the therapeutic or pharmaceutical composition
which is effective in treatment of a neurodegenerative disease will
depend on the nature of the disease, condition or disorder and can
be determined by standard clinical techniques. Illustratively,
dosage levels of the administered active ingredients can be:
intravenous, 0.01 to about 20 mg/kg; intraperitoneal, 0.01 to about
100 mg/kg; subcutaneous, 0.01 to about 100 mg/kg; intramuscular,
0.01 to about 100 mg/kg; orally 0.01 to about 200 mg/kg and
preferably about 1 to 100 mg/kg; intranasal instillation, 0.01 to
about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal
(body) weight.
[0038] Once improvement of the subject's condition has occurred, a
maintenance dose is administered if necessary. Subsequently, the
dosage or the frequency of administration, or both, may be reduced
as a function of the symptoms to a level at which the improved
condition is retained. When the symptoms have been alleviated to
the desired level, treatment should cease. Subjects may however
require intermittent treatment on a long-term basis upon any
recurrence of disease symptoms.
[0039] In addition, in vitro assays may optionally be employed to
help identify optimal dosage ranges. The precise dose to be
employed in the formulation/composition will also depend on the
route of administration, and the seriousness of the disease,
condition or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0040] The compounds and compositions of the present invention can
be used to treat or ameliorate neurodegenerative diseases
including, but not limited to, Alzheimer's disease, Parkinson's
disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease,
multiple sclerosis, Pick's disease, fronto temporal dementia,
cortico-basal degeneration, progressive supranuclear palsy,
Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,
and Kuru.
[0041] The compounds and compositions of the present invention can
also be used to treat or ameliorate neurodegenerative diseases
including, but not limited to, Down's syndrome, Argyrophilic grain
disease, parkinsonism dementia complex of Guam, non-Guamanian motor
neurone disease with NFT, Niemann-Pick disease type C, subacute
sclerosing panencephalitis, postencephalitic parkinsonism, dementia
pugilistica, myotonic dystrophy, prion protein amyloid antipathy,
and Hallervorden-Spatz disease.
[0042] The compounds and compositions of the present invention are
particularly useful to treat or ameliorate a neurodegenerative
disease involving tau pathologies (i.e., tauopathies) including,
but not limited to, Alzheimer's disease, Parkinson's disease,
frontotemporal dementia, frontotemporal dementia with Parkinsonism,
frontotemporal lobe dementia, pallidopontonigral degeneration,
progressive supranuclear palsy, multiple system tauopathy, multiple
system tauopathy with presenile dementia, Wilhelmsen-Lynch disease,
Pick's disease, and Pick's disease-like dementia.
[0043] Specifically, the compounds and compositions of the present
invention are particularly useful to treat or ameliorate a disease
or condition arising, at least in part, from abnormally high levels
of protein tau in the nervous system, such as in cytoplasm of
neuronal and glial cells and in neuronal and glial cell processes.
Thus, the subject invention is particularly useful for treatment of
neurodegenerative diseases and disorders, in which reduction of
levels of protein tau in the nervous system would be
beneficial.
[0044] In addition, the compounds and compositions of the subject
invention are useful for alleviating or attenuating symptoms
arising from or associated with neurodegenerative diseases,
including cognitive dysfunction, impaired memory, impaired mental
capacities, emotional disturbances, speech dysfunction,
incontinence, tremor, postural instability, rigidity or stiff
movement, muscle paralysis, and pain.
[0045] In additional aspects, the invention provides pharmaceutical
compositions comprising a therapeutically effective amount of any
of compounds A and/or B and a pharmaceutically acceptable carrier
or adjuvant.
[0046] The terms "pharmaceutically acceptable", "physiologically
tolerable" and grammatical variations thereof, as used herein,
include compositions, carriers, diluents and reagents, are used
interchangeably, and represent that the materials are capable of
administration to or upon a subject such as mammal.
[0047] The term "carrier" refers to an adjuvant, excipient, or
vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum oil such as mineral oil,
vegetable oil such as peanut oil, soybean oil, and sesame oil,
animal oil, or oil of synthetic origin. Particularly preferred
pharmaceutical carriers for treatment of or amelioration of a
neurodegenerative disease are carriers that can penetrate the
blood/brain barrier.
[0048] Suitable carriers also include ethanol, dimethyl sulfoxide,
glycerol, silica, alumina, starch, sorbitol, inosital, xylitol,
D-xylose, mannitol, powdered cellulose, microcrystalline cellulose,
talc, colloidal silicon dioxide, calcium carbonate, calcium
phosphate, calcium aluminium silicate, aluminium hydroxide, sodium
starch phosphate, lecithin, and equivalent carriers and diluents.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions.
[0049] Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The therapeutic composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents.
[0050] The amount of active ingredient (i.e., one or more of
compounds A or B) that may be combined with the carrier materials
to produce a single dosage form will vary, depending on the type of
the condition and the subject to be treated. In general, a
therapeutic composition contains from about 5% to about 95% active
ingredient (w/w). More specifically, a therapeutic composition
contains from about 20% (w/w) to about 80% or about 30% to about
70% active ingredient (w/w).
[0051] The compounds and compositions can be formulated according
to known methods for preparing pharmaceutically useful
compositions. Formulations are described in detail in a number of
sources which are well known and readily available to those skilled
in the art. For example, Remington's Pharmaceutical Science by E.
W. Martin describes formulations which can be used in connection
with the present invention. In general, the compositions of the
present invention will be formulated such that an effective amount
of the bioactive compound(s) is/are combined with a suitable
carrier in order to facilitate effective administration of the
composition.
[0052] The therapeutic or pharmaceutical compositions of the
invention can also be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free
amino groups such as those derived from hydrochloric, phosphoric,
acetic, oxalic, tartaric acids, etc., and those formed with free
carboxyl groups such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0053] The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
understood in the art and need not be limited based on formulation.
Typically such compositions are prepared as injectables, either as
liquid solutions or suspensions; however, solid forms suitable for
solution, or suspensions, in liquid prior to use also can be
prepared. The preparation also can be emulsified, such as
oil-in-water emulsion.
[0054] The compounds and compositions of the present invention in
prescription amounts can be readily made into any form of drug,
suitable for administering to humans or other animals. Suitable
forms include, for example, tinctures, decoctions, and dry
extracts. These can be taken orally, applied through venous
injection, mucous membranes or inhalation. The active ingredient(s)
can also be formulated into capsules, powder, pallets, pastille,
suppositories, oral solutions, pasteurized gastroenteric suspension
injections, small or large amounts of injection, frozen powder
injections, pasteurized powder injections and the like.
EXAMPLES
[0055] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0056] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Example 1
Palmadorin M Extraction and Characterization
[0057] 600 g of freeze-dried Antarctic nudibranchs, Austrodoris
kerguelenensis, collected by hand via SCUBA off the Palmer Station
in the Western Antarctic Peninsula (3-40 m depth range), were first
extracted by DCM/MeOH (1:1) then EtOAc/H.sub.2O (1:1). Both
resulting extracts were combined and fractionated by normal phase
MPLC using gradient conditions from 100% hexanes to 100% ethyl
acetate to 100% methanol. The separation provided 10 MPLC
fractions. The last 8 fractions were combined and purified by
reversed-phase semi-preparative HPLC (40-100% MeOH--H.sub.2O over
70 minutes) to provide 16 fractions. Fraction 13 (47 min) yielded
10.1 mg of palmadorin M (Maschek J. A., 2011 et Maschek J. A. et
al., 2012).
##STR00008##
Colorless oil; [.alpha.].sub.D.sup.20-18 (c 0.1, CHCl.sub.3); UV
(MeOH) .lamda.max (.epsilon.) 230 (4.38) nm; IR (thin film) 3429
(br), 2935, 1696, 1645, 1214 cm.sup.-1; .sup.1H NMR (500 MHz,
CDCl3) 5.72 (1H, s, H-14), 4.93 (1H, s, H-2'), 4.71 (1H, t, H-17b),
4.52 (1H, t, H-17a), 3.83 (2H, m, H.sub.2-1'), 3.83 (2H, m,
H.sub.2-3'), 2.18 (1H, m, H-7b), 2.15 (3H, s, H.sub.3-16), 2.04
(1H, m, H-7a), 2.04 (1H, m, H-12b), 1.87 (1H, m, H-12a), 1.70 (1H,
m, H-11b), 1.63 (1H, m, H-2b), 1.62 (1H, m, H-6b), 1.53 (1H, m,
H-1b), 1.51 (1H, m, H-9), 1.47 (1H, m, H-11a), 1.44 (1H, m, H-2a),
1.42 (1H, m, H-3b), 1.29 (1H, m, H-6a), 1.28 (1H, m, H-5), 1.18
(1H, m, H-3a), 1.06 (1H, m, H-1a), 0.92 (3H, s, H.sub.3-20), 0.88
(3H, s, H.sub.3-19), 0.81 (3H, s, H.sub.3-18); .sup.13C NMR (125
MHz, CDCl3) 166.8 (C-15), 163.2 (C-13), 148.7 (C-8), 114.5 (C-14),
109.9 (C-17), 74.2 (C-2'), 62.3 (C-1'), 62.3 (C-3'), 57.8 (C-9),
45.8 (C-5), 42.6 (C-3), 39.8 (C-12), 38.0 (C-10), 36.7 (C-1), 33.4
(C-19), 33.2 (C-4), 31.5 (C-7), 24.7 (C-11), 23.5 (C-6), 22.3
(C-20), 22.1 (C-18), 19.4 (C-16), 19.1 (C-2); HRESIMS m/z 401.2648
[M+Na].sup.+ (C.sub.23H.sub.38NaO.sub.4 requires 401.2668).
Example 2
Beauvericin Extraction and Characterization
[0058] Fungi Collection, Culture Preparation, Isolation and Scale
Up:
[0059] Segments of approximately 1-2 mm.sup.2 of mangrove samples
were plated on malt extract freshwater agar. Fungal growth was
examined every day for two weeks and then twice a week for at least
one month for isolation of pure fungal strain. Pure fungal cultures
were grown for scale up to 2 L total volume in liquid medium which
contained 1% w/v glucose, 0.1% w/v yeast extract and 0.2% w/v
peptone (3 weeks) for extraction, separation and pure metabolite
identification (Calcul et al. 2013)
[0060] Beauvericin:
[0061] 2.3 g from a freeze-dried scale up of fungus strain
collection # EG09-15B-2, isolated from the Florida Everglades bark
of Coccoloba uvifera, was extracted by MeOH. The methanolic extract
was separated by reverse phase MPLC using gradient from
H.sub.2O/10% MeOH to 100% MeOH to give 6 fractions. Fraction 5 was
selected to be further chromatographied by normal phase MPLC using
a gradient from 100% hexane to 100% EtOAc and provided 6 fractions.
Fraction 4 was purified on reverse phase semi-preparative HPLC
using isocratic H.sub.2O/55% ACN yielding 206.2 mg of Beauvericin
(trimer). (Beau J. 2012)
##STR00009##
Colorless film; .sup.1H NMR (500 MHz, CDCl3) 7.26-7.14 (3H, m,
H-12), 7.26-7.14 (6H, m, H-11), 7.26-7.14 (6H, m, H-10), 5.62 (3H,
s, H-6), 4.81 (3H, d, H-3), 3.39 (3H, dd, H-8a), 3.03 (9H, s, H-7),
2.93 (3H, dd, H-8b), 1.93 (3H, dq, H-13), 0.78 (9H, d, H-14), 0.35
(9H, d, H-15); .sup.13C NMR (125 MHz, CDCl3) 170.0 (C-1), 169.9
(C-4), 136.4 (C-9), 128.7 (C-11), 128.5 (C-10), 126.8 (C-12), 75.7
(C-6), 56.8 (C-3), 34.7 (C-7), 31.9 (C-8), 29.7 (C-13), 18.4
(C-14), 17.1 (C-15); LRESIMS m/z 784.4 [M+H].sup.+--Note: the
.sup.1H and .sup.13C NMR data matched to the data from the
literature (Gupta, S. et al. 1991);
Example 3
Palmadorin M and Beauvericin Preferentially Reduce Phosphorylated
Tau
[0062] Compounds effects on tau activity/level were determine by a
previously described method (Jinwal et al J. Neurosci. 2009 Sep.
30; 29(39):12079-88). In brief, human tau transfected HeLa Cells
were treated with Compounds for 24 hours. Cells were harvested by
using MPER reagent containing protease inhibitor and phosphatase
inhibitor. Samples were analyzed by western blot. pS199-202
antibody was used to detect phospho tau level. Total tau levels
were detected by tau H-150 &/or tau 46 antibodies.
[0063] HeLa cells were transfected with wild-type human tau (A) and
mutant P301L human tau (B) (FIG. 1). After 24 hours, cells were
treated with either palmadorin M or beauvericin for 24 hours.
Western blot analysis of samples showed potent reduction in
phosphorylated (pS199-202) wild type and mutant P301L tau at
various doses of palmadorin M and beauvericin. Tau H-150 antibody
tested for N-terminal tau showed increase in tau level. Tau 46
antibody tested for C-terminal tau showed moderate change in tau
level.
[0064] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
REFERENCES
[0065] Maschek J. A. "Chemical Investigation of the Antarctic
Marine Invertebrates Austrodoris kerguelenensis & Dendrilla
membranosa and the Antarctic Red Alga Gigartina skottsbergii" Ph.D.
Thesis, Department of Chemistry, College of Art and Sciences,
University of South Florida, Tampa, Fla., USA, 7 Jun. 2011. [0066]
Maschek J. A. 2012, Tetrahedron, 9095-9104. [0067] Beau J. "Drug
Discovery from Floridian Mangrove Endophytes" PhD. Thesis,
Department of Chemistry, College of Art and Sciences, University of
South Florida, Tampa, Fla., USA, 12 Jul. 2012. [0068] Gupta S. et
al. Mycopathologia, 1991, 185-189 [0069] Calcul L. et al. Mar.
Drugs, 2013, 5036-5050.
Sequence CWU 1
1
61352PRTHomo sapiens 1Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met
Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp
Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr
Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45 Gly Ile Gly Asp Thr
Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60 Thr Gln Ala
Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 65 70 75 80 Asp
Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90
95 Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg
100 105 110 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser
Ser Gly 115 120 125 Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser
Ser Pro Gly Ser 130 135 140 Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr
Pro Ser Leu Pro Thr Pro 145 150 155 160 Pro Thr Arg Glu Pro Lys Lys
Val Ala Val Val Arg Thr Pro Pro Lys 165 170 175 Ser Pro Ser Ser Ala
Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190 Pro Asp Leu
Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 195 200 205 Lys
His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val 210 215
220 Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His
225 230 235 240 His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu
Lys Leu Asp 245 250 255 Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser
Leu Asp Asn Ile Thr 260 265 270 His Val Pro Gly Gly Gly Asn Lys Lys
Ile Glu Thr His Lys Leu Thr 275 280 285 Phe Arg Glu Asn Ala Lys Ala
Lys Thr Asp His Gly Ala Glu Ile Val 290 295 300 Tyr Lys Ser Pro Val
Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser 305 310 315 320 Asn Val
Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu 325 330 335
Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 340
345 350 2441PRTHomo sapiens 2Met Ala Glu Pro Arg Gln Glu Phe Glu
Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg
Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly
Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro
Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp
Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70
75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr
Glu 85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly
Asp Thr Pro 100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr
Gln Ala Arg Met Val 115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser
Asp Asp Lys Lys Ala Lys Gly 130 135 140 Ala Asp Gly Lys Thr Lys Ile
Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly
Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175 Pro Ala
Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190
Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195
200 205 Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro
Lys 210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser
Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Pro Met
Pro Asp Leu Lys Asn Val 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu
Asn Leu Lys His Gln Pro Gly Gly 260 265 270 Gly Lys Val Gln Ile Ile
Asn Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285 Ser Lys Cys Gly
Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300 Ser Val
Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315
320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln
325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val
Gln Ser 340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro
Gly Gly Gly Asn 355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe
Arg Glu Asn Ala Lys Ala 370 375 380 Lys Thr Asp His Gly Ala Glu Ile
Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro
Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser 405 410 415 Ile Asp Met
Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430 Ser
Ala Ser Leu Ala Lys Gln Gly Leu 435 440 3383PRTHomo sapiens 3Met
Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10
15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His
20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu
Glu Ala 35 40 45 Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala
Ala Gly His Val 50 55 60 Thr Gln Ala Arg Met Val Ser Lys Ser Lys
Asp Gly Thr Gly Ser Asp 65 70 75 80 Asp Lys Lys Ala Lys Gly Ala Asp
Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95 Arg Gly Ala Ala Pro Pro
Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110 Ile Pro Ala Lys
Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125 Glu Pro
Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140
Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 145
150 155 160 Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro
Pro Lys 165 170 175 Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala
Pro Val Pro Met 180 185 190 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile
Gly Ser Thr Glu Asn Leu 195 200 205 Lys His Gln Pro Gly Gly Gly Lys
Val Gln Ile Ile Asn Lys Lys Leu 210 215 220 Asp Leu Ser Asn Val Gln
Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys 225 230 235 240 His Val Pro
Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 245 250 255 Leu
Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 260 265
270 Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe
275 280 285 Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile
Thr His 290 295 300 Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His
Lys Leu Thr Phe 305 310 315 320 Arg Glu Asn Ala Lys Ala Lys Thr Asp
His Gly Ala Glu Ile Val Tyr 325 330 335 Lys Ser Pro Val Val Ser Gly
Asp Thr Ser Pro Arg His Leu Ser Asn 340 345 350 Val Ser Ser Thr Gly
Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 355 360 365 Thr Leu Ala
Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380
4758PRTHomo sapiens 4Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met
Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp
Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr
Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu
Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys
Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp
Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90
95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro
100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Glu Pro
Glu Ser 115 120 125 Gly Lys Val Val Gln Glu Gly Phe Leu Arg Glu Pro
Gly Pro Pro Gly 130 135 140 Leu Ser His Gln Leu Met Ser Gly Met Pro
Gly Ala Pro Leu Leu Pro 145 150 155 160 Glu Gly Pro Arg Glu Ala Thr
Arg Gln Pro Ser Gly Thr Gly Pro Glu 165 170 175 Asp Thr Glu Gly Gly
Arg His Ala Pro Glu Leu Leu Lys His Gln Leu 180 185 190 Leu Gly Asp
Leu His Gln Glu Gly Pro Pro Leu Lys Gly Ala Gly Gly 195 200 205 Lys
Glu Arg Pro Gly Ser Lys Glu Glu Val Asp Glu Asp Arg Asp Val 210 215
220 Asp Glu Ser Ser Pro Gln Asp Ser Pro Pro Ser Lys Ala Ser Pro Ala
225 230 235 240 Gln Asp Gly Arg Pro Pro Gln Thr Ala Ala Arg Glu Ala
Thr Ser Ile 245 250 255 Pro Gly Phe Pro Ala Glu Gly Ala Ile Pro Leu
Pro Val Asp Phe Leu 260 265 270 Ser Lys Val Ser Thr Glu Ile Pro Ala
Ser Glu Pro Asp Gly Pro Ser 275 280 285 Val Gly Arg Ala Lys Gly Gln
Asp Ala Pro Leu Glu Phe Thr Phe His 290 295 300 Val Glu Ile Thr Pro
Asn Val Gln Lys Glu Gln Ala His Ser Glu Glu 305 310 315 320 His Leu
Gly Arg Ala Ala Phe Pro Gly Ala Pro Gly Glu Gly Pro Glu 325 330 335
Ala Arg Gly Pro Ser Leu Gly Glu Asp Thr Lys Glu Ala Asp Leu Pro 340
345 350 Glu Pro Ser Glu Lys Gln Pro Ala Ala Ala Pro Arg Gly Lys Pro
Val 355 360 365 Ser Arg Val Pro Gln Leu Lys Ala Arg Met Val Ser Lys
Ser Lys Asp 370 375 380 Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Thr
Ser Thr Arg Ser Ser 385 390 395 400 Ala Lys Thr Leu Lys Asn Arg Pro
Cys Leu Ser Pro Lys His Pro Thr 405 410 415 Pro Gly Ser Ser Asp Pro
Leu Ile Gln Pro Ser Ser Pro Ala Val Cys 420 425 430 Pro Glu Pro Pro
Ser Ser Pro Lys Tyr Val Ser Ser Val Thr Ser Arg 435 440 445 Thr Gly
Ser Ser Gly Ala Lys Glu Met Lys Leu Lys Gly Ala Asp Gly 450 455 460
Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys 465
470 475 480 Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro
Ala Pro 485 490 495 Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser
Gly Asp Arg Ser 500 505 510 Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr
Pro Gly Ser Arg Ser Arg 515 520 525 Thr Pro Ser Leu Pro Thr Pro Pro
Thr Arg Glu Pro Lys Lys Val Ala 530 535 540 Val Val Arg Thr Pro Pro
Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu 545 550 555 560 Gln Thr Ala
Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys 565 570 575 Ile
Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly Gly Lys Val 580 585
590 Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys
595 600 605 Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser
Val Gln 610 615 620 Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr
Ser Lys Cys Gly 625 630 635 640 Ser Leu Gly Asn Ile His His Lys Pro
Gly Gly Gly Gln Val Glu Val 645 650 655 Lys Ser Glu Lys Leu Asp Phe
Lys Asp Arg Val Gln Ser Lys Ile Gly 660 665 670 Ser Leu Asp Asn Ile
Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile 675 680 685 Glu Thr His
Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp 690 695 700 His
Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr 705 710
715 720 Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp
Met 725 730 735 Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val
Ser Ala Ser 740 745 750 Leu Ala Lys Gln Gly Leu 755 5776PRTHomo
sapiens 5Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His
Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly
Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly
Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser
Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro
Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp Glu Gly Ala
Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95 Ile Pro
Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110
Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Glu Pro Glu Ser 115
120 125 Gly Lys Val Val Gln Glu Gly Phe Leu Arg Glu Pro Gly Pro Pro
Gly 130 135 140 Leu Ser His Gln Leu Met Ser Gly Met Pro Gly Ala Pro
Leu Leu Pro 145 150 155 160 Glu Gly Pro Arg Glu Ala Thr Arg Gln Pro
Ser Gly Thr Gly Pro Glu 165 170 175 Asp Thr Glu Gly Gly Arg His Ala
Pro Glu Leu Leu Lys His Gln Leu 180 185 190 Leu Gly Asp Leu His Gln
Glu Gly Pro Pro Leu Lys Gly Ala Gly Gly 195 200 205 Lys Glu Arg Pro
Gly Ser Lys Glu Glu Val Asp Glu Asp Arg Asp Val 210 215 220 Asp Glu
Ser Ser Pro Gln Asp Ser Pro Pro Ser Lys Ala Ser Pro Ala 225 230 235
240 Gln Asp Gly Arg Pro Pro Gln Thr Ala Ala Arg Glu Ala Thr Ser Ile
245 250 255 Pro Gly Phe Pro Ala Glu Gly Ala Ile Pro Leu Pro Val Asp
Phe Leu 260 265 270 Ser Lys Val Ser Thr Glu Ile Pro Ala Ser Glu Pro
Asp Gly Pro Ser 275 280 285 Val Gly Arg Ala Lys Gly Gln Asp Ala Pro
Leu Glu Phe Thr Phe His 290 295 300 Val Glu Ile Thr Pro Asn Val Gln
Lys Glu Gln Ala His Ser Glu Glu 305 310 315 320 His Leu Gly Arg Ala
Ala Phe Pro Gly Ala Pro Gly Glu Gly Pro Glu 325 330 335 Ala Arg Gly
Pro Ser Leu Gly Glu Asp Thr Lys Glu Ala Asp Leu Pro 340 345 350 Glu
Pro Ser Glu Lys Gln Pro Ala Ala Ala Pro Arg Gly Lys
Pro Val 355 360 365 Ser Arg Val Pro Gln Leu Lys Ala Arg Met Val Ser
Lys Ser Lys Asp 370 375 380 Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys
Thr Ser Thr Arg Ser Ser 385 390 395 400 Ala Lys Thr Leu Lys Asn Arg
Pro Cys Leu Ser Pro Lys His Pro Thr 405 410 415 Pro Gly Ser Ser Asp
Pro Leu Ile Gln Pro Ser Ser Pro Ala Val Cys 420 425 430 Pro Glu Pro
Pro Ser Ser Pro Lys Tyr Val Ser Ser Val Thr Ser Arg 435 440 445 Thr
Gly Ser Ser Gly Ala Lys Glu Met Lys Leu Lys Gly Ala Asp Gly 450 455
460 Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys
465 470 475 480 Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro
Pro Ala Pro 485 490 495 Lys Thr Pro Pro Ser Ser Ala Thr Lys Gln Val
Gln Arg Arg Pro Pro 500 505 510 Pro Ala Gly Pro Arg Ser Glu Arg Gly
Glu Pro Pro Lys Ser Gly Asp 515 520 525 Arg Ser Gly Tyr Ser Ser Pro
Gly Ser Pro Gly Thr Pro Gly Ser Arg 530 535 540 Ser Arg Thr Pro Ser
Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys 545 550 555 560 Val Ala
Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser 565 570 575
Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys 580
585 590 Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly
Gly 595 600 605 Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn
Val Gln Ser 610 615 620 Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val
Pro Gly Gly Gly Ser 625 630 635 640 Val Gln Ile Val Tyr Lys Pro Val
Asp Leu Ser Lys Val Thr Ser Lys 645 650 655 Cys Gly Ser Leu Gly Asn
Ile His His Lys Pro Gly Gly Gly Gln Val 660 665 670 Glu Val Lys Ser
Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys 675 680 685 Ile Gly
Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys 690 695 700
Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys 705
710 715 720 Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val
Ser Gly 725 730 735 Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser
Thr Gly Ser Ile 740 745 750 Asp Met Val Asp Ser Pro Gln Leu Ala Thr
Leu Ala Asp Glu Val Ser 755 760 765 Ala Ser Leu Ala Lys Gln Gly Leu
770 775 6412PRTHomo sapiens 6Met Ala Glu Pro Arg Gln Glu Phe Glu
Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg
Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly
Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro
Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp
Ala Lys Ser Thr Pro Thr Ala Glu Ala Glu Glu Ala Gly Ile Gly 65 70
75 80 Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln
Ala 85 90 95 Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp
Asp Lys Lys 100 105 110 Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala
Thr Pro Arg Gly Ala 115 120 125 Ala Pro Pro Gly Gln Lys Gly Gln Ala
Asn Ala Thr Arg Ile Pro Ala 130 135 140 Lys Thr Pro Pro Ala Pro Lys
Thr Pro Pro Ser Ser Gly Glu Pro Pro 145 150 155 160 Lys Ser Gly Asp
Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr 165 170 175 Pro Gly
Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 180 185 190
Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser 195
200 205 Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp
Leu 210 215 220 Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu
Lys His Gln 225 230 235 240 Pro Gly Gly Gly Lys Val Gln Ile Ile Asn
Lys Lys Leu Asp Leu Ser 245 250 255 Asn Val Gln Ser Lys Cys Gly Ser
Lys Asp Asn Ile Lys His Val Pro 260 265 270 Gly Gly Gly Ser Val Gln
Ile Val Tyr Lys Pro Val Asp Leu Ser Lys 275 280 285 Val Thr Ser Lys
Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly 290 295 300 Gly Gly
Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg 305 310 315
320 Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly
325 330 335 Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg
Glu Asn 340 345 350 Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val
Tyr Lys Ser Pro 355 360 365 Val Val Ser Gly Asp Thr Ser Pro Arg His
Leu Ser Asn Val Ser Ser 370 375 380 Thr Gly Ser Ile Asp Met Val Asp
Ser Pro Gln Leu Ala Thr Leu Ala 385 390 395 400 Asp Glu Val Ser Ala
Ser Leu Ala Lys Gln Gly Leu 405 410
* * * * *