U.S. patent application number 12/030849 was filed with the patent office on 2008-12-04 for alpha-synuclein kinase.
This patent application is currently assigned to ELAN PHARMACEUTICALS, INC.. Invention is credited to John P. Anderson, Kelly Banducci, Guriqbal S. Basi, David Chereau, Tamie J. Chilcote, Normand L. Frigon, JR., Jason Goldstein, Irene Griswold-Prenner.
Application Number | 20080300206 12/030849 |
Document ID | / |
Family ID | 38328036 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080300206 |
Kind Code |
A1 |
Anderson; John P. ; et
al. |
December 4, 2008 |
Alpha-Synuclein Kinase
Abstract
The invention provides agents and methods for treatment of
diseases associated with Lewy body diseases (LBDs) in the brain of
a patient. Preferred agents include inhibitors of PLK2 kinase.
Inventors: |
Anderson; John P.; (San
Francisco, CA) ; Banducci; Kelly; (Pleasanton,
CA) ; Basi; Guriqbal S.; (Palo Alto, CA) ;
Chereau; David; (San Mateo, CA) ; Chilcote; Tamie
J.; (San Francisco, CA) ; Frigon, JR.; Normand
L.; (South San Francisco, CA) ; Goldstein; Jason;
(Decatur, GA) ; Griswold-Prenner; Irene; (San
Francisco, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
ELAN PHARMACEUTICALS, INC.
South San Francisco
CA
|
Family ID: |
38328036 |
Appl. No.: |
12/030849 |
Filed: |
February 13, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11669093 |
Jan 30, 2007 |
|
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12030849 |
|
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60764000 |
Jan 31, 2006 |
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Current U.S.
Class: |
514/44A ; 435/15;
435/375; 514/312; 514/338; 514/339; 514/394; 514/414; 800/3 |
Current CPC
Class: |
A61P 25/16 20180101;
C12N 15/1137 20130101; G01N 2800/28 20130101; A61P 25/28 20180101;
C12N 2310/11 20130101; C12Q 2600/136 20130101; C12Q 1/6883
20130101; C12Y 207/11016 20130101; C12N 2740/15043 20130101; C12Q
2600/158 20130101; C12N 2310/14 20130101; C12N 15/86 20130101; C12Q
1/485 20130101; A61P 43/00 20180101; A61P 25/00 20180101; C12Y
207/11021 20130101; G01N 2500/04 20130101 |
Class at
Publication: |
514/44 ; 435/375;
514/339; 514/338; 514/394; 514/312; 514/414; 435/15; 800/3 |
International
Class: |
A61K 31/70 20060101
A61K031/70; C12N 5/06 20060101 C12N005/06; A61K 31/44 20060101
A61K031/44; A61K 31/415 20060101 A61K031/415; A61K 31/47 20060101
A61K031/47; A61K 31/40 20060101 A61K031/40; C12Q 1/48 20060101
C12Q001/48; A01K 67/027 20060101 A01K067/027; A61P 25/00 20060101
A61P025/00 |
Claims
1. A method for inhibiting phosphorylation of alpha-synuclein in a
mammalian cell, the method comprising reducing polo-like kinase 2
(PLK2) activity in the cell such that phosphorylation of synuclein
is reduced.
2. A method for inhibiting phosphorylation of alpha-synuclein in a
mammalian cell, the method comprising contacting the cell with a
compound that reduces PLK2 activity in the cell such that
phosphorylation of alpha-synuclein is reduced.
3. The method of claim 2 wherein the agent reduces expression of
the PLK2 protein.
4. The method of claim 2 wherein the cell is a neuronal cell.
5. The method of claim 4 wherein the agent has a molecular weight
less than 4000.
6. The method of claim 5 wherein the agent is a synthetic
compound.
7. The method of claim 4 wherein the agent preferentially reduces
PLK2 activity activity relative to reduction of PLK1 activity, PLK2
activity, or PLK3 activity.
8. The method of claim 7 wherein the agent is a polynucleotide that
inhibits expression or translation of a PLK2 RNA transcript.
9. The method of claim 8 wherein the agent is an siRNA comprising a
double stranded region.
10. The method of claim 9 wherein one strand of the double stranded
region is perfectly complementary to a PLK2 transcript and lacks
perfect complementarity to a PLK1 transcript or a PLK3
transcript.
11. The method of claim 5 wherein the compound is
4-[[(7r)-8-cyclopentyl-7-ethyl-5,6,7,8-tetrahydro-5-methyl-6-oxo-2-pterid-
inyl]amino]-3-methoxy-n-(1-methyl-4-piperidinyl)-benzamide.
12. A method of treating a patient diagnosed with Parkinson's
Disease comprising administering a therapeutically effective amount
of an agent that inhibits PLK2 activity.
13. The method of claim 12 wherein the agent preferentially
inhibits PLK2 activity relative to inhibition of PLK1 activity or
PLK3 activity.
14. The method of claim 13 wherein the agent preferentially
inhibits PLK2 activity relative to inhibition of PLK1 activity and
PLK3 activity.
15. The method of claim 14 wherein the agent is an siRNA.
16. The method of claim 15 wherein the agent is an siRNA comprising
a double stranded region.
17. The method of claim 16 wherein one strand of the double
stranded region is perfectly complementary to a PLK2 transcript and
lacks perfect complementarity to a PLK1 transcript or a PLK3
transcript.
18. The method of claim 12 wherein the agent is a synthetic
compound with a molecular weight less than 4000.
19. The method of claim 12 wherein the compound is
4-[[(7r)-8-cyclopentyl-7-ethyl-5,6,7,8-tetrahydro-5-methyl-6-oxo-2-pterid-
inyl]amino]-3-methoxy-n-(1-methyl-4-piperidinyl)-benzamide.
20. The method of claim 12 wherein the patient is not diagnosed or
under treatment for cancer.
21. The method of claim 12 wherein the patient is not diagnosed or
under treatment for Alzheimer's disease.
22. A method of identifying an agent reduces alpha-synuclein
phosphorylation in a mammalian cell expressing alpha-synuclein
comprising selecting an agent that a) reduces activity of PLK2 in a
cell expressing PLK2; and b) does not reduce activity of PLK1 in a
cell expressing PLK1, or reduces activity of PLK1 at a higher
EC.sub.50 than for PLK2; and/or c) does not reduce activity of PLK3
in a cell expressing PLK3, or reduces activity of PLK3 at a higher
EC.sub.50 than for PLK2; and/or d) does not reduce activity of PLK4
in a cell expressing PLK4, or reduces activity of PLK4 at a higher
EC.sub.50 than for PLK2.
23. The method of claim 22 wherein the cell is a mammalian cell
overexpressing alpha-synuclein.
24. The method claim 23 comprising selecting an agent that a)
reduces activity of PLK2 in a cell expressing PLK2; b) does not
reduce activity of PLK1 in a cell expressing PLK1, or reduces
activity of PLK1 at a higher EC.sub.50 than for PLK2; c) does not
reduce activity of PLK3 in a cell expressing PLK3, or reduces
activity of PLK3 at a higher EC.sub.50 than for PLK2; and d) does
not reduce activity of PLK4 in a cell expressing PLK4, or reduces
activity of PLK4 at a higher EC.sub.50 than for PLK2.
25. A method of identifying an agent for treatment of Parkinson's
disease comprising selecting an agent according to the method of
claim 22, and further comprising determining whether the selected
agent shows activity useful in treating Lewy Body Disease in an
animal model of the disease.
26. A method of identifying an agent for treatment of Parkinson's
disease comprising selecting an agent according to the method of
claim 22, and further comprising determining whether the selected
agent shows activity useful in treating Lewy Body Disease in a
cellular model of the disease.
27. The method of claim 26 wherein the cellular model of the
disease is a neuronally-derived cell culture.
28. The method of claim 27 wherein the cellular model of the
disease is a mammalian cell over-expressing alpha-synuclein.
29. The method of claim 27 wherein the activity is reduction of the
proportion of total alpha-synuclein that is phosphorylated at of
serine-129.
30. The method of claim 27 wherein said activity is a reduction in
aggregation of alpha-synuclein in the cell.
31. The method of claim 22, further comprising identifying an agent
that modulates PLK2 in the presence of synphilin.
Description
RELATED APPLICATIONS
[0001] This application claims priority as a continuation-in-part
of U.S. patent application Ser. No. 11/669,093, filed Jan. 30,
2007, which claims benefit of U.S. provisional application No.
60/764,000, filed Jan. 31, 2006, each of which is herein
incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Lewy body diseases (LBDs) are characterized by degeneration
of the dopaminergic system, motor alterations, cognitive
impairment, and formation of Lewy bodies (LBs). (McKeith et al,
Clinical and pathological diagnosis of dementia with Lewy bodies
(DLB): Report of the CDLB International Workshop, Neurology (1996)
47:1113-24). LBDs include Parkinson's disease, Diffuse Lewy body
disease (DLBD), Lewy body variant of Alzheimer's disease (LBV),
combined Parkinson's disease (PD) and Alzheimer's disease (AD), and
the syndromes identified as multiple system atrophy (MSA). Dementia
with Lewy bodies (DLB) is a term coined to reconcile differences in
the terminology of LBDs. Disorders with LBs continue to be a common
cause for movement disorders and cognitive deterioration in the
aging population (Galasko et al., Clinical-neuropathological
correlations in Alzheimer's disease and related dementias. Arch.
Neurol. (1994) 51:888-95). Although their incidence continues to
increase, creating a serious public health problem, to date these
disorders lack approved treatments (Tanner et al., Epidemiology of
Parkinson's disease and akinetic syndromes, Curr. Opin. Neurol.
(2000) 13:427-30). The cause for LBDs is controversial and multiple
factors have been proposed to play a role, including various
neurotoxins and genetic susceptibility factors.
[0003] In recent years, new hope for understanding the pathogenesis
of LBDs has emerged. Specifically, several studies have shown that
the synaptic protein alpha-synuclein plays a central role in PD
pathogenesis because: (1) this protein accumulates in LBs
(Spillantini et al., Nature (1997) 388:839-40; Takeda et al., J.
Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett.
(1997) 239:45-8), (2) mutations in the alpha-synuclein gene
co-segregate with rare familial forms of parkinsonism (Kruger et
al., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science
(1997) 276:2045-7) and, (3) overexpression of alpha-synuclein in
transgenic mice (Masliah et al., Science (2000) 287:1265-9) and
Drosophila (Feany et al, Nature (2000) 404:394-8) mimics several
pathological aspects of PD.
[0004] Many scientists believe that PD is a relatively late
development in a systemic synucleinopathy and that "parkinsonism is
just the tip of the iceberg" (Langston, Annals of Neurology (2006)
59:591-596). For example, Lewy bodies have been described in
sympathetic ganglia and in the myenteric plexus of the gut (Herzog
E., Dtch Z Nervenheilk (1928) 107:75-80; Kupsky et al., Neurology
(1987) 37:1253-1255). Various disorders have been associated with
the presence of Lewy bodies. For example, Lewy bodies have been
found in the brain stem of a patient with rapid eye movement sleep
behavioral disorder (Uchiyama et al., Neurology (1995) 45:709-712).
Olfactory dysfunction has been reported in many PD patients long
before the development of parkinsonism. Examination of cardiac
tissue from patients with incidental Lewy body disease and typical
PD revealed synuclein-positive neuritis in the myocardium (Iwanaga
et al., Neurology (1999) 52:1269-1271). There is also evidence that
esophageal, lower bowel and bladder dysfunction are early
manifestations of PD-related pathology in the peripheral autonomic
system (Qualman et al., Gastroenterology (1984) 87:848-856; Castell
et al., Neurogasdtroenterol Motil (2001) 13:361-364; Hague et al.,
Acta Neuropathol (Berl) (1997) 94:192-196). Thus, the fact that
accumulation of alpha-synuclein in the brain and other tissues is
associated with similar morphological and neurological alterations
in species as diverse as humans, mice, and flies suggests that this
molecule contributes to the development of PD.
BRIEF SUMMARY OF THE INVENTION
[0005] In one aspect, the invention provides methods of screening
an agent for activity for treating a Lewy Body disease (LBD). Such
diseases include Parkinson's disease (PD), Diffuse Lewy body
disease (DLBD), Lewy body variant of Alzheimer's disease (LBV),
combined PD and Alzheimer's disease (AD), and the syndromes
identified as multiple system atrophy (MSA). Some methods entail
identifying an agent that modulates the activity or expression of a
kinase shown in Table 1A, B; C, Table 2, Table 11 or Table 12, and
determining whether the agent shows activity useful in treating LBD
in an animal model of the disease. In some methods the modulation
is inhibition. In some methods, step (a) involves identifying
whether the agent inhibits the kinase. In some methods, step (a) is
performed in a cell transformed with a nucleic acid expressing the
kinase and/or alpha-synuclein. In some methods, step (a) is
performed in vitro. In some methods, step (b) is performed in a
transgenic animal model of LBD disease, and the transgenic animal
may have a transgene expressing human alpha-synuclein. Preferably,
the kinase is at least one of: APEG1, PLK2, CDC7L1, PRKG1, MAPK13,
GAK, RHOK, ADRBK1, ADRBK2, GRK2L, GRK5, GRK6, GRK7, IKBKB, CKII and
MET and the modulation is inhibition. More preferably, the kinase
is PLK2 or GRK6 and the modulation is inhibition. More preferably,
the kinase is PLK2. Preferably in some methods, the kinase is
PRKG1, MAPK13, or GAK and the modulation is activation. In some
aspects, step (b) involves contacting the transgenic animal with
the agent and determining whether the agent inhibits formation of
deposits of alpha-synuclein relative to a control transgenic animal
not treated with the agent.
[0006] In another aspect, the invention provides methods of
effecting treatment or prophylaxis of a LBD. Some examples of the
method involve administering to a patient suffering from or at risk
of the disease, an effective regime of an agent effective to
modulate activity or expression of a kinase. The kinase can be one
of those shown in Table 1A, B or C, Table 2, Table 11 or Table 12.
Preferably, the agent is an antibody to the kinase, a zinc finger
protein that modulates expression of the kinase, or an antisense
RNA, siRNA, ribozyme or RNA having a sequence complementary to a
nucleic acid sequence of the kinase. In some methods, the
modulation is inhibition, and preferably, the kinase is at least
one of the following: APEG1, PLK2, CDC7L1, RHOK, ADRBK1, ADRBK2,
GRK2L, GRK5, GRK6, GRK7, IKBKB, CKII and MET. More preferably, the
kinase is PLK2 or GRK6. More preferably, the kinase is PLK2. In
some of the methods, the kinase is at least one of: PRKG1, MAPK13,
and GAK, and the modulation is activation.
[0007] In one aspect the invention provides method of treating a
patient diagnosed with Parkinson's Disease by administering a
therapeutically effective amount of an agent that inhibits PLK2
activity. In an embodiment the agent preferentially inhibits PLK2
activity relative to inhibition of PLK1 activity and/or PLK3
activity and/or PLK4 activity. The agent may be, for example, an
siRNA. In one embodiment the patient is not diagnosed or under
treatment for cancer and/or is not diagnosed or under treatment for
Alzheimer's disease.
[0008] In related aspects, the invention provides a method for
inhibiting phosphorylation of alpha-synuclein in a mammalian cell
by reducing polo-like kinase 2 (PLK2) activity in the cell such
that phosphorylation of synuclein is reduced. In a related aspect,
the invention provides a method for inhibiting phosphorylation of
alpha-synuclein in a mammalian cell (e.g., a neuronal cell) by
contacting the cell with a compound that reduces PLK2 activity in
the cell such that phosphorylation of alpha-synuclein is reduced.
For example, the agent may reduce expression of a PLK2 gene
product.
[0009] In certain embodiments the agent preferentially reduces PLK2
activity relative to reduction of PLK1 activity, PLK2 activity, or
PLK3 activity. In certain embodiments the agent has a molecular
weight less than 4000. In one embodiment the agent is a synthetic
compound. In some embodiments the agent is a polynucleotide that
inhibits expression or translation of a PLK2 RNA transcript, such
as an siRNA. In an embodiment, one strand of the double stranded
region of the siRNA is perfectly complementary to a PLK2 transcript
but not to a PLK1 transcript or a PLK3 transcript.
[0010] In another aspect, the invention provides methods of
identifying a kinase that phosphorylates alpha-synuclein by
transfecting a cell expressing alpha-synuclein with a nucleic acid
having a sequence complementary to a gene encoding a kinase or zinc
finger protein that specifically binds to the gene. The transfected
nucleic acid or zinc finger protein inhibits expression of the
kinase; and an amount of phosphorylated alpha-synuclein the cell
can then be measured relative to a control cell not transfected
with the siRNA or nucleic acid encoding the same. In this case, a
reduction in phosphorylated alpha-synuclein will provide an
indication that the kinase phosphorylates alpha-synuclein. Some
methods also include measuring an amount of alpha-synuclein
produced by the cell relative to a control cell not transfected
with the nucleic acid. In some methods, the nucleic acid is an
siRNA or a DNA molecule encoding the same.
[0011] The invention provides a method of identifying an agent
reduces alpha-synuclein phosphorylation in a mammalian cell
expressing alpha-synuclein. The method includes selecting an agent
that a) reduces activity of PLK2 in a cell expressing PLK2 (and
optionally expressing synuclein), and b) does not reduce activity
of PLK1 in a cell expressing PLK1, or reduces activity of PLK1 at a
higher EC.sub.50 than for PLK2; and/or c) does not reduce activity
of PLK3 in a cell expressing PLK3, or reduces activity of PLK3 at a
higher EC.sub.50 than for PLK2; and/or d) does not reduce activity
of PLK4 in a cell expressing PLK4, or reduces activity of PLK4 at a
higher EC.sub.50 than for PLK2. The cell can be a mammalian cell
overexpressing alpha-synuclein. In one embodiment the agent a)
reduces activity of PLK2 in a cell expressing PLK2; b) does not
reduce activity of PLK1 in a cell expressing PLK1, or reduces
activity of PLK1 at a higher EC.sub.50 than for PLK2; c) does not
reduce activity of PLK3 in a cell expressing PLK3, or reduces
activity of PLK3 at a higher EC.sub.50 than for PLK2; and d) does
not reduce activity of PLK4 in a cell expressing PLK4, or reduces
activity of PLK4 at a higher EC.sub.50 than for PLK2. In a further
step, the method involves determining whether the selected agent
shows activity useful in treating Lewy Body Disease in an animal
model of the disease or a cellular model of the disease. Animal
models include transgenic animals. Cellular models include
neuronally-derived cell cultures and mammalian cells
over-expressing alpha-synuclein. Activities that can be assayed
include reduction of the proportion of total alpha-synuclein that
is phosphorylated at serine-129 or a reduction in aggregation of
alpha-synuclein in the cell.
[0012] In other aspects, the invention provides methods of method
of screening an agent for activity for treating a Lewy Body disease
(LBD), by identifying an agent that modulates the activity or
expression of synphilin, and determining whether the agent shows
activity useful in treating LBD in an animal model of the
disease.
[0013] In other aspects, the invention provides methods for
producing Ser-129 phosphorylated-alpha synuclein, by providing a
plasmid encoding alpha-synuclein and a plasmid encoding PLK2 in a
bacterial cell, culturing the cell so that the plasmids are
co-expressing to produce alpha synuclein and PLK2 so that the PLK2
phosphorylates the alpha-synuclein in a bacterial cell, and
isolating phosphorylated alpha-synuclein from the cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-C show the results of the in vitro phosphorylation
assay for alpha-synuclein phosphorylation by a variety of
recombinant kinases. FIG. 1A shows total alpha-synuclein, FIG. 1B
shows phosphorylation of the pser-129 (phospho-serine-129) of
alpha-synuclein and FIG. 1C shows phosphorylation of the pser-87
(phospho-serine-87) of alpha-synuclein.
[0015] FIGS. 1D-F show a study with recombinant kinases, including
kinases from the GPCR-receptor kinase (GRK) family and PLK2. FIG.
1D shows total alpha-synuclein, FIG. 1E shows phosphorylation of
the pser-129 of alpha-synuclein and FIG. 1F shows phosphorylation
of the pser-87 of alpha-synuclein.
[0016] FIGS. 2A and B show the results of kinase activity in vitro
for various kinases.
[0017] FIG. 2A shows the total (AS). FIG. 2B shows phosphor-serine
129.
[0018] FIGS. 3A-C show the results of kinase activity in vitro for
various kinases. FIG. 2A shows the total AS. FIG. 3B shows Serine
129. FIG. 3C shows phospho-serine 87.
[0019] FIGS. 4A and B show the effect of phospholipid on the assay
results in FIGS. 3A and 3B. FIG. 4A shows the total AS. FIG. 4B
shows Serine 129.
[0020] FIG. 5 shows the results of transfection of cDNA to PLK2
into 293-synuclein cells. Cells were analyzed by ELISA for total
and phospho-synuclein levels.
[0021] FIG. 6 shows the results of transfection of cDNA to GPRK6
and PLK2 into HEK-Synuclein cells.
[0022] FIG. 7 shows that knockdown of the PLK2 using siRNA from a
second source causes a reduction in the proportion of
alph-synuclein that is phosphorylated.
[0023] FIGS. 8A and 8B show the in vitro phosphorylation of
alpha-synuclein by putative kinase targets in alpha-synuclein KO
mouse brain.
[0024] FIGS. 9A and 9B show the in vitro phosphorylation of
alpha-synuclein by putative kinase targets in alpha-synuclein KO
mouse brain.
[0025] FIG. 10 shows that siRNA knockdown of PLK2, but not PLK3 or
PLK4, reduced alpha-synuclein phosphorylation.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0026] The term "agent" is used to describe a compound that has or
may have a pharmacological activity. Agents include compounds that
are known drugs, compounds for which pharmacological activity has
been identified but which are undergoing further therapeutic
evaluation, and compounds that are members of collections and
libraries that are to be screened for a pharmacological
activity.
[0027] A "pharmacological" activity means that an agent exhibits an
activity in a screening system that indicates that the agent is or
may be useful in the prophylaxis or treatment of a disease. The
screening system can be in vitro, cellular, animal or human. Agents
can be described as having pharmacological activity notwithstanding
that further testing may be required to establish actual
prophylactic or therapeutic utility in treatment of a disease.
[0028] A Lewy-like body is a deposit of alpha-synuclein found in a
transgenic animal that resembles some or all of the characteristics
of a Lewy body found in human patients. The preferred
characteristics are a compact alpha-synuclein positive inclusion.
These inclusions preferably form in an age-dependent manner. The
formation of alpha-synuclein positive inclusions preferably results
in observable cellular pathology, leading to loss of functionality
of affected neurons. Loss of function of affected neurons can be
determined through behavioral tests, neuropharmacological response
evaluation and electrophysiology.
[0029] The phrase "specifically binds" refers to a binding reaction
which is determinative of the presence of the protein in the
presence of a heterogeneous population of proteins and other
biologics. Thus, under designated conditions, a specified ligand
binds preferentially to a particular protein and does not bind in a
significant amount to other proteins present in the sample. A
molecule such as an antibody that specifically binds to a protein
often has an association constant of at least 10.sup.6 M.sup.-1 or
10.sup.7 M.sup.-1, preferably 10.sup.8 M.sup.-1 to 10.sup.9
M.sup.-1, and more preferably, about 10.sup.10 M.sup.-1 to
10.sup.11 M.sup.-1 or higher. A variety of immunoassay formats may
be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immunoreactive with a protein. See, e.g., Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for examples of immunoassay formats and conditions that
can be used to determine specific immunoreactivity.
[0030] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequent coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0031] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., supra).
[0032] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al., J. Mol.
Biol. 215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al, supra.).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. For identifying whether a nucleic acid or polypeptide
is within the scope of the invention, the default parameters of the
BLAST programs are suitable. The BLASTN program (for nucleotide
sequences) uses as defaults a word length (W) of 11, an expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a word length
(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring
matrix. The TBLATN program (using protein sequence for nucleotide
sequence) uses as defaults a word length (W) of 3, an expectation
(E) of 10, and a BLOSUM 62 scoring matrix. (see Henikoff &
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
[0033] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0034] For purposes of classifying amino acids substitutions as
conservative or non-conservative, amino acids are grouped as
follows: Group I (hydrophobic side chains): norleucine, met, ala,
val, leu, ile; Group II (neutral hydrophilic side chains): cys,
ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic
side chains): asn, gln, his, lys, arg; Group V (residues
influencing chain orientation): gly, pro; and Group VI (aromatic
side chains): trp, tyr, phe. Conservative substitutions involve
substitutions between amino acids in the same class.
Non-conservative substitutions constitute exchanging a member of
one of these classes for a member of another.
[0035] Therapeutic agents of the invention are typically
substantially pure from undesired contaminant. This means that an
agent is typically at least about 50% w/w (weight/weight) purity,
as well as being substantially free from interfering proteins and
contaminants. Sometimes the agents are at least about 80% w/w and,
more preferably at least about 90%, at least about 95%, or at least
about 99% w/w purity. However, using conventional protein
purification techniques, homogeneous peptides of at least 99% w/w
can be obtained.
[0036] The term "antibody" or "immunoglobulin" is used to include
intact antibodies and binding fragments thereof. Typically,
fragments compete with the intact antibody from which they were
derived for specific binding to an antigen fragment including
separate heavy chains, light chains Fab, Fab'F(ab')2, Fabc, and Fv.
Fragments are produced by recombinant DNA techniques, or by
enzymatic or chemical separation of intact immunoglobulins. The
term "antibody" also includes one or more immunoglobulin chains
that are chemically conjugated to, or expressed as, fusion proteins
with other proteins. The term "antibody" also includes bispecific
antibody. A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0037] A symptom of a disorder means a phenomenon experienced by an
individual having the disorder indicating a departure from normal
function, sensation or appearance.
[0038] A sign of a disorder is any bodily manifestation that serves
to indicate presence or risk of a disorder.
[0039] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0040] As used herein, "treating" a condition (e.g., Parkinson's
Disease) or patient refers to taking steps to obtain beneficial or
desired result. For purposes of this invention, beneficial or
desired results include, but are not limited to, alleviation or
amelioration of one or more symptoms of Parkinson's Disease,
diminishment of extent of disease, delay or slowing of disease
progression, amelioration, palliation or stabilization of the
disease state.
[0041] As used herein, a "therapeutically effective amount" of a
drug is an amount of a drug that, when administered to a subject
diagnosed with Parkinson's disease, or diagnosed as being at high
risk for developing Parkinson's disease will have the intended
therapeutic effect, e.g., alleviation, amelioration, palliation or
elimination of one or more manifestations of the disease in the
subject. The full therapeutic effect does not necessarily occur by
administration of one dose, and may occur only after administration
of a series of doses. Thus, a therapeutically effective amount may
be administered in one or more administrations.
[0042] Compositions or methods "comprising" one or more recited
elements may include other elements not specifically recited. For
example, a composition that comprises alpha-synuclein peptide
encompasses both an isolated alpha-synuclein peptide and
alpha-synuclein peptide as a component of a larger polypeptide
sequence.
[0043] Unless otherwise apparent from the context, each embodiment,
element, step or feature of the invention can be used in
combination with any other.
II. General
[0044] The invention is premised in part on the insight that Lewy
Body diseases (LBDs) can be inhibited by inhibiting one or more
kinases that phosphorylate alpha-synuclein and/or inhibit its
production. Although practice of the invention is not dependent on
an understanding of mechanism, it is believed that phosphorylation
of alpha-synuclein at serine-129 is one of a series of molecular
events leading to formation of intracellular deposits of
alpha-synuclein. Alpha-synuclein phosphorylated at ser-129 is
highly enriched in Lewy bodies (LBs) in Diffuse Lewy body disease
(DLBD), multiple system atrophy (MSA) and familial forms of
Parkinson's Disease (PD). The abnormal accumulation of
phospho-alpha-synuclein in LBs indicates that phospho-synuclein may
be a pathogenic species that drives LB formation, and that the
kinase(s) responsible for its phosphorylation or which regulates
production of alpha-synuclein itself are therapeutic target(s) for
treatment of multiple synucleinopathies. Other events in this
series likely include proteolytic cleavages following
phosphorylation (see WO 2005/013889, filed May 19, 2004).
[0045] Identification of the kinase(s) primarily responsible for
phosphorylation of alpha-synuclein allows compounds that reduce
activity of the relevant kinase(s) to be identified. For
convenience, reference herein to "phosphorylation of
alpha-synuclein" refers to phosphorylation at Serine-129 (but does
not exclude additional phosphorylation elsewhere, e.g.,
Serine-87).
[0046] The present application reports identification of several
kinases where reduction in kinase activity is accompanied by a
reduction of phosphorylation of alpha-synuclein and/or a reduction
in total alpha-synuclein level. In particular, the kinase PLK2 can
be inhibited to reduce phosphorylation of alpha-synuclein. The
invention provides methods of (i) identifying modulators of the
activity and expression of these kinases, (ii) methods of treating
Lewy body diseases using kinase inhibitors, and (iii) exemplary
kinase inhibitors for use in treating Lewy body diseases.
[0047] As discussed in the Examples, infra, we have carried out a
variety of experiments to identify kinases important in
phosphorylation of alpha-synuclein. Section III outlines a strategy
for identifying alpha-synuclein kinases. Section IV summarizes
results of screening assays used to identify likely alpha-synuclein
kinases. Section V describes agents that reduce synuclein kinase
activity or expression and may be used therapeutically. Section VI
describes methods for treating Parkinson's Disease and other Lewy
Body Diseases. Section VII describes Lewy Body Diseases. Section
VIII describes transgenic animal and cellular models of Lewy Body
Disease. Section IX describes method for identification of
modulators of PLK2 and other kinases. Section X describes methods
for alpha-synuclein isolation. Section XI provides experimental
results including the aforementioned screening assays.
III. Identification of Target Kinases
[0048] Kinases that directly or indirectly modulate phosphorylation
of alpha-synuclein can be identified as shown in the Examples. In
general, a library of potential inhibitors is designed based on the
known sequences of a collection of kinase genes. The members of the
library can be any of the types of molecule described above.
Members of the library are then introduced into cells expressing
alpha-synuclein. Preferably both the cells and the alpha-synuclein
are human. Usually, such cells are transfected with both DNA
encoding human alpha-synuclein and DNA encoding the library member
to be tested. Library members can be screened individually or en
masse. After introduction of a library member, and culturing for a
period sufficient for the library member to be expressed and effect
repression of its kinase, the levels of total alpha-synuclein and
phosphorylated alpha-synuclein are measured and compared with
corresponding levels in an otherwise similar control cell not
treated with a library member to suppress expression of a kinase.
Measurements can be made by immunoassay using an antibody specific
for alpha-synuclein (preferably human alpha-synuclein) to measure
total levels of alpha-synuclein, and an antibody specific for
phosphorylated alpha-synuclein to measure the level of
phosphorylated alpha-synuclein. Exemplary antibodies are described
in WO05047860, incorporated herein by reference. A reduction in
level of phosphorylated alpha-synuclein between the treated and
control cell that is significant in the sense of being outside the
typical margin of error for measurements, indicates that the
inhibitor introduced into the cell inhibited a kinase, which
directly or indirectly affected phosphorylation of alpha-synuclein.
The identity of the kinase can be determined from the identity of
inhibitor, either by screening inhibitors individually, or if
inhibitors are screened en masse, by sequencing the nucleic acid
encoding the inhibitor. Likewise a reduction in the total level of
alpha-synuclein between treated and control cells that is outside
the margin of typical experimental error in measuring such levels
provides an indication that the inhibitor inhibits a kinase that
indirectly affects the expression level of alpha-synuclein.
[0049] Kinases identified by the initial screen, particularly,
kinases known to be serine kinases, can then be tested for their
capacity to phosphorylate alpha-synuclein in vitro, in cells or in
transgenic animal models. An in vitro assay is an indication of
whether a kinase directly phosphorylates alpha-synuclein and is
therefore only useful for the kinases identified in the initial
screen which are thought to be capable of directly phosphorylating
alpha-synuclein. Cellular and transgenic assays can be used to
screen kinases that affect phosphorylation either directly or
indirectly. In vitro assays may be performed by contacting
alpha-synuclein with the kinase under test and ATP in a suitable
buffer. Preferably, the ATP is .gamma.-32P ATP, in which case
phosphorylated alpha-synuclein is radiolabeled and can be detected
on a gel. Phosphorylation can also be measured using an antibody
specific to phosphorylated alpha-synuclein as described before.
Alternatively, phosphorylation can be measured indirectly by
measuring ATP consumption using a coupled assay, in which ADP is
detected as described for example by Nature 78, 632 (1956); Mol.
Pharmacol. 6, 31-40 (1970). The extent of phosphorylation can be
compared with a control in which the kinase or ATP or both is/are
omitted. An increase in phosphorylation is an indication that the
kinase directly phosphorylates alpha-synuclein. Cellular assays are
performed on cells expressing alpha-synuclein, preferably human
alpha-synuclein transfected into the cells. A nucleic acid capable
of expressing the kinase is also transfected into the cells. The
level of phosphorylated alpha-synuclein in the cells is measured
relative to that in similar control cells lacking the transfected
kinase. An increase in phosphorylation is an indication that the
kinase directly or indirectly phosphorylates alpha-synuclein.
Transgenic assays can be performed by comparing a transgenic animal
expressing human alpha-synuclein disposed to develop Lewy body-like
deposits with a similar animal also expressing a kinase transgene.
A reduction in phosphorylated alpha-synuclein and/or in Lewy
body-like deposits in the transgenic animal with the additional
kinase transgene relative to the transgenic animal with just the
alpha-synuclein transgene is an indication that the kinase is
directly or indirectly involved in phosphorylating
alpha-synuclein.
IV. Target Kinases
[0050] Tables 1A, 1B and 1C show proteins whose inhibition
modulates the phosphorylation at position ser-129. Table 1A shows
kinases that can phosphorylate serine and/or threonine residues and
sometimes tyrosine. Table 1B shows tyrosine kinases that cannot (so
far as is known) modify serine residues. Table 1C shows kinases
that phosphorylate non-protein targets but are not known to
phosphorylate proteins. Kinases from the upper portion of Table 1A
are candidates for direct phosphorylation of ser-129 of
alpha-synuclein. Kinases from the upper part of Table 1B are also
useful therapeutic targets via roles indirectly phosphorylating
alpha-synuclein. Proteins in the upper part of Table 1C are also
useful therapeutic targets for the same reason. Cols. 1, 2 and 3 of
each table indicate the gene name, kinase name and Genbank
accession number of kinases. The next column indicates whether
treatment of cells with siRNA to that kinase decreased ("down") or
increased ("up") phosphorylation of ser-129. The next three columns
indicate the number of standard deviations the measured level of
phosphorylation departs from the mean in three independent
experiments. The final two columns indicate the kinase family
(i.e., amino acid specificity) and group.
[0051] Table 2 shows kinases whose inhibition modulates the overall
levels of human alpha-synuclein without changing the percentage of
phosphorylation. Table 2 shows all of the kinases with the
strongest reduction in levels of human alpha-synuclein. The columns
are labeled similarly to Tables 1A, 1B and 1C.
[0052] Tables 3 and 4 show kinases from Tables 1 and 2 that were
confirmed in the Examples to modulate overall levels of human
alpha-synuclein. The kinases that were verified include PLK2, APEG
1, CDC7L1, MET, GRK1, 2, 6, and 7 as kinases that phosphorylate
alpha-synuclein directly or indirectly. The kinases that were found
to increase alpha-synuclein phosphorylation when inhibited, PRKG1,
MAPK13, and GAK, are likely to function as negative regulators of
alpha-synuclein phosphorylation. Further data from phosphorylation
studies in vitro identified PKL2, GRK2, 5, 6, and 7 as capable of
phosphorylating alpha-synuclein in vitro and also identified CKII
and IKBKB. Further studies in cell culture showed that PLK2 and
GPRK6 could directly phosphorylate alpha-synuclein in cell culture.
These data were substantiated with immunohistochemistry. In
summary, PLK2 and, to a lesser extent, GRK6 are particularly
preferred targets for therapeutic intervention in Lewy body
diseases because they can directly phosphorylate alpha-synuclein.
Agents that inhibit PLK2 and GRK6 also inhibit phosphorylation of
alpha-synuclein and thus can be used in treatment or prophylaxis of
Lewy body disease.
[0053] In the Examples, below, transfection of cells with siRNA and
knockdown of specific kinase targets was employed to identify
kinases that modulated alpha-synuclein phosphorylation directly or
indirectly. Subsequent experiments in vitro and in cell culture
showed that two of these kinases, PLK2 directly and specifically
phosphorylated the serine 129 of alpha-synuclein. Further
experiments showed that PLK2 phosphorylated the serine 129 of
alpha-synuclein to a much greater extent than GRK6 and other
kinases described herein under the experimental conditions used.
Thus, PLK2 is very likely a synuclein kinase. Additional evidence
that PLK2 is a synuclein kinase is provided in Examples 11-16.
Synuclein phosphorylation is reduced in cells treated with siRNA
directed to PLK2, inhibitors of PLK2 activity reduce synuclein
phosphorylation in a variety of cell types including primary
neuronal cultures and cells over expressing PLK2, inhibitors affect
endogenous kinase in with an EC.sub.50 consistent with the
EC.sub.50 observed for their effect on PLK2.
[0054] PLK2 is a Polo like kinase that is a G1 cell cycle protein,
has a rapid turnover in cells, and is expressed in brain where it
is involved in synaptic plasticity. The PLK family members are
serine/threonine kinases, and contains four members that have an
N-terminal kinase domain and a C-terminal regulatory domain
consisting of two (PLKs 1-3) or one (PLK4) polo-box domains. The
polo-box domain serves to bind to scaffolding proteins that then
target the PLKs to specific sub-cellular locations and to
phosphorylate their target proteins (Seeburg, D. P. et al,
Oncogene, 2005). The polo-box also serves to negatively regulate
the kinase domain by adopting a conformation that prevents kinase
activity. Upon binding of the polo-box to a scaffolding protein,
the polo-box is removed from the kinase domain, whereupon the
kinase becomes active and is able to phosphorylate its substrate/s.
Polo-like kinases are described in Seeburg et al., 2005, "Polo-like
kinases in the nervous system" Oncogene 24:292-8; Lowery et al.,
2005, "Structure and function of Polo-like kinases" Oncogene
24:248-59; and Winkles et al., 2005, "Differential regulation of
polo-like kinase 1, 2, 3, and 4 gene expression in mammalian cells
and tissues" Oncogene 24:260-6. DNA and protein sequences can be
found at the accession numbers below:
TABLE-US-00001 GenBank Accession Entrez UniProt Kinase number Gene
ID ID PLK1 NM_005030 5347 P53350 PLK2 NM_006622 10769 Q9NYY3 PLK3
AJ293866, NM_004073 1263 Q9H4B4 PLK4 Y13115, NM_014264 10733
O00444
[0055] When PLK2 is activated, it is targeted to dendrites of
activated neurons, where it is believed to phosphorylate proteins
in the synaptic terminals. An exemplary accession number for PLK2
is provided Table 1A. The sequence for PLK2 can also be found in
any one of Ma, et al. Mol. Cell. Biol. 23 (19), 6936-6943 (2003),
Burns, et al. Mol. Cell. Biol. 23 (16), 5556-5571 (2003), Matsuda,
et al. Oncogene 22 (21), 3307-3318 (2003), Shimizu-Yoshida et al.
Biochem. Biophys. Res. Commun. 289 (2), 491-498 (2001), Liby, et
al. DNA seq. 11 (6), 527-533 (2001), Holtrich, et al. Oncogene 19
(42), 4832-4839, Ouyang, et al. Oncogene 18 (44), 6029-6036 (1999),
and Kauselmann, et al. EMBO J. 18 (20), 5528-5539; reference to an
amino acid or nucleic acid sequence of PLK2 includes the sequences
of any of these references or allelic variants thereof. PLK2 is
also called SNK; for consistency, the name PLK2 is used throughout
the present patent application.
[0056] APEG1, CDC7L1, MET, IKBKB, CKII, GRK1, GRK2, GRK6 and GRK7
are also targets for therapeutic intervention in Lewy body diseases
because they are likely to be indirect activators of the direct
kinase(s). Thus, agents that inhibit APEG1, CDC7L1, MET, IKBKB,
CKII, GRK1, GRK2, GRK6 and GRK7 also inhibit phosphorylation of
alpha-synuclein and can be used for treatment or prophylaxis of
Lewy body disease. PRKG1, MAPK13, and GAK are negative regulators
of the phosphorylation of alpha-synuclein. Thus, agents that
activate these kinases decrease phosphorylation of alpha-synuclein
and can be used in treatment or prophylaxis of Lewy body
disease.
[0057] GRK6, also called GPRK6, is a G protein-coupled receptor
kinase and is involved in signal transduction. G protein-coupled
receptor kinases phosphorylate and desensitize ligand-activated G
protein-coupled receptors. GRK6 expression has previously been
shown to be significantly elevated in the MPTP-lesioned group in
most brain regions. For the purposes of consistency, the name GRK6
will be used throughout the present patent application. An
exemplary accession number is provided in Table 1A. The sequence
for GRK6 can be found in any one of Teli, et al., Anesthesiology 98
(2), 343-348 (2003); Miyagawa, et al., Biophys. Res. Commun. 300
(3), 669-673 (2003); Gaudreau, et al., J. Biol. Chem. 277 (35),
31567-31576 (2002); Grange-Midroit, et al., Brain Res. Mol Brain
Res. 101 (1-2), 39-51 (2002); Willets, et al., J. Biol. Chem. 277
(18), 15523-15529 (2002); Blaukat, et al., J. Biol. Chem. 276 (44),
40431-40440 (2001); Zhou, et al., J. Pharmacol. Exp. Ther. 298 (3),
1243-1251 (2001); Pronin, et al., J. Biol. Chem. 275 (34),
26515-26522 (2000); Tiruppathi, Proc. Natl. Acad. Sci. U.S.A., 97
(13), 7440-7445 (2000); Premont, et al. J. Biol. Chem., 274 (41),
29381-29389 (1999); Brenninkmeijer, et al., J. Endocrinol. 162 (3),
401-408 (1999); Hall, et al., J. Biol. Chem. 274 (34), 24328-24334
(1999); Lazari, et al., Mol. Endocrinol. 13 (6), 866-878 (1999);
Milcent, et al., Biochem. Biophys. Res. Commun. 259 (1), 224-229
(1999); Premont, Proc. Natl. Acad. Sci. U.S.A. 95 (24), 14082-14087
(1998); Stoffel, et al., Biochemistry 37 (46), 16053-16059 (1998);
Loudon, et al., J. Biol. Chem. 272 (43), 27422-27427 (1997);
Freedman, et al., J. Biol. Chem. 272 (28), 17734-17743 (1997);
Bullrich, et al., Cytogenet. Cell Genet. 70 (3-4), 250-254 (1995);
Stoffel, et al.; J. Biol. Chem. 269 (45), 27791-27794 (1994);
Loudon, et al., J. Biol. Chem. 269 (36), 22691-22697 (1994);
Haribabu and Snyderman, Proc. Natl. Acad. Sci. U.S.A. 90 (20),
9398-9402 (1993); and Benovic and Gomez, J. Biol. Chem. 268 (26),
19521-19527 (1993); reference to the amino acid or nucleic acid
sequence of GRK6 includes the amino acid or nucleic acid sequence
of any of these references and allelic variants thereof.
[0058] Casein kinase 2 (also called Casein kinase II, CKII, CSNK2
and CSNKII) has been reported to phosphorylate alpha-synuclein. For
consistency, the name CKII will be used herein. The sequence for
CKII has been provided in Genbank under the following accession
numbers: NM.sub.--001896, NM.sub.--001320 and/or can be found in
any one of: Panasyu, et al. J. Biol. Chem. 281 (42), 31188-31201
(2006); Salvi, et al. FEBS Lett. 580 (16), 3948-3952 (2006); Lim et
al. Cell 125 (4), 801-814 (2006); Llorens, et al. Biochem. J. 394
(Pt. 1), 227-236, (2006); Bjorling-Poulsen, et al. Oncogene 24
(40), 6194-6200 (2005); Schubert, et al. Eur. J. Biochem. 204 (2),
875-883 (1991); Voss, et al. J. Biol. Chem. 266 (21), 13706-13711
(1991); Yang-Feng, et al. Genomics 8 (4), 741-742 (1990);
Heller-Harrison, et al. Biochemistry 28 (23), 9053-9058 (1989);
Ackermann, et al. Mol. Cell. Biochem. 274 (1-2), 91-101 (2005);
Barrios-Rodiles, et al. Science 307 (5715), 1621-1625 (2005);
Andersen, et al. Nature 433 (7021), 77-83 (2005); Ballif, et al.
Mol. Cell. Proteomics, 3 (11), 1093-1101 (2004); Beausoleil, et al.
PNAS, USA 101 (33), 12130-12135 (2004); Marais, et al. EMBO J. 11
(1), 97-105 (1992). Reference to the amino acid or nucleic acid
sequence of CKII includes the amino acid or nucleic acid sequence
of any of these references and allelic variants thereof.
[0059] IKBKB and the related IKBKA are positive regulators of the
NFkB inflammatory pathway. The sequence for IKBKB has been provided
in Genbank under the following accession number: NM.sub.--001556
and/or can be found in any one of: Caterino, et al. FEBS Lett. 580
(28-29), 6527-6532 (2006); Castle, et al. Genome Biol. 4 (10), R66
(2003); Satoh, et al. Biochim, Biophys. Acta 1600 (103), 61-67
(2002), Caohuy, and Pollard, J. Biol. Chem. 277 (28), 25217-25225
(2002); Yu, et al. J. Biol. Chem. 277 (18), 15819-15827 (2002);
Selbert, et al. J. Cell. Sci. 108 (Pt.1), 85095 (1995); Shirvan, et
al. Biochemistry 33 (22), 6888-6901 (1994); Creutz, et al. Biochem.
Biophys. Res. Commun. 184 (1), 347-352 (1992); Megendzo, et al. J.
Biol. Chem. 266 (5), 3228-3232 (1991); Burns, et al. PNAS, USA 86
(10), 3798-3802 (1989). Reference to the amino acid or nucleic acid
sequence of IKBKB includes the amino acid or nucleic acid sequence
of any of these references and allelic variants thereof.
[0060] Synphilin is a synuclein-associated protein that has been
shown to bind alpha-synuclein. Although not a kinase itself,
Synphilin was found herein to promote phosphorylation of synuclein
particularly in combination with PLK2. The synphilin appeared to
promote phosphorylation of synuclein in a PLK2-dependent manner.
The sequence for synphilin has been provided in Genbank under the
following accession number: NM.sub.--005460 and/or can be found in
any one of: Tanji, et al. Am. J. Pathol. 169 (2), 553-565 (2006);
Eyal, et al. PNAS, USA 103 (15), 5917-5922 (2006); Avraham, et al.
J. Biol. Chem. 280 (52), 42877-42886 (2005); Bandopadhyay, et al.
Neurobiol. Dis. 20 (2), 401-411 (2005); Lim et al. J. Neurosci. 25
(8), 2002-2009 (2005); Ribeiro, et al. J. Biol. Chem. 277 (26),
23927-23933 (2002); Chung, et al. Nat. Med. 7 (10), 1144-1150
(2001); Engelender, et al. Mamm. Genome 11 (9), 763-766 (2000);
Engelender, et al. Nat. Genet. 22 (1), 110-114 (1999). Reference to
the amino acid or nucleic acid sequence of synphilin includes the
amino acid or nucleic acid sequence of any of these references and
allelic variants thereof
V. Agents to Modulate Synuclein Kinase Activity or Expression
[0061] In one aspect, the invention provides methods of effecting
treatment or prophylaxis of an LBD by administering an agent that
modulates activity or expression of a kinase described herein. A
number of agents of well-characterized general classes can be used.
Without limitation these include inhibitory nucleic acids (e.g.,
siRNA, antisense RNA, ribozymes), inhibitory proteins (e.g., zinc
finger proteins), antibodies, and small molecule inhibitors.
[0062] Preferably the gene to be inhibited is PLK2 or GRK6 because
the kinases encoded by these genes directly phosphorylate
alpha-synuclein, and particularly PLK2. APEG1, CDC7L1, MET GRK1,
GRK2, GRK6, IKBKB, CKII and GRK7 genes are also preferred targets
for inhibition because they are likely to encode indirect
activators of the direct kinase(s). PRKG1, MAPK13, and GAK are
preferred candidates for activation in Lewy body diseases because
they encode negative regulators of the phosphorylation of
alpha-synuclein. The synphilin gene is a preferred target for
inhibition because, although synphilin is not a kinase, it is
associated with increased phosphorylation of alpha-synuclein
(typically in the presence of a kinase such as PLK2).
[0063] Inhibitors that show specificity for PLK2 over one or more
other polo-like kinase family members (i.e., PLK1, PLK3, and PLK4)
are preferred. Inhibitors especially suited for therapeutic use may
be identified by selecting for at least one of the following
properties: [0064] I. Inhibits PLK2 activity and has no, or
reduced, effect on PLK1. [0065] II. Inhibits PLK2 activity and has
no, or reduced, effect on PLK3. [0066] III. Inhibits PLK2 activity
and has no, or reduced, effect on PLK4. [0067] IV. Inhibits PLK2
activity and has no, or reduced, effect on PLK1 and PLK3. [0068] V.
Inhibits PLK2 activity and has no, or reduced, effect on PLK1 and
PLK4. [0069] VI. Inhibits PLK2 activity and has no, or reduced,
effect on PLK1, PLK3 and PLK4.
[0070] As used in this context, "no effect" means administration of
the agent does not reduce expression, or reduces expression by a
physiologically insignificant degree. "Reduced effect" means that
the EC.sub.50 or K.sub.i values for inhibiting PLK2 is lower than
the EC.sub.50 for the reference PLK(s). In some embodiments the
EC.sub.50 may be at least 2-fold lower, and is sometimes at least
10-fold lower, and may be at least 100-fold, or even at least
1000-fold lower.
[0071] As used in this context, inhibition of PLK2 "activity" can
result from reducing protein expression (e.g., reducing expression
of the PLK2 gene, interfering with processing of PLK2 RNA, reducing
the half-life of PLK2 mRNA or protein) or by competitive or
noncompetitive inhibition of the PLK2 kinase activity.
[0072] Inhibitors that show specificity for PLK2 over non-polo
kinases, especially other kinases expressed in the tissues to which
the agent is delivered are especially preferred. In preferred
embodiments the agent does not inhibit non-polo kinases (or has a
EC.sub.50 greater than 10-times higher, sometimes 100-times
greater, and sometimes greater than 1000-times higher) for non-polo
kinases compared to PLK2. However, inhibition of other kinases may
be tolerated depending on the role and expression of the kinase.
For example, a kinase that functions in the gut may not be affected
by an inhibitor delivered to the brain.
[0073] To further guide the reader, inhibitory nucleic acids (e.g.,
siRNA, antisense RNA, ribozymes), inhibitory proteins (e.g., zinc
finger proteins), antibodies, and small molecule inhibitors are
discussed below.
A. Inhibitory Polynucleotides
[0074] Several examples of inhibitors of target kinases, including
PLK2, are described below. Polynucleotide inhibitors are designed
to bind specific target sequences within a target transcript.
Preferably the inhibitors bind to a target site in a PLK2 RNA
without binding a target site in: PLK1 and/or PLK3 and/or PLK4.
Suitable target sites are identified by selecting segments of PLK2
that have no exact corresponding segment in other PLKs. Preferably,
a selected segment of PLK2 lacks a corresponding segment having
substantial sequence identity (for example, a selected segment from
PLK2 should show less than 95, 90, 75 or 50% sequence identity with
the closest corresponding segment in PLK4). A selected target
segment is also preferably screened against a gene database to
ensure that it does not show significant sequence identity with
unrelated genes by chance.
[0075] Polynucleotide inhibitors of PLK2 preferably show at least
30, 50, 75, 95, or 99% inhibition of levels of PLK2 mRNA or protein
with little or no detectable reduction in levels of PLK1 and/or
PLK3 and/or PLK4 mRNA or protein (i.e, less than 10, 5 or 1%
inhibition). Protein expression can be quantified by forming
immunological analyses using an antibody that specifically binds to
the protein followed by detection of complex formed between the
antibody and protein. mRNA levels can be quantified by, for
example, dot blot analysis, in-situ hybridization, RT-PCR,
quantitative reverse-transcription PCR (i.e., the so-called
"TaqMan" methods), Northern blots and nucleic acid probe array
methods.
[0076] i) Short Inhibitory RNAs
[0077] siRNAs are relatively short, at least partly double
stranded, RNA molecules that serve to inhibit expression or
translation of a complementary mRNA transcript, such as a kinase
transcript. Although an understanding of mechanism is not required
for practice of the invention, it is believed that siRNAs act by
inducing degradation of a complementary mRNA transcript. Principles
for design and use of siRNAs generally are described by WO
99/32619, Elbashir, EMBO J. 20, 6877-6888 (2001) and Nykanen et
al., Cell 107, 309-321 (2001); WO 01/29058. siRNAs are formed from
two strands of at least partly complementary RNA, each strand
preferably of 10-30, 15-25, or 17-23 or 19-21 nucleotides long. The
strands can be perfectly complementary to each other throughout
their length or can have single stranded 3'-overhangs at one or
both ends of an otherwise double stranded molecule. Single stranded
overhangs, if present, are usually of 1-6 bases with 1 or 2 bases
being preferred. The antisense strand of an siRNA is selected to be
substantially complementary (e.g., at least 80, 90, 95% and
preferably 100% complementary) to a segment of a transcript from a
gene of the invention. Any mismatched bases preferably occur at or
near the ends of the strands of the siRNA. Mismatched bases at the
ends can be deoxyribonucleotides. The sense strand of an siRNA
shows an analogous relationship with the complement of the segment
of the gene transcript of interest. siRNAs having two strands, each
having 19 bases of perfect complementarity, and having two
unmatched bases at the 3' end of the sense strand and one at the 3'
end of the antisense strand are particularly suitable.
[0078] If an siRNA is to be administered as such, as distinct from
the form of DNA encoding the siRNA, then the strands of an siRNA
can contain one or more nucleotide analogs. The nucleotide analogs
are located at positions at which inhibitor activity is not
substantially affected, e.g. in a region at the 5'-end and/or the
3'-end, particularly single stranded overhang regions. Preferred
nucleotide analogues are sugar- or backbone-modified
ribonucleotides. Nucleobase-modified ribonucleotides, i.e.
ribonucleotides, containing a non-naturally occurring nucleobase
instead of a naturally occurring nucleobase such as uridines or
cytidines modified at the 5-position, e.g. 5-(2-amino)propyl
uridine, 5-bromo uridine; adenosines and guanosines modified at the
8 position, e.g. 8-bromo guanosine; deaza nucleotides, e.g.
7-deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6-methyl
adenosine are also suitable. In preferred sugar-modified
ribonucleotides, the 2' OH-group is replaced by a group selected
from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is
C1-C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I. In
preferred backbone-modified ribonucleotides the phosphoester group
connecting to adjacent ribonucleotides is replaced by a modified
group, e.g. of phosphothioate group. A further preferred
modification is to introduce a phosphate group on the 5' hydroxide
residue of an siRNA. Such a group can be introduced by treatment of
an siRNA with ATP and T4 kinase. The phosphodiester linkages of
natural RNA can also be modified to include at least one of a
nitrogen or sulfur heteroatom. Modifications in RNA structure can
be tailored to allow specific genetic inhibition while avoiding a
general panic response in some organisms which is generated by
dsRNA. Likewise, bases can be modified to block the activity of
adenosine deaminase.
[0079] One example of such an agent is siRNA specific for PLK2.
siRNAs to the gene encoding PLK2 can be specifically designed using
methods described below. An exemplary accession number for PLK2 is
[NM.sub.--006622] as provided in Table 1A. The amino acid sequence
of human PLK2 is also set forth as SEQ ID NO:2 and SEQ ID NO:1 is
the nucleic acid sequence encoding the amino acid sequence. For
convenience exemplary sequences are provided below.
TABLE-US-00002 Human PLK2 (SEQ ID NO:1) 1 gcacaagtgg accggggtgt
tgggtgctag tcggcaccag aggcaagggt gcgaggacca 61 cggccggctc
ggacgtgtga ccgcgcctag ggggtggcag cgggcagtgc ggggcggcaa 121
ggcgaccatg gagcttttgc ggactatcac ctaccagcca gccgccagca ccaaaatgtg
181 cgagcaggcg ctgggcaagg gttgcggagc ggactcgaag aagaagcggc
cgccgcagcc 241 ccccgaggaa tcgcagccac ctcagtccca ggcgcaagtg
cccccggcgg cccctcacca 301 ccatcaccac cattcgcact cggggccgga
gatctcgcgg attatcgtcg accccacgac 361 tgggaagcgc tactgccggg
gcaaagtgct gggaaagggt ggctttgcaa aatgttacga 421 gatgacagat
ttgacaaata acaaagtcta cgccgcaaaa attattcctc acagcagagt 481
agctaaacct catcaaaggg aaaagattga caaagaaata gagcttcaca gaattcttca
541 tcataagcat gtagtgcagt tttaccacta cttcgaggac aaagaaaaca
tttacattct 601 cttggaatac tgcagtagaa ggtcaatggc tcatattttg
aaagcaagaa aggtgttgac 661 agagccagaa gttcgatact acctcaggca
gattgtgtct ggactgaaat accttcatga 721 acaagaaatc ttgcacagag
atctcaaact agggaacttt tttattaatg aagccatgga 781 actaaaagtt
ggggacttcg gtctggcagc caggctagaa cccttggaac acagaaggag 841
aacgatatgt ggtaccccaa attatctctc tcctgaagtc ctcaacaaac aaggacatgg
901 ctgtgaatca gacatttggg ccctgggctg tgtaatgtat acaatgttac
tagggaggcc 961 cccatttgaa actacaaatc tcaaagaaac ttataggtgc
ataagggaag caaggtatac 1021 aatgccgtcc tcattgctgg ctcctgccaa
gcacttaatt gctagtatgt tgtccaaaaa 1081 cccagaggat cgtcccagtt
tggatgacat cattcgacat gacttttttt tgcagggctt 1141 cactccggac
agactgtctt ctagctgttg tcatacagtt ccagatttcc acttatcaag 1201
cccagctaag aatttcttta agaaagcagc tgctgctctt tttggtggca aaaaagacaa
1261 agcaagatat attgacacac ataatagagt gtctaaagaa gatgaagaca
tctacaagct 1321 taggcatgat ttgaaaaaga cttcaataac tcagcaaccc
agcaaacaca ggacagatga 1381 ggagctccag ccacctacca ccacagttgc
caggtctgga acacccgcag tagaaaacaa 1441 gcagcagatt ggggatgcta
ttcggatgat agtcagaggg actcttggca gctgtagcag 1501 cagcagtgaa
tgccttgaag acagtaccat gggaagtgtt gcagacacag tggcaagggt 1561
tcttcgggga tgtctggaaa acatgccgga agctgattgc attcccaaag agcagctgag
1621 cacatcattt cagtgggtca ccaaatgggt tgattactct aacaaatatg
gctttgggta 1681 ccagctctca gaccacaccg tcggtgtcct tttcaacaat
ggtgctcaca tgagcctcct 1741 tccagacaaa aaaacagttc actattacgc
agagcttggc caatgctcag ttttcccagc 1801 aacagatgct cctgagcaat
ttattagtca agtgacggtg ctgaaatact tttctcatta 1861 catggaggag
aacctcatgg atggtggaga tctgcctagt gttactgata ttcgaagacc 1921
tcggctctac ctccttcagt ggctaaaatc tgataaggcc ctaatgatgc tctttaatga
1981 tggcaccttt caggtgaatt tctaccatga tcatacaaaa atcatcatct
gtagccaaaa 2041 tgaagaatac cttctcacct acatcaatga ggataggata
tctacaactt tcaggctgac 2101 aactctgctg atgtctggct gttcatcaga
attaaaaaat cgaatggaat atgccctgaa 2161 catgctctta caaagatgta
actgaaagac ttttcgaatg gaccctatgg gactcctctt 2221 ttccactgtg
agatctacag ggaagccaaa agaatgatct agagtatgtt gaagaagatg 2281
gacatgtggt ggtacgaaaa caattcccct gtggcctgct ggactggttg gaaccagaac
2341 aggctaaggc atacagttct tgactttgga caatccaaga gtgaaccaga
atgcagtttt 2401 ccttgagata cctgttttaa aaggtttttc agacaatttt
gcagaaaggt gcattgattc 2461 ttaaattctc tctgttgaga gcatttcagc
cagaggactt tggaactgtg aatatacttc 2521 ctgaagggga gggagaaggg
aggaagctcc catgttgttt aaaggctgta attggagcag 2581 cttttggctg
cgtaactgtg aactatggcc atatataatt ttttttcatt aatttttgaa 2641
gatacttgtg gctggaaaag tgcattcctt gttaataaac tttttattta ttacagccca
2701 aagagcagta tttattatca aaatgtcttt ttttttatgt tgaccatttt
aaaccgttgg 2761 caataaagag tatgaaaacg cagaaaaaaa aaaaa Human PLK2
(SEQ ID NO:2)
MELLRTITYQPAASTKMCSQALGKGCGADSKKKRPPQPPEESQPPQSQAQVPPAAPHHHHHHSHSGPEIS
RIIVDPTTGKRYCRGKVLGKGGFAKCYEMTDLTNNKVYAAKIIPHSRVAKPHQREKIDKEIELHRILHHK
HVVQFYHYFEDKENIYILLEYCSRRSMAHILKARKVLTEPEVRYYLRQIVSGLKYLHEQEILHRDLKLGN
FFINEAMELKVGDFGLAARLEPLEHRRRTICGTPNYLSPEVLNKQGHGCESDIWALGCVMYTMLLGRPPF
ETTNLKETYRCIREARYTMPSSLLAPAKHLIASMLSKNPEDRPSLDDIIRHDFFLQGFTPDRLSSSCCHT
VPDFHLSSPAKNFFKKAAAALFGGKKDKARYIDTHNRVSKEDEDIYKLRHDLKKTSITQQPSKHRTDEEL
QPPTTTVARSGTPAVENKQQIGDAIRMIVRGTLGSCSSSSECLEDSTMGSVADTVARVLRGCLENMPEAD
CIPKEQLSTSFQWVTKWVDYSNKYGFGYQLSDHTVGVLFNNGAHMSLLPDKKTVHYYAELGQCSVFPATD
APEQFISQVTVLKYFSHYMEENLMDGGDLPSVTDIRRPRLYLLQWLKSDKALMMLFNDGTFQVNFYHDHT
KIIICSQNEEYLLTYINEDRISTTFRLTTLLMSGCSSELKNRMEYALNMLLQRCN
[0080] ii) Design and Production of siRNA
[0081] An advantage of inhibitory polynucleotides is that they can
be designed to be highly target specific. For example, siRNAs
specific for PLK2 can be designed using target sequences that
distinguish PLK2 from other PLKs. The program "siDESIGN"
(Dharmacon, Inc., Lafayette, Colo.) can be used to predict siRNA
sequences for any nucleic acid sequence, and is available on the
World Wide Web at dharmacon.com. A number of other programs for
designing siRNAs are available from others, including Genscript
(available on the Web at genscript.com/ssl-bin/app/mai) and from
the Whitehead Institute for Biomedical Research
jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/. Guidelines
for designing siRNA are available in the scientific literature
(see, e.g., Elbashir et al., 2001, "Duplexes of 21-nucleotide RNAs
mediate RNA interference in cultured mammalian cells" Nature.
411:494-8.; and Elbashir et al., 2001, "RNA interference is
mediated by 21- and 22-nucleotide RNAs" Genes Dev. 15:188-200) and
published on the Web (see, e.g., "maiweb.com/RNAi/siRNA Design/"
and
"protocol-online.org/prot/Protocols/Rules-of-siRNA-design-for-RNA-interfe-
rence-RNAi-3210.html").
[0082] There are a variety of ways to produce siRNAs. siRNAs can be
generated using kits which generate siRNA from the kinase (e.g.,
PLK2) gene. For example, the "Dicer siRNA Generation" kit (catalog
number T510001, Gene Therapy Systems, Inc., San Diego, Calif.) uses
the recombinant human enzyme "dicer" in vitro to cleave long double
stranded RNA into 22 bp siRNAs. By producing a mixture of siRNAs,
the kit permits a high degree of success in generating siRNAs that
will reduce expression of the target gene. Similarly, the
Silencer.TM. siRNA Cocktail Kit (RNase III) (catalog no. 1625,
Ambion, Inc., Austin, Tex.) generates a mixture of siRNAs from
dsRNA using RNase III instead of dicer. Like dicer, RNase III
cleaves dsRNA into 12-30 bp dsRNA fragments with 2 to 3 nucleotide
3' overhangs, and 5'-phosphate and 3'-hydroxyl termini. According
to the manufacturer, dsRNA is produced using T7 RNA polymerase, and
reaction and purification components included in the kit. The dsRNA
is then digested by RNase III to create a population of siRNAs. The
kit includes reagents to synthesize long dsRNAs by in vitro
transcription and to digest those dsRNAs into siRNA-like molecules
using RNase III. The manufacturer indicates that the user need only
supply a DNA template with opposing T7 phage polymerase promoters
or two separate templates with promoters on opposite ends of the
region to be transcribed.
[0083] The siRNAs of the invention can also be expressed from
vectors. Typically, such vectors are administered in conjunction
with a second vector encoding the corresponding complementary
strand. Once expressed, the two strands anneal to each other and
form the functional double stranded siRNA. One exemplar vector
suitable for use in the invention is pSuper, available from
OligoEngine, Inc. (Seattle, Wash.). In some embodiments, the vector
contains two promoters, one positioned downstream of the first and
in antiparallel orientation. The first promoter is transcribed in
one direction, and the second in the direction antiparallel to the
first, resulting in expression of the complementary strands. In yet
another set of embodiments, the promoter is followed by a first
segment encoding the first strand, and a second segment encoding
the second strand. The second strand is complementary to the
palindrome of the first strand. Between the first and the second
strands is a section of RNA serving as a linker (sometimes called a
"spacer") to permit the second strand to bend around and anneal to
the first strand, in a configuration known as a "hairpin."
[0084] The formation of hairpin RNAs, including use of linker
sections, is well known in the art. Typically, an siRNA expression
cassette is employed, using a Polymerase III promoter such as human
U6, mouse U6, or human H1. The coding sequence is typically a
19-nucleotide sense siRNA sequence linked to its reverse
complementary antisense siRNA sequence by a short spacer.
Nine-nucleotide spacers are typical, although other spacers can be
designed. Further, 5-6 T's are often added to the 3' end of the
oligonucleotide to serve as a termination site for Polymerase III.
See also, Yu et al., Mol Ther 7(2):228-36 (2003); Matsukura et al.,
Nucleic Acids Res 31(15):e77 (2003).
[0085] The siRNA targets can be targeted by hairpin siRNA as
follows. To attack the same targets by short hairpin RNAs, produced
by a vector (permanent RNAi effect), sense and antisense strand can
be put in a row with a loop forming sequence in between and
suitable sequences for an adequate expression vector to both ends
of the sequence.
[0086] The siRNA of the invention can be made using any suitable
method for producing a nucleic acid, such as the chemical synthesis
and recombinant methods disclosed herein and known to one of skill
in the art. It will be appreciated that the oligonucleotides can be
made using nonstandard bases (e.g., other than adenine, cytidine,
guanine, and uridine) or nonstandard backbone structures to provide
desirable properties (e.g., increased nuclease-resistance,
tighter-binding, stability or a desired Tm). Techniques for
rendering oligonucleotides nuclease-resistant include those
described in PCT Publication WO 94/12633. A wide variety of useful
modified oligonucleotides may be produced, including
oligonucleotides having a peptide-nucleic acid (PNA) backbone
(Nielsen et al., 1991, Science 254:1497) or incorporating
2'-O-methyl ribonucleotides, phosphorothioate nucleotides, methyl
phosphonate nucleotides, phosphotriester nucleotides,
phosphorothioate nucleotides, phosphoramidates.
[0087] For example and not limitation, the following are examples
of siRNA sequences that can be used to inhibit PLK2. "Start
position" refers to the sequence at accession number
NM.sub.--006622.
TABLE-US-00003 Sense Strand Sequence Region Start Pos 1
CCGGAGATCTCGCGGATTA ORF 326 2 GGGGCAAAGTGCTGGGAAA ORF 378 3
TCACAGCAGAGTAGCTAAA ORF 469 4 GGGAAAAGATTGACAAAGA ORF 498 5
GATTGTGTCTGGACTGAAA ORF 691 6 GCACAGAGATCTCAAACTA ORF 733 7
ACACAGAAGGAGAACGATA ORF 829 8 AGGAGAACGATATGTGGTA ORF 836 9
CATAAGGGAAGCAAGGTAT ORF 1000 10 GCTAGTATGTTGTCCAAAA ORF 1061 11
GAAGACATCTACAAGCTTA ORF 1304 12 CATCAATGAGGATAGGATA ORF 2062 13
GACATGTGGTGGTACGAAA 3' UTR 2281 14 CAGAACAGGCTAAGGCATA 3' UTR 2335
15 GTGCATTCCTTGTTAATAA 3' UTR 2660
[0088] The siRNAs above were designed using the siDESIGN.RTM.
center at www followed by
dharmacon.com/DesignCenter/DesignCenterPage.aspx. Each will be
double stranded and have a "TT" overhang.
[0089] As additional examples, siRNAs from the Ambion Kinase siRNA
Library (Ambion, Austin, Tex.) can be used to inhibit PLK2.
Exemplary sequences are provided below. Each siRNA is
double-stranded with the final TT's (present on both strands) as
overhangs:
TABLE-US-00004 16. GGUAUACAAUGCCGUCCUCTT 17. GGACUUUGGAACUGUGAAUTT
18. GGGAAAAGAUUGACAAAGATT
[0090] iii) Antisense Polynucleotides
[0091] Antisense polynucleotides can cause suppression by binding
to, and interfering with the translation of sense mRNA, interfering
with transcription, interfering with processing or localization of
RNA precursors, repressing transcription of mRNA or acting through
some other mechanism (see, e.g., Sallenger et al. Nature 418, 252
(2002). The particular mechanism by which the antisense molecule
reduces expression is not critical. Typically antisense
polynucleotides comprise a single-stranded antisense sequence of at
least 7 to 10 to typically 20 or more nucleotides that specifically
hybridize to a sequence from mRNA of a kinase gene of the
invention. Some antisense polynucleotides are from about 10 to
about 50 nucleotides in length or from about 14 to about 35
nucleotides in length. Some antisense polynucleotides are
polynucleotides of less than about 100 nucleotides or less than
about 200 nucleotides. In general, the antisense polynucleotide
should be long enough to form a stable duplex but short enough,
depending on the mode of delivery, to administer in vivo, if
desired. The minimum length of a polynucleotide required for
specific hybridization to a target sequence depends on several
factors, such as G/C content, positioning of mismatched bases (if
any), degree of uniqueness of the sequence as compared to the
population of target polynucleotides, and chemical nature of the
polynucleotide (e.g., methylphosphonate backbone, peptide nucleic
acid, phosphorothioate), among other factors.
[0092] iv) Ribozymes
[0093] Ribozymes are RNA molecules that act as enzymes and can be
engineered to cleave other RNA molecules at specific sites. The
ribozyme itself is not consumed in this process, and can act
catalytically to cleave multiple copies of mRNA target molecules.
General rules for the design of ribozymes that cleave target RNA in
trans are described in Haseloff & Gerlach, (1988) Nature
334:585-591 and Hollenbeck, (1987) Nature 328:596-603 and U.S. Pat.
No. 5,496,698. Ribozymes typically include two flanking segments
that show complementarity to and bind to two sites on a transcript
(target subsites) of a gene encoding a kinase of the invention and
a catalytic region between the flanking segments. The flanking
segments are typically 5-9 nucleotides long and optimally 6 to 8
nucleotides long. The catalytic region of the ribozyme is generally
about 22 nucleotides in length. The mRNA target contains a
consensus cleavage site between the target subsites having the
general formula NUN, and preferably GUC. (Kashani-Sabet and
Scanlon, (1995) Cancer Gene Therapy 2:213-223; Perriman, et al.,
(1992) Gene (Amst.) 113:157-163; Ruffner, et al., (1990)
Biochemistry 29: 10695-10702); Birikh, et al., (1997) Eur. J.
Biochem. 245:1-16; Perrealt, et al., (1991) Biochemistry
30:4020-4025). The specificity of a ribozyme can be controlled by
selection of the target subsites and thus the flanking segments of
the ribozyme that are complementary to such subsites. Ribozymes can
be delivered either as RNA molecules or in the form of DNA encoding
the ribozyme as a component of a replicable vector or in
nonreplicable form as described below.
[0094] Expression of a target kinase gene can also be reduced by
delivering nucleic acids having sequences complementary to the
regulatory region of the target gene (i.e., the target gene
promoter and/or enhancers) to form triple helical structures which
prevent transcription of the target gene in target cells in the
body. See generally, Helene, (1991), Anticancer Drug Des.,
6(6):569-584; Helene, et al., (1992), Ann. N.Y. Acad. Sci.,
60:27-36; and Maher, (1992), Bioassays 14(12):807-815.
[0095] v) Administration of siRNA and Other Inhibitory Nucleic
Acids
[0096] The brain is the therapeutic target of kinase inhibitors of
the invention. Therapeutic polynucleotides such as siRNAs,
ribozymes and antisense polynucleotides can be administered in a
number of ways. Methods of introduction include but are not limited
to intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
may be administered by any convenient route, for example by
infusion, by bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral, rectal and intestinal mucosa,
etc.), and may be administered together with other biologically
active agents. In general, therapeutic polynucleotides can be
administered remotely (e.g., by i.v. injection) with a carrier that
facilitates transfer to the brain or they can be delivered directly
to the brain.
[0097] For direct administration to the brain, siRNAs (i.e.,
pharmaceutical compositions containing siRNAs) can be administered
by any suitable route, including intraventricular and intrathecal
injection; intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir, direct injection or perfusion at the
lesion site, injection into the brain arterial system, or by
chemical or osmotic opening of the blood-brain barrier.
[0098] In one approach the therapeutic polynucleotide (e.g., siRNA)
is delivered as a peptide conjugate. Kumar et al. exploited the
fact that neurotropic viruses, such as rabies viruses, that
preferentially infect the nervous system can penetrate the brain.
The rabies virus achieves this through glycoprotein on its lipid
envelope. To transfer siRNA to the neural cells in the brain, Kumar
et al. identified a 29-residue peptide from the rabies virus
glycoprotein (RVG) envelope that selectively binds to the
acetylcholine receptor expressed by neurons. They fused this
peptide with a sequence of 9 arginine residues that binds to
siRNAs. After intravaneous injection into mice with this
peptide-conjugated siRNA, they found that the peptide not only
enabled the transvascular delivery of siRNA to the brain but also
resulted in efficient gene silencing (Kumar et al., (2007) Nature
448:39-43).
[0099] The therapeutic polynucleotide (e.g., siRNA) can be
delivered using a liposome and targeted monoclonal antibody system.
Pardridge reported that chemically modified liposomes conjugated to
monoclonal antibodies raised against epidermal growth factor can
penetrate mouse brain. Plasmid DNA encoding for short hairpin RNA
(shRNA) was delivered to the brain following intravenous
administration with pegylated immunoliposomes (PILs). The plasmid
DNA is encapsulated in liposome, which is pegylated, and conjugated
with receptor specific targeting monoclonal antibodies. Intravenous
RNAi with PILs enables a 90% knockdown of the human epidermal
growth factor receptor, which results in a 90% increase in survival
time in mice with intra-cranial brain cancer (Pardridge, (2007)
Adv. Drug Deliv. Rev. 59:141-152).
[0100] Similarly, Boado disclosed the use of the "Trojan Horse
Liposome" (THL) technology as an effective delivery system to
deliver shRNA to the brain. The tissue target specificity of THL is
given by conjugation of approximately 1% of the PEG residues in the
liposome to peptidomimetic monoclonal antibodies that bind to
specific endogenous receptors (i.e. insulin and transferrin
receptors) located on both the BBB and the brain cellular
membranes, respectively. (Boado (2007) Pharm. Res.
24:1772-1787).
[0101] The therapeutic polynucleotide (e.g., siRNA) can be
delivered by the combined use of receptor specific antibody
delivery systems and avidin-biotin technology. The siRNA was
mono-biotinylated on either terminus of the sense strand, in
parallel with the production of a conjugate of the targeting
monoclonal antibody and streptavidin. Rat glial cells permanently
transfected with the luciferase gene were implanted in the brain of
adult rats. Following the formation of intra-cranial tumors, the
rats were treated with a single intravenous injection of
biotinylated siRNA attached to a transferrin receptor antibody via
a biotin-streptavidin linker. The intravenous administration of the
siRNA caused a 69-81% decrease in luciferase gene expression in the
intracranial brain cancer in vivo (Xia et al., (2007) Pharm. Res.
24:2309-2316).
[0102] The therapeutic polynucleotide (e.g., siRNA) can be
delivered by stereotactic surgery or injection. Davidson and
Boudreau reviewed in their article that siRNA can be delivered into
the brain using neurosurgical method of stereotaxis and showed that
a decrease in the transcription of certain genes alleviated
symptoms of neuronal diseases (Davidson and Boudreau, (2007) Neuron
53:781-788).
[0103] Xia et al. reported that upon intracerebellar injection,
recombinant adeno-associated virus vectors expressing short hairpin
RNAs, which once expressed are processed into siRNAs, improved
motor coordination, restored cerebellar morphology and resolved
characteristic ataxin-1 inclusions in Purkinje cells of
spinocerebellar ataxia type 1 mice (Xia et al., (2004) Nature Med.
10:816-820).
[0104] Further, DiFiglia et al. reported injecting intrastriatally
cholesterol-conjugated siRNA that targets human huntingtin mRNA.
The authors found that a single administration into the adult mouse
striatum of the siRNA effected silencing of the gene, attenuated
neuronal pathology, and delayed the abnormal behavioral phenotype
observed in a rapid-onset, viral transgenic mouse model of
Huntington's disease (DiFiglia et al. (2007) Proc. Natl. Acad. Sci.
USA 104:17204-17209). It is noted that such method results only in
localized delivery around the injection site, with no widespread
effect within the brain.
[0105] Singer et al. disclosed using modified lentiviral vectors to
deliver siRNAs into the brain cells of the transgenic mice that
were producing vast amounts of human beta-amyloid and whose brains
where littered with plaques. They found that lentiviral vector
delivery of beta-secretase siRNA specifically reduced the cleavage
of amyloid precursor protein and neurodegeneration in vivo and
indicated that this approach could have potential therapeutic value
for treatment of Alzheimer disease (Singer et al. (2005) Nature
Neurosci. 8:1343-1349; reviewed in Orlacchio et al. (2007) Mini.
Rev. Med. Chem. 7:1166-1176).
[0106] Koutsilieri et al. reviewed the literature in the field of
siRNA, disclosed different siRNA target strategies aiming for an
allele-specific degradation of disease-inducing mRNA and its
application in animal models of neurodegenerative diseases,
including Alzheimer's disease (AD), amyotrophic lateral sclerosis
(ALS), Huntington's disease (HD) and spinocerebellar ataxia (SCA1)
(Koutsilieri et al. (2007) J. Neural Transm Suppl. (72):43-49).
[0107] Hassani et al. demonstrated that cationic lipids and
polyethylenimine (PEI) based polyplexes provided efficient delivery
of siRNAs into the brains of new born mice, producing >80%
inhibition of an exogenous gene with only picomolar levels of siRNA
(Hassani et al. (2005) J. Gene Med. 7:198-207).
[0108] Kateb et al. employed nanotechnology as a method for
delivering drugs to the brain for treatment of brain cancers.
Specifically, the authors disclosed the use Multi-Walled Carbon
Nanotubes (MWCNTs) as nanovectors for transporting siRNA (Kateb et
al. (2007) Neuroimage 37 Suppl 1:S9-17).
[0109] The therapeutic polynucleotide (e.g., siRNA) can be used in
combination with other agents to improve or enhance the therapeutic
effect of either. This process can involve administering both
agents to the patient at the same time, either as a single
composition or pharmacological formulation that includes both
agents, or by administering two distinct compositions or
formulations, wherein one composition includes the siRNA of the
invention and the other includes the second agent(s). The siRNA
therapy also can precede or follow the other agent treatment by
intervals ranging from minutes to weeks.
[0110] Polynucleotides can be delivered via a controlled release
system. As an example, a pump may be used (Langer, supra; Sefton,
1987, CRC Crit. Ref. Biomed. Eng. 14:201-240; Buchwald et al.,
1980, Surgery 88:507-516; Saudek et al., 1989, N. Engl. J. Med.
321:574-579). Alternatively, polymeric materials can be used
(Medical Applications of Controlled Release, Langer and Wise, eds.,
CRC Press, Boca Raton, Fla., 1974; Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball, eds., Wiley,
N.Y., 1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol.
Chem. 23:61; Levy et al., 1985, Science 228:190-192; During et al.,
1989, Ann. Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg.
71:858-863). In yet another alternative, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(e.g., Goodson, 1984, In: Medical Applications of Controlled
Release, supra, Vol. 2, pp. 115-138). Other controlled release
systems are discussed in the review by Langer (1990, Science
249:1527-1533).
[0111] Other approaches can include the use of various transport
and carrier systems, for example though the use of viral and/or
non-viral delivery systems. For example, siRNA can be introduced
into the brain in a virus modified to serve as a vehicle without
causing pathogenicity. The virus can be, e.g., adenovirus, fowlpox
virus, vaccinia virus, lentivirus, or neurotropic virus such as
HIV, herpes simplex virus, flavivirus, or rabies virus. (Li et al.,
Methods Mol. Biol. 309:261-272 (2005); Davidson et al., Neuron
53:781-788 (2007); Xia et al., Nature Med 10:816-820 (2004); Kumar
et al., Nature 448:39-43 (2007); U.S. Pat. Nos. 6,344,445,
6,924,144, 6,521,457). Furthermore, gene therapy approaches, for
example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and
Davidson, WO 04/013280, can be used to express nucleic acid
molecules in the brain.
[0112] Various non-viral delivery systems are known and can be used
to administer a therapeutic polynucleotide (e.g., siRNA) to the
brain, e.g., encapsulation in liposomes, microparticles,
microcapsules, by iontophoresis, or by incorporation into other
vehicles, such as biodegradable polymers, hydrogels, cyclodextrins
(see for example Gonzalez et al., 1999, Bioconjugate Chem., 10,
1068-1074; Wang et al., International PCT publication Nos. WO
03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and
PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US
Patent Application Publication No. US 2002130430), biodegradable
nanocapsules, and bioadhesive microspheres, or by proteinaceous
vectors (O'Hare and Normand, International PCT Publication No. WO
00/53722) or by the use of conjugates. For example, chemically
modified liposomes encapsulating small hairpin RNA can be
conjugated to monoclonal antibodies specific for specific
endogenous receptors (e.g., such as insulin and transferrin)
located on the blood brain barrier and brain cellular membranes
(see Boado, Pharm. Res. 24(9):1772-1787 (2007)). As a further
example, siRNA duplexes can be delivered with the combined use of
receptor specific antibody delivery systems and avidin-biotin
technology. The siRNA can be mono-biotinylated in parallel with the
production of a conjugate of the targeting monoclonal antibody and
streptavidin (see Xia et al., Pharm Res. 24(12):2309-16 (2007)).
Other methods to deliver siRNA across plasma membranes in vivo
include chemically modified siRNAs such as cholesterol-conjugated
siRNAs (see DiFiglia et al., Proc Natl Acad Sci USA.
104(43):17204-9 (2007); Wolfrum et al., Nature Biotech.
25(10):1149-1157 (2007); Soutschek et al., Nature 432:173-178
(2003)).
C. Zinc Finger Proteins
[0113] Zinc finger proteins can be engineered or selected to bind
to any desired target site within a kinase gene of known sequence
including, for example, PLK2. An exemplary motif characterizing one
class of these proteins (C.sub.2H.sub.2 class) is
-Cys-(X).sub.2-4-Cys-(X).sub.12-His-(X).sub.3-5-His (where X is any
amino acid). A single finger domain is about 30 amino acids in
length, and several structural studies have demonstrated that it
contains an alpha helix containing the two invariant histidine
residues and two invariant cysteine residues in a beta turn
co-ordinated through zinc. In some methods, the target site is
within a promoter or enhancer. In other methods, the target site is
within the structural gene. In some methods, the zinc finger
protein is linked to a transcriptional repressor, such as the KRAB
repression domain from the human KOX-1 protein (Thiesen et al., New
Biologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad.
Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res.
22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA
91, 4514-4518 (1994)). In some methods, the zinc finger protein is
linked to a transcriptional activator, such as VIP16. Zinc finger
proteins can also be used to activate expression of desired genes.
Methods for selecting target sites suitable for targeting by zinc
finger proteins, and methods for designing zinc finger proteins to
bind to selected target sites are described in WO 00/00388. Methods
for selecting zinc finger proteins to bind to a target using phage
display are described by EP.95908614.1. The target site used for
design of a zinc finger protein is typically of the order of 9-19
nucleotides.
[0114] For example, proteins have been described that have the
ability to translocate desired nucleic acids across a cell
membrane. Typically, such proteins have amphiphilic or hydrophobic
subsequences that have the ability to act as membrane-translocating
carriers. For example, homeodomain proteins have the ability to
translocate across cell membranes. The shortest internalizable
peptide of a homeodomain protein, Antennapedia, was found to be the
third helix of the protein, from amino acid position 43 to 58 (see,
e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996).
Another subsequence, the h (hydrophobic) domain of signal peptides,
was found to have similar cell membrane translocation
characteristics (see, e.g., Lin et al., J. Biol. Chem.
270:14255-14258 (1995)). Such subsequences can be used to
translocate oligonucleotides across a cell membrane.
Oligonucleotides can be conveniently derivatized with such
sequences. For example, a linker can be used to link the
oligonucleotides and the translocation sequence. Any suitable
linker can be used, e.g., a peptide linker or any other suitable
chemical linker.
D. Antibodies
[0115] Kinase activity can be reduced by administering anti-kinase
(e.g., anti-PLK2) antibodies, both intact and binding fragments
thereof, such as Fabs, Fvs, which specifically bind to a kinase of
the invention. Usually the antibody is a monoclonal antibody
although polyclonal antibodies can also be expressed recombinantly
(see, e.g., U.S. Pat. No. 6,555,310). Examples of antibodies that
can be expressed include mouse antibodies, chimeric antibodies,
humanized antibodies, veneered antibodies and human antibodies.
Chimeric antibodies are antibodies whose light and heavy chain
genes have been constructed, typically by genetic engineering, from
immunoglobulin gene segments belonging to different species (see,
e.g., Boyce et al., Annals of Oncology 14:520-535 (2003)). For
example, the variable (V) segments of the genes from a mouse
monoclonal antibody may be joined to human constant (C) segments. A
typical chimeric antibody is thus a hybrid protein consisting of
the V or antigen-binding domain from a mouse antibody and the C or
effector domain from a human antibody. Humanized antibodies have
variable region framework residues substantially from a human
antibody (termed an acceptor antibody) and complementarity
determining regions substantially from a mouse-antibody, (referred
to as the donor immunoglobulin). See Queen et al., Proc. Natl.
Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat. No.
5,693,762, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,585,089, U.S.
Pat. No. 5,530,101 and Winter, U.S. Pat. No. 5,225,539. The
constant region(s), if present, are also substantially or entirely
from a human immunoglobulin. Antibodies can be obtained by
conventional hybridoma approaches, phage display (see, e.g., Dower
et al., WO 91/17271 and McCafferty et al., WO 92/01047), use of
transgenic mice with human immune systems (Lonberg et al.,
WO93/12227 (1993)), among other sources. Nucleic acids encoding
immunoglobulin chains can be obtained from hybridomas or cell lines
producing antibodies, or based on immunoglobulin nucleic acid or
amino acid sequences in the published literature.
[0116] Antibodies can be administered by intravenous injection. A
portion of the injected antibodies will cross the blood-brain
barrier. Alternatively antibodies can be administered directly to
the brain (e.g., by intraventricular or intrathecal injection).
Antibodies may be internalized by synuclein-expressing cells by
endocytosis. Alternatively, antibodies may be linked to carrier
moiety that facilitates transport across the cell membrane.
[0117] In one embodiment an intrabody is used to reduce PLK2
activity. The term "intrabody" or "intrabodies" refers to
intracellularly expressed antibody constructs, usually single-chain
Fv antibodies, directed against a target inside a cell. Nam, et al.
(2002) Methods Mol. Biol. 193:301; der Maurr et al. (2002) J. Biol
Chem November 22; 277(47):45075; Cohen (2002) Methods Mol Biol
178:367. The scFv gene can be transferred into cells, where scFv
protein expression can modulate the properties of its target, e.g.
PLK2, sometimes extinguishing protein function and causing a
phenotypic knockout. Indeed, the scFv intrabody can be expressed in
the cytoplasm and directed to any cellular compartment where it can
target intracellular proteins and elicit specific biological
effects. Intrabodies thus provide effective means for blocking or
modulating the activity of proteins such as PLK2.
E. Kinase Inhibitors to Modulate Activity
[0118] In addition to the biological molecules discussed above,
small molecule compounds can be used to modulate (usually inhibit)
expression or activity of kinases. As discussed below, known kinase
inhibitors can be screened for desired target specificity and other
properties, and additional inhibitors can be identified based on
target specificity. PLK2 or GRK6 are preferred kinases for
inhibition because they are candidates for directly phosphorylating
alpha-synuclein. PLK2 is a particularly preferred kinase because it
has been shown to phosphorylate alpha-synuclein to a much higher
level than GRK6 or other kinases tested herein.
[0119] Other kinase targets include APEG1, CDC7 .mu.l, MET GRK1,
GRK2, GRK6, IKBKB, CKII and GRK7 are also preferred kinases for
inhibition because they are likely to be indirect activators of the
direct kinase(s). PRKG1, MAPK13, and GAK are preferred kinases for
activation in Lewy body diseases because they are negative
regulators of the phosphorylation of alpha-synuclein.
Alternatively, these kinases themselves or fragments or mimetics
thereof having similar activity can be used directly as inhibitors
of alpha-synuclein phosphorylation. Synphilin can be used as an
alternative therapeutic target for inhibition of alpha-synuclein
phosphorylation. For example, synphilin can be added to an assay
having alpha-synuclein and PLK-2 expression and inhibitors of
synphilin identified.
[0120] Compounds to be screened for capacity to modulate expression
or activity of kinases include the modulators of expression
described in Section IV. These compounds also include many known
examples of kinase inhibitors, some of which are already approved
for therapeutic uses or in clinical trials, usually for treatment
of cancer. Lead structures include quinazolines, pyrido[d]- and
pyrimidol[d]pyrimidines, pyrazolo[d]-pyrimidienes,
pyrrolo[d]pyrimidines, pheylamino-pyrimidines,
1-oxo-3-aryl-1H-indene-2-carboxylic acid derivatives, and
substituted indolin-2-ones and natural products such as
strauosporine (see Traxler et al., 2001, Medicinal Research Reviews
21:499-512). Some such compounds are commercially available from
Calbiochem-Novabiochem Corp. (La Jolla, Calif.) including H89,
Y27632, AT877 (fasudil hydrochloride), rottlerin, KN62, U0123,
PD184352, PD98059, SB203580, SB202190, wortmannin, Li.sup.+, Ro
318220, chelerythrein and
10-[3-(1-piperazinyl)propyl]-2-trifluoromethyl-phenothiazine (see
Davies, Biochem. J. 351, 95-105 (2000)). Other compounds currently
in clinical trials include STI571 (Glivec.TM., a
phenylamino-pyrimidine derivative) (Novartis), ZD1839 (Iressa)
(AstraZeneca), OSI-774 (Roche/OSI), PK1166 (Novartis), CI1033
(Pfizer/Wamer-Lambert), EKB-569 (Wyeth-Ayerst), SU5416 (SUGEN),
PTK787/ZK224584, aniline-phthalazine derivative (Novartis/Schering
AG), SU6668 (Sugen), ZD6474 (AstraZeneca), and CEP2583 (Cephalon).
Caveolin-1 is an example of a compound known to modulate the
activity of the GRK kinases. Examples of compounds known to
modulate the activity of the PLK2/SNK kinases include the RING-H2
domain of hVPS18 (human vacuolar protein sorting 18), and calcium-
and integrin-binding protein CIB.
[0121] In certain embodiments the therapeutic agent is a small
molecule that is a thiazolidone, a quinazoline, a pyrimidine (e.g.,
a pyrido[d]-pyrimidine, pyrimidol[d]pyrimidine, a
pyrazolo[d]-pyrimidiene, a pyrrolo[d]pyrimidine, a
pheylamino-pyrimidines, or a phenylamino-pyrimidine derivative); an
indazole-pyridine derivative, a carboxylic acid derivative (e.g., a
1-oxo-3-aryl-1H-indene-2-carboxylic acid derivative), a substituted
indolin-2-one, an aniline-phthalazine derivative; a quinolinone
derivative, a benzthiazole-3 oxide compound, an azaindazole
compound, or a dihydropteridinone. In certain embodiments the
therapeutic agent is a small molecule protein kinase inhibitor
described in US 20070203143; US 2007/0179177; US 2007/0135387; US
2007/0010565; US 2007/0037862; US 2007/0010566; US 2006/0074119; US
2006/0079503; US 2006/0223833; US 2005/0014761; US 2004/0176380; US
2006/0040997; U.S. Pat. No. 6,861,422; US 2005/0014760; US
2006/0025411; US 2004/0176380; US 2005/0014761; US 2007/0203143; US
2007/0072833; US 2007/0135387 or WO 03087095. Each of the
aforelisted publications is incorporated herein by reference.
[0122] Compounds may be synthetic or can be obtained from natural
sources, such as, e.g., marine microorganisms, algae, plants, and
fungi. Other compounds that can be tested include compounds known
to interact with alpha-synuclein, such as synphilin. Alternatively,
compounds can be from combinatorial libraries of agents, including
peptides or small molecules, or from existing repertories of
chemical compounds synthesized in industry, e.g., by the chemical,
pharmaceutical, environmental, agricultural, marine, cosmeceutical,
drug, and biotechnological industries. Combinatorial libraries can
be produced for many types of compounds that can be synthesized in
a step-by-step fashion. Such compounds include polypeptides,
proteins, nucleic acids, beta-turn mimetics, polysaccharides,
phospholipids, hormones, prostaglandins, steroids, aromatic
compounds, heterocyclic compounds, benzodiazepines, oligomeric
N-substituted glycines and oligocarbamates. Large combinatorial
libraries of compounds can be constructed by the encoded synthetic
libraries (ESL) method described in Affymax WO 95/12608, Affymax WO
93/06121, Columbia University WO 94/08051, Pharmacopeia WO 95/35503
and Scripps WO 95/30642 (each of which is incorporated herein by
reference in its entirety for all purposes). Peptide libraries can
also be generated by phage display methods. See, e.g., Devlin, WO
91/18980. Compounds to be screened can also be obtained from
governmental or private sources, including, e.g., the National
Cancer Institute's (NCI) Natural Product Repository, Bethesda, Md.,
the NCl Open Synthetic Compound Collection, Bethesda, Md., NCl's
Developmental Therapeutics Program, or the like. Compounds can
include, e.g., pharmaceuticals, therapeutics, environmental,
agricultural, or industrial agents, pollutants, cosmeceuticals,
drugs, organic compounds, lipids, glucocorticoids, antibiotics,
peptides, proteins, sugars, carbohydrates, and chimeric
molecules.
[0123] As discussed above, kinase inhibitors that preferentially
inhibit PLK2 are of particular value. As is shown in Examples
13-16, below, we assayed the effect of several kinase inhibitors
levels of phosphorylation of alpha-synuclein in rat ventral
mesencephalon and mouse cortical cell cultures, and other
cells.
[0124] An exemplary compound for use according to the invention is
the compound BI2536 having the structure:
##STR00001##
[0125] As demonstrated in the Examples below, BI 2536
(4-[[(7r)-8-cyclopentyl-7-ethyl-5,6,7,8-tetrahydro-5-methyl-6-oxo-2-pteri-
dinyl]amino]-3-methoxy-n-(1-methyl-4-piperidinyl)-benzamide; also
called ELN-481574-2;) reduced phosphorylation of alpha synuclein in
a variety of cell types. BI 2536 inhibits PLK1, PLK2 and PLK3 (see,
Steegmaier et al., 2007, Current Biology, 17:316-322) and does not
inhibit PLK4 (Johnson et al., 2007, Biochemistry 46:9551-9563).
Steegmaier et al. reported an IC.sub.50 of 0.83 nM for PLK1, 3.5 nM
for PLK2 and 9 nM for PLK3. In tests described in Example 15,
below, BI2536 was shown to have 16-fold selectivity for PLK2
(IC.sub.50 11 nM) and 13-fold selectivity for PLK3 (IC.sub.50 14
nM). BI 2536 has category IV PLK2 specificity (Inhibits PLK2
activity and has no, or reduced, effect on PLK4) and is a candidate
for Parkinson's disease therapeutics.
[0126] Accordingly, in one aspect the invention provides a method
for inhibiting phosphorylation of alpha-synuclein in a mammalian
cell by contacting the cell with an amount of BI 2536 that reduces
PLK2 activity in the cell such that phosphorylation of
alpha-synuclein is reduced. In a related aspect, the invention
provides a method of treating a patient diagnosed with Parkinson's
disease by administering a therapeutically effective amount of BI
2536.
[0127] U.S. Pat. No. 6,861,422, incorporated herein by reference,
describes BI 2536 and structurally related dihydropteridinone
kinase inhibitors. Dihydropteridinone compounds that inhibit PLK2
are useful for inhibiting phosphorylation of synuclein.
[0128] Another exemplary compound for use according to the
invention is the compound ELN-481080 having the structure:
##STR00002##
See, McInnes et al., 2006, Current Topics in Medicinal Chemistry
5:181-97 (Compound 8). Also see US 2006/0040997, "Benzthiazole-3
oxides useful for the treatment of proliferative disorders"
incorporated herein by reference. See Example 14, below.
[0129] Several inhibitors of polo-like kinases are described in
Johnson et al., 2007, "Pharmacological and functional comparison of
the polo-like kinase family: insight into inhibitor and substrate
specificity" Biochemistry 46:9551-63. Of those characterized in
that study, several preferentially inhibit PLK2 and may be used as
therapeutic agents.
[0130] For example, CHIR-258 (3) is a multitarget growth factor
kinase inhibitor developed for treatment of t(4; 14) multiple
myeloma (Trudel et al., 2005, Blood. 105:2941-8).
##STR00003##
K.sub.i values for CHIR-258 for PLK1, PLK2, PLK3 and PLK4 are
>20, 0.85, >20, and 1.4 uM respectively meaning CHR-258 is a
Type VI inhibitor (inhibits PLK2 activity and has reduced effect on
PLK1, PLK3 and PLK4). Although CHIR-258 is an inhibitor of receptor
tyrosine kinases, warranting study of the side effect profile,
targeted delivery to the brain and/or particular treatment regimens
can be investigated to determine whether the compound has an
acceptable therapeutic index for treatment and prevention of
Parkinson's Disease, and a clinically acceptable side effect
profile. CHIR-258 and related quinolinone derivatives are described
in WO03087095, incorporated herein by reference.
[0131] Sunitinib (SU11248) (4) is an example of a Type IV
inhibitor, based on the Johnson et al., 2007, studies (inhibits
PLK2 activity with reduced effect on PLK1 or PLK3). Sunitinib was
identified as an inhibitor of FLT3 receptor tyrosine kinase (RTK)
approved for treatment of advanced renal cell carcinoma and
gastrointestinal stromal tumors that are refractory or intolerant
to imatinib (Gleevec). O'Farrell et al., 2003, Blood
101:3597-605.
##STR00004##
[0132]
5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-{[2-(trifluoromethyl)-benz-
yl]oxy}thiophene-2-carboxamide (5) is an example of a Type III
agent (inhibits PLK2 activity and has reduced effect on PLK4). The
compound was initially identified as a selective thiophene
benzimidazole ATP-competitive inhibitor of PLK1 and PLK3 for
treatment of neoplasms. Lansing et al., 2007, Mol Cancer Ther.
6:450-9. Also see US 20060074119, incorporated herein by reference,
describing other inhibitors of PLKs.
##STR00005##
[0133] Compounds 6 and 7 are indazole-pyridine based inhibitors of
protein kinase B/Akt proposed as antitumor agents (see Woods et
al., 2006, Bioorg Med. Chem. 14:6832-46). Each has a lower K.sub.i
for PLK2 than for PLK1 or PLK3 (Type IV inhibitor) and may be used
for treatment of Parkinson's disease.
##STR00006##
[0134] In some embodiments the agent is a naturally occurring
agent. In some embodiments the agent has a molecular weight less
than 4000, sometimes less than 3000, sometimes less than 2000,
usually less than 1000, and sometimes less than 500.
[0135] In certain embodiments the therapeutic agent is a small
molecule other than a thiazolidone. In certain embodiments the
therapeutic agent is a small molecule other than X, where X is one
of, or an independently selected one or more of, (i) a
thiazolidone, (ii) a quinazoline, (iii) a pyrimidine, (iv)
pyrido[d]-pyrimidine, (v) a pyrimidol[d]pyrimidine, (vi) a
pyrazolo[d]-pyrimidiene, (vii) a pyrrolo[d]pyrimidine, (viii) a
pheylamino-pyrimidines, (ix) phenylamino-pyrimidine derivative, (x)
a indazole-pyridine derivative, (xi) a carboxylic acid derivative,
(xii) a 1-oxo-3-aryl-1H-indene-2-carboxylic acid derivative, (xiii)
a substituted indolin-2-one, (xiv) an aniline-phthalazine
derivative; (xv) a quinolinone derivative, (xvi) a thiazolidinone
compound, (xvii) a benzthiazole-3 oxide (xviii) a
dihydropteridinone or (xix) an azaindazole compound.
[0136] In certain embodiments the therapeutic agent is a small
molecule with the proviso it is not a compound described in US
2007/0179177. In certain embodiments the therapeutic agent is a
small molecule with the proviso it is not a compound described in
US 2007/0010565. In certain embodiments the therapeutic agent is a
small molecule with the proviso it is not a compound described in
US 2007/0037862. In certain embodiments the therapeutic agent is a
small molecule with the proviso it is not a compound described in
2007/0010566. In certain embodiments the therapeutic agent is a
small molecule with the proviso it is not a compound described in
2006/0079503. In certain embodiments the therapeutic agent is a
small molecule with the proviso it is not a compound described in
US 2006/0223833. Each of the aforelisted publications is
incorporated herein by reference.
VI. Methods of Treatment
[0137] The invention provides several methods of preventing or
treating Lewy Body disease in patients suffering from or at risk of
such disease. Therapeutic agents include any of the agents
described above that inhibit phosphorylation of alpha-synuclein
and/or reduce total levels of alpha-synuclein.
[0138] The experimental examples below provide strong evidence that
PLK2 is a synuclein kinase. Thus, in a particular aspect, the
invention provides a method of effecting treatment or prophylaxis
of an LB disease by administering to a patient suffering from or at
risk of the disease an effective regime of an agent effective to
suppress activity or expression of PLK2. It is preferred that the
agent shows a high level of specificity for PLK2.
[0139] Patients amenable to treatment include individuals at risk
of disease of a LBD but not showing symptoms, as well as patients
presently showing symptoms. Therefore, the present methods can be
administered prophylactically to individuals who have a known
genetic risk of a LBD. Such individuals include those having
relatives who have experienced this disease, and those whose risk
is determined by analysis of genetic or biochemical markers.
Genetic markers of risk toward PD include mutations in the
alpha-synuclein or Parkin, UCHLI, LRRK2, and CYP2D6 genes;
particularly mutations at positions 30 and 53 of the
alpha-synuclein gene. Another genetic marker of risk toward PD
includes measuring the levels or SNCA dosage or transcription.
Individuals presently suffering from Parkinson's disease can be
recognized from its clinical manifestations including resting
tremor, muscular rigidity, bradykinesia and postural
instability.
[0140] In some methods, the patient is free of clinical symptoms or
risk factors for any amyloidogenic disease other than one
characterized by Lewy bodies. In some methods, the patient is free
of clinical symptoms or risk factors of any disease characterized
by extracellular amyloid deposits.
[0141] In some methods the patient is not diagnosed with cancer
and/or Alzheimer's disease.
[0142] Treatment typically entails multiple dosages over a period
of time. Treatment can be monitored by assaying signs or symptoms
of the disease being treated relative to base line measurements
before initiating treatment. In some methods, administration of an
agent results in reduction of intracellular levels of aggregated
alpha-synuclein. In some methods, administration of the agent
results in a reduction in levels of phosphorylated synuclein. In
some methods, administration of an agent results in improvement in
a clinical symptom of a LBD, such as motor or cognitive function in
the case of Parkinson's disease.
[0143] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of a LBD in regime comprising an amount and
frequency of administration of the composition or medicament
sufficient to eliminate or reduce the risk, lessen the severity, or
delay the outset of the disease, including physiological,
biochemical, histologic and/or behavioral symptoms of the disease,
its complications and intermediate pathological phenotypes
presenting during development of the disease. In therapeutic
applications, compositions or medicates are administered to a
patient suspected of, or already suffering from such a disease in a
regime comprising an amount and frequency of administration of the
composition sufficient to cure, or at least partially arrest, the
symptoms of the disease (physiological, biochemical, histologic
and/or behavioral), including its complications and intermediate
pathological phenotypes in development of the disease. An amount
adequate to accomplish therapeutic or prophylactic treatment is
defined as a therapeutically- or prophylactically-effective dose. A
combination of amount and dosage frequency adequate to accomplish
therapeutic or prophylactic treatment is defined as a
therapeutically or prophylactically-effective regime.
[0144] Effective doses of the compositions of the present
invention, for the treatment of the above described conditions vary
depending upon many different factors, including means of
administration, target site, physiological state of the patient,
whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
Usually, the patient is a human but nonhuman mammals including
transgenic mammals can also be treated. Treatment dosages need to
be titrated to optimize safety and efficacy.
[0145] The dosage and frequency of administration can vary
depending on whether the treatment is prophylactic or therapeutic.
Guidance can be obtained from the dosing schedules of kinase
inhibitors currently approved or in clinical trials for other
indications. Dosages in the range of 0.1-1000 mg, preferably 10-500
mg, may be used. Frequency of dosing (e.g., daily, weekly or
monthly) depends on the half-life of the drug. In prophylactic
applications, a relatively low dosage is administered at relatively
infrequent intervals over a long period of time. Some patients
continue to receive treatment for the rest of their lives. In
therapeutic applications, a relatively high dosage at relatively
short intervals is sometimes required until progression of the
disease is reduced or terminated, and preferably until the patient
shows partial or complete amelioration of symptoms of disease.
Thereafter, the patent can be administered a prophylactic
regime.
[0146] Therapeutic agents can be administered by parenteral,
topical, intravenous, oral, subcutaneous, intraarterial,
intracranial, intrathecal, intraperitoneal, intranasal or
intramuscular means for prophylactic and/or therapeutic treatment.
In some methods, agents are injected directly into a particular
tissue where deposits have accumulated, for example intracranial
injection. In some methods, agents are administered as a sustained
release composition or device, such as a Medipad.TM. device. Small
molecules that pass through the blood brain barrier sufficiently
are usually administered orally, but can also be administered
intravenously.
[0147] Agents of the invention can optionally be administered in
combination with other agents that are at least partly effective in
treatment of LBD. Agents of the invention can also be administered
in conjunction with other agents that increase passage of the
agents of the invention across the blood-brain barrier.
[0148] Agents of the invention are often administered as
pharmaceutical compositions comprising an active therapeutic agent
and a variety of other pharmaceutically acceptable components. See
Remington's Pharmaceutical Science (15th ed., Mack Publishing
Company, Easton, Pa., 1980). The preferred form depends on the
intended mode of administration and therapeutic application. The
compositions can also include, depending on the formulation
desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent is selected so as not to affect the biological activity of
the combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0149] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized Sepharose(TM), agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes).
[0150] For parenteral administration, agents of the invention can
be administered as injectable dosages of a solution or suspension
of the substance in a physiologically acceptable diluent with a
pharmaceutical carrier that can be a sterile liquid such as water,
oils, saline, glycerol, or ethanol. Additionally, auxiliary
substances, such as wetting or emulsifying agents, surfactants, pH
buffering substances and the like can be present in compositions.
Other components of pharmaceutical compositions are those of
petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, and mineral oil. In general, glycols such
as propylene glycol or polyethylene glycol are preferred liquid
carriers, particularly for injectable solutions. Antibodies can be
administered in the form of a depot injection or implant
preparation which can be formulated in such a manner as to permit a
sustained release of the active ingredient. An exemplary
composition comprises monoclonal antibody at 5 mg/mL, formulated in
aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl,
adjusted to pH 6.0 with HCl. Compositions for parenteral
administration are typically substantially sterile, substantially
isotonic and manufactured under GMP conditions of the FDA or
similar body.
[0151] Compositions may be prepared as injectables, either as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection can also
be prepared. The preparation also can be emulsified or encapsulated
in liposomes or micro particles such as polylactide, polyglycolide,
or copolymer for enhanced adjuvant effect, as discussed above (see
Langer, Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery
Reviews 28, 97-119 (1997). The agents of this invention can be
administered in the form of a depot injection or implant
preparation which can be formulated in such a manner as to permit a
sustained or pulsatile release of the active ingredient.
[0152] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications. For
suppositories, binders and carriers include, for example,
polyalkylene glycols or triglycerides; such suppositories can be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0153] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins
(See Glenn et al., Nature 391, 851 (1998)). Co-administration can
be achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein. Alternatively, transdermal delivery can be achieved
using a skin patch or using transferosomes (Paul et al., Eur. J.
Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta
1368, 201-15 (1998)).
VII. Lewy Body Diseases
[0154] Lewy Body Diseases (LBD) are characterized by degeneration
of the dopaminergic system, motor alterations, cognitive
impairment, and formation of Lewy bodies (LBs). (McKeith et al.,
Clinical and pathological diagnosis of dementia with Lewy bodies
(DLB): Report of the CDLB International Workshop, Neurology (1996)
47:1113-24). Lewy Bodies are spherical protein deposits found in
nerve cells. Their presence in the brain may disrupt the brain's
normal function interrupting the action of chemical messengers
including acetylcholine and dopamine. Lewy Body diseases include
Parkinson's disease (including idiopathic Parkinson's disease
(PD)), Diffuse Lewy Body Disease (DLBD) also known as Dementia with
Lewy Bodies (DLB), Combined Alzheimer's and Parkinson disease and
multiple system atrophy (MSA). DLBD shares symptoms of both
Alzheimer's and Parkinson's disease. DLBD differs from Parkinson's
disease mainly in the location of Lewy Bodies. In DLBD Lewy Bodies
form mainly in the cortex. In Parkinson's disease, they form in
selected regions throughout the brain stem, midbrain, and in
advanced disease, cerebral cortex. See, Braak et al., 2003,
"Staging of brain pathology related to sporadic Parkinson's
disease" Neurobiology of Aging 24:197-211. Other Lewy Body diseases
include Pure Autonomic Failure, Lewy body dysphagia, Incidental
LBD, Inherited LBD (e.g., mutations of the alpha-synuclein gene,
PARK3 and PARK4) and Multiple System Atrophy (e.g.,
Olivopontocerebellar Atrophy, Striatonigral Degeneration and
Shy-Drager Syndrome).
VIII. Identification of Modulators of Kinases that Directly or
Indirectly Phosphorylate Alpha-Synuclein
[0155] Agents that modulate expression or activity of a kinase that
directly or indirectly phosphorylates alpha-synuclein can be
identified by a variety of assays. Particularly preferred are
agents that inhibit kinases PLK2 or GRK6, or APEG1, CDC7L1, MET
GRK1, GRK2, GRK6, IKBKB, CKII and GRK7, or agents that activate
PRKG1, MAPK13, and GAK. Agents that modulate expression can be
identified in cell-based assays in which an agent under test is
introduced into a cell expressing alpha-synuclein and a kinase that
directly or indirectly effects phosphorylation of the
alpha-synuclein or modulates levels of total alpha-synuclein.
Optionally, particularly for PLK2, synphilin can be expressed as
well to augment activity of the kinase. The agent can be introduced
directly or in the form of a nucleic acid encoding the agent and
capable of expressing the agent. The cell can naturally express the
alpha-synuclein and kinase or one or both of these can be
introduced into the cell by transfection of a suitable nucleic
acid. The effect of the agent on expression of the kinase can be
measured directly from the level of the kinase or its mRNA, or
indirectly by measuring the level of phosphorylated alpha-synuclein
or total alpha-synuclein as described above. The level of kinase
mRNA can be assayed by a hybridization type assay. The level of the
kinase can be assayed by an immunoassay. Optionally, the kinase is
tagged with a peptide label such as Flag.TM. (Hopp et al.,
BioTechnology 6, 1204-1210 (1988)) to facilitate detection. An
agent that decrease the level of the kinase, decrease the level of
phosphorylation of alpha-synuclein and/or decrease the level of
total alpha-synuclein relative to similar control cells not treated
with an agent have a pharmacological activity potentially useful
for treatment of Lewy body diseases.
[0156] Agents are also screened for activity to modulate activity
of a kinase suspected of phosphorylating alpha-synuclein or
increasing total levels of alpha-synuclein. An initial screen can
be performed to select a subset of agents capable of specifically
binding to a kinase. Such an assay can be performed in vitro using
an isolated kinase or fragment thereof having kinase activity.
[0157] In one embodiment an agent that reduces alpha-synuclein
phosphorylation in a mammalian cell expressing alpha-synuclein, and
which may be used as a therapeutic agent, is identified based on
the following criteria: a) reduces activity of PLK2 in a cell
expressing PLK2; and b) does not reduce activity of PLK1 in a cell
expressing PLK1, or reduces activity of PLK1 at a higher EC.sub.50
than for PLK2; and/or c) does not reduce activity of PLK3 in a cell
expressing PLK3, or reduces activity of PLK3 at a higher EC.sub.50
than for PLK2; and/or d) does not reduce activity of PLK4 in a cell
expressing PLK4, or reduces activity of PLK4 at a higher EC.sub.50
than for PLK2. In one embodiment an agent is selected that meets
all of criteria a-d. The mammalian cell may be a cell
overexpressing alpha-synuclein (e.g., transfected with a vectors
expressing exogenous, e.g., human, alpha-synuclein). The mammalian
cell may be a neuronally-derived cell such as, for example, cells
from mouse cortical cell cultures, rat ventral mesencephalon cell
cultures, or other neuronal cells from humans or non-human
mammals.
[0158] Agents identified by such a screen can then be assayed
functionally. Agents can also be directly assayed functionally
without the binding assay. For a kinase that directly
phosphorylates alpha-synuclein, modulators can be screened by an in
vitro assay combining the kinase, alpha-synuclein, ATP and the
modulator in comparison with a control in which the modulator is
omitted. Optionally, synphilin can be included as well to increase
phosphorylation particularly if the kinase is PLK2. The modulator
has potentially useful pharmacological activity if it reduces the
level of phosphorylation beyond the margin of typical experimental
error relative to the control.
[0159] Agents can also be screened in cells expressing
alpha-synuclein and the kinase under test, and optionally,
particularly if the kinase is PLK2, synphilin. Such screens are
effective regardless of whether the kinase phosphorylates
alpha-synuclein directly or indirectly, or otherwise affects levels
of alpha-synuclein. Cells are contacted with the agent and levels
of total alpha-synuclein and phosphorylated alpha-synuclein are
measured, as above, relative to a control cell not treated with the
agent. A reduction in the level of phosphorylated alpha-synuclein
or total alpha-synuclein relative to the corresponding level in a
control cell not treated with the agent, beyond the margin of
typical experimental error, is an indication that the compound has
pharmacological activity potential useful in treating Lewy body
diseases. Agents may also be screened in cells for ability to
reduce aggregation of alpha-synuclein in the cell.
[0160] Agents can also be screened in transgenic animal models of
Lewy body disease, alone or in combination with the other assays
described above. Total levels of alpha-synuclein, phosphorylated
alpha-synuclein or Lewy-like bodies or other indica of Lewy Body
pathology or symptoms are measured in a transgenic animal treated
with an agent under test relative to corresponding levels in a
similar control animal not treated with the agent. A reduction in
one or more of these levels is an indication, the agent has
pharmacological activity potentially useful in treating Lewy body
diseases.
[0161] The kinase used in the above assays and cellular and
transgenic models is preferably a human kinase having a sequence in
one of the references or accession numbers provided in this
disclosure. However, allelic (variants within a species) and
species variants (variants between species) of such a kinase can
also be used, as can variants having at least 90% sequence identity
to such a kinase. For subsequent clinical use, agents identified by
such assays are capable of modulating the activity or expression of
a natural kinase, preferably a form occurring in humans.
IX. Transgenic Animal and Cellular Models of Lewy Body Disease
[0162] Transgenic animal models are useful for testing the capacity
of kinases to effect phosphorylation of alpha-synuclein and
formation of Lewy-like bodies as described above. Transgenic
animals are also useful for screening agents for activity in
modulating phosphorylation or production of alpha-synuclein.
Particularly preferred are agents that inhibit or are suspected of
inhibiting kinases including PLK2 and GRK6, or APEG1, CDC7L1, MET
GRK1, GRK2, GRK6, IKBKB, CKII and GRK7. Also preferred are agents
that activate or are suspected of activating kinases PRKG1, MAPK13,
and GAK. Further, knockout animals (i.e., animals in which an
endogenous kinase is inactivated either by insertional inactivation
or trans inhibition by an siRNA, zinc finger protein or the like)
are useful for identifying the effect of eliminating activity of a
kinase on an animal. For example, analysis of a PLK2-knockout mouse
can indicate whether inhibitors of PLK2 have any side effects.
Analogous knockouts can reveal similar information for other
kinases.
[0163] In general, transgenic models have a genome comprising an
alpha-synuclein transgene in operable linkage with one or more
regulatory sequences to ensure its expression. Expression of the
transgene leads to Lewy-body like deposits of alpha-synuclein in
the brain of the animal. Several such transgenic animals have been
described in the scientific and patent literature (see Masliah et
al., Am. J. Pathol. (1996) 148:201-10 and Feany et al., Nature
(2000) 404:394-8)), U.S. Pat. No. 5,811,633 (for transgenic animals
with a mutant form of APP). Some transgenic animals express variant
or mutant alpha-synuclein, such as familial mutants A30P, A53T, and
E46K of alpha synuclein. Some transgenic animals have an additional
transgene, such as a transgene encoding a kinase as described
above. Transgenic animals bearing a transgene expressing
alpha-synuclein protein can also be crossed with other transgenic
models of neurogenic disease, such as models of Alzheimer's
disease. For example, transgenic animals bearing a transgene
expressing a truncated alpha-synuclein protein can be crossed with
transgenic animals bearing a transgene expressed APP with a FAD
mutation as described by e.g., Games et al., Nature 373, 523 (1995)
McConlogue et al., U.S. Pat. No. 5,612,486, Hsiao et al., Science
274, 99 (1996); Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94,
13287-13292 (1997); Sturchler-Pierrat et al., Proc. Natl. Acad.
Sci. USA 94, 13287-13292 (1997); Borchelt et al., Neuron 19,
939-945 (1997)). The procedure for performing such a cross is
described by e.g., Masliah et al., PNAS USA 98:12245-12250 (2001),
which reports a cross between transgenic mice expressing a full
length alpha-synuclein with PDAPP mice as described by Games et al
Transgenic animals of the invention are preferably rodents, such as
mice or rats, or insects, such as Drosophila. Transgenic animals
can be produced by introduction of a transgene at the germline
stage in which case all or substantially all (except for rare loss
through somatic mutation) of the cells of the transgenic animal
include the transgene integrated into the genome. Transgenes can be
introduced by microinjection, nuclear transfer or viral infection
into cells or animals. Adeno Associated Viruses and Lentiviruses
are particularly suitable for the latter. Alternatively, transgenes
can be introduced by viral infection into the brain of the animal.
Such transgenes are not part of the germline of recipient animals
but can be targeted to regions of the brain responsible for disease
(e.g., the substantia nigra). Such animal models incorporate an
alpha-synuclein into the genome of brain cells and are disposed to
develop at least one characteristic of LBD disease. Lentiviruses
provide a suitable vehicle for so introducing an alpha-synuclein
transgene into the brain (see Brain Pathology 13, 364-372 (2003);
Bjorklund, Trends Neurosci. 26, 386-92 (2003), Lotharius et al., J.
Biol. Chem. 277, 38884-94 (2002), Zhou et al., Brain Research 866,
33-43 (2000)). Transgenic animals can also include a transgene
capable of expressing one of the kinases of the invention (e.g., a
nucleic acid encoding the kinase in operable linkage with
regulatory elements to ensure its expression in the brain of an
animal), instead of or as well as a transgene expressing alpha
synuclein. Optionally, a transgene expressing synphilin can be
included as well. Some cellular models express variant or mutant
alpha-synuclein, such as familial mutants A30P, A53T, and E46K of
alpha synuclein. Some cells have an additional transgene, such as a
transgene encoding a kinase as described above, e.g., PLK2.
[0164] Cellular models of Lewy body disease can also be used in the
screening methods of the invention. Cells transfected with
alpha-synuclein form inclusion bodies containing aggregated
alpha-synuclein. The transformed cells are preferably neuronal
cells, such as GT1-7 neuronal cells (Hsue et al. Am. J. Pathol.
157:401-410 (2000)), PC12 cells or SY5Y neuroblastoma cells. PEAK
and/or HCC cells can also be used (see Example 10). The cells are
preferably human cells. A vector comprising a segment encoding a
form of alpha-synuclein operably linked to one or more regulatory
sequences that ensure expression of the expression is transfected
into the cells. Cells can also be transfected with a nucleic acid
encoding a kinase of the invention as described above. Transfected
cells can be used to screen agents for activity in clearing
alpha-synuclein inclusions. An exemplary cellular model is
identified in Example 10 in which HCC neuronal cells are
transfected with synuclein and PLK2 with the result that
aggregation and phosphorylation of the synuclein matching LB
formation occurs. In order to identify inhibitors of the kinase,
the cell is contacted with the inhibitor and a reduction in the
amount of phosphorylation and/or aggregation is identified.
X. Alpha-Synuclein Isolation and Phosphorylation
[0165] Human alpha-synuclein is a polypeptide of 140 amino acids
having the following amino acid sequence:
TABLE-US-00005 (SEQ ID NO:1) MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA
GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA
ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
[0166] (Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6);
GenBank accession number: P37840). The protein has three recognized
domains, a KTKE repeat domain covering amino acids 1-61, a NAC
(Non-Amyloid Component) domain running from about amino acids
60-95, and a C-terminal acidic domain running from about amino acid
98 to 140.
[0167] Unless otherwise apparent from the context, reference to
alpha-synuclein includes the natural human amino acid sequence
indicated above as well as analogs including allelic, species and
induced variants (e.g., E83Q, A90V, A76T) having at least 90%
sequence identity to natural human alpha-synuclein. Amino acids of
analogs are assigned the same numbers as corresponding amino acids
in the natural human sequence when the analog and human sequence
are maximally aligned. Analogs typically differ from naturally
occurring peptides at one, two or a few positions, often by virtue
of conservative substitutions. Some natural allelic variants are
genetically associated with hereditary LBD. The term "allelic
variant" is used to refer to variations between genes of different
individuals in the same species and corresponding variations in
proteins encoded by the genes. Allelic variants include familial
mutants or variants, such as E46K, A30P and A53T (the first letter
indicates the amino acid in SEQ ID NO:1, the number is the residue
position in SEQ ID NO:1, and the second letter is the amino acid in
the allelic variant). Analogs can include any combination of
allelic variants. The A53T variation is associated with enhanced
levels of phosphorylation at position 129 of alpha-synuclein in an
individual having the mutation relative to the norm of
phosphorylation in undiseased individuals who lack the
mutation.
[0168] Alpha-synuclein, its fragments, and analogs can be
synthesized by solid phase peptide synthesis or recombinant
expression, or can be obtained from natural sources. Automatic
peptide synthesizers are commercially available from numerous
suppliers, such as Applied Biosystems, Foster City, Calif.
Recombinant expression can be in bacteria, such as E. coli, yeast,
insect cells or mammalian cells. Procedures for recombinant
expression are described by Sambrook et al., Molecular Cloning: A
Laboratory Manual (C.S.H.P. Press, NY 2d ed., 1989).
[0169] A method was developed herein to prepare large amounts of
wild-type phospho-S129 alpha-synuclein, mutant and/or familial
forms in a bacterial expression system. When recombinant hPLK2 was
co-expressed with alpha-synuclein in bacteria, the phospho-S129
alpha-synuclein that was produced in the cell was recovered with a
very high yield and purity. This is because, unlike most E. coli
proteins, the alpha-synuclein could resist heating. After boiling
of the bacterial lysate, alpha-synuclein purity reached about 95%
before chromatography.
[0170] To co-express recombinant protein in a bacterial system, the
plasmids harboring each gene of interest were chosen be compatible
within the bacterial cell by ensuring that they possessed a
different origin of replication and a different antibiotic
selection. The alpha-synuclein gene was sub-cloned into a pDEST24
compatible vector, pCDF1b. BL21-DE3 bacteria were then
co-transformed with both the pDEST24 containing wild-type hPLK2 or
hPLK2 constitutive mutant constructs without a GST tag and the
pCDF1b/AS plasmid. The bacterial lysates were boiled and the
supernatant, expected to contain alpha-synuclein was analyzed by
Western blot with an anti-phospho-S129-alpha-synuclein antibody
(11A5), by using the SYPRO Ruby.TM. and ProZDiamond.TM. dyes (total
protein and phospho-Ser/Thr specific dye respectively), by SDS-PAGE
and by mass spectrometry, with the results that a fairly pure
phospho-S129-alpha-synuclein product was isolated that, upon
analysis by mass spectrometry was revealed to be more than 95%
phosphorylated. To ensure that the final product was 100%
phosphorylated and highly pure, the supernatant of the last
centrifugation was passed through an 11A5-sepharose-affinity
purification column one or more times. Any contamination was
removed using HPLC.
XI. Examples
Example 1
Screen for Kinases that Modulate Alpha-Synuclein
Phosphorylation
[0171] To identify the kinase or kinases that phosphorylates
.alpha.-synuclein at serine-129 an siRNA kinase library (Ambion)
was screened on cells containing a quantifiable amount of
phosphorylated .alpha.-synuclein. Human embryonic kidney cell line
HEK293 cells (PEAK cells) stably transfected with human wild-type
.alpha.-synuclein under control of a CMV promoter (PEAK-Syn cells)
were transfected with 100 nM siRNAs targeting 597 human kinases and
were assayed by ELISA assays to quantitate total and
phospho-synuclein levels. Ninety-five kinases with siRNAs that
altered the percentage of phosphorylated alpha-synuclein were
identified (see Tables 1-2). Of those, 28 belonged to the class of
kinases that phosphorylate serine residues and hence were capable
of directly phosphorylating .alpha.-synuclein at serine-129. Others
were tyrosine kinases. Although tyrosine kinases do not
phosphorylate .alpha.-synuclein at ser-129 directly, they can act
as upstream regulators of the alpha-synuclein kinase. Two of these
ser/thr kinases, casein kinase 2 and calcium/calmodulin dependent
protein kinase II, have been reported to phosphorylate
.alpha.-synuclein in vitro (Pronin et al, J. Biol. Chem. 275:
26515-26522 (2000), Okochoa et al, J. Biol. Chem. 275: 390-397
(2000); Nakajo et al, Eur. J. Biochem. 217: 1057-1063 (1993) and a
casein kinase 2 inhibitor has been reported to decrease
phospho-synuclein levels in cells (Okochoa et al, 2000). Several of
the GRK family members (although not GRK6) have been reported to
phosphorylate alpha-synuclein in vitro (Pronin et al, 2000). GRK2
expression in flies has been reported to increase phospho-synuclein
levels (Chen and Feany, Nature Neurosci. 8: 657-663 (2005)).
[0172] In addition to kinases that lower phospho-synuclein levels,
99 kinases whose siRNAs altered total .alpha.-synuclein levels in
the PEAK-Syn cells were identified in Table 2 and included
fucokinase (FUK), Genbank number NM.sub.--145059; Protein Kinase N1
(PRKCL1, PKN1), Genbank number NM.sub.--002741; Doublecortin and
CaM kinase-like 1 (DCAMKL1) NM.sub.--004734; Branched chain
Ketoacid dehydrogenase kinase (BCKDK) NM.sub.--005881; Aurora
Kinase C (AURKC, STK13); NM.sub.--003160, Kinase suppressor of ras
2 (FLJ25965), NM.sub.--173598; FLJ32704; MAP2K6; and Tousled-like
kinase 2 (TLK2) NM.sub.--006842. The mechanism of action may
involve either regulation of alpha-synuclein turnover or synthesis
(See Table 2).
[0173] Tables 1A, 1B and 1C show kinases whose inhibition modulates
the phosphorylation at position ser-129. Table 1A, B, and C differ
in the type of kinase. Table 1A shows kinases that can
phosphorylate serine residues and often tyrosine and/or threonine
as well. Table 1B shows tyrosine kinases that cannot (so far as is
known) modify serine residues. Table 1C shows kinases not known to
have phosphorylate proteins. Kinases from the upper portion of
Table 1A are candidates for direct phosphorylation of ser-129 of
alpha-synuclein. Kinases from the upper part of Table 1B are also
useful therapeutic targets via roles indirectly phosphorylating
alpha-synuclein. Proteins in the upper part of Table 1C are also
useful therapeutic targets for the same reason. Cols. 1, 2 and 3 of
each table indicate the gene name, kinase name and Genbank
accession number of kinases. The next column indicates whether
inhibiting expression of the kinase decreased ("down") or increased
("up") phosphorylation. The next three columns indicate the number
of standard deviations the measured level of phosphorylation
departs from the mean in three independent experiments. The final
two columns indicate the kinase family (i.e., amino acid
specificity) and group.
[0174] Tables 2 and 3 show kinases whose inhibition modulates the
overall levels of human alpha-synuclein without changing the
percentage of phosphorylation. Table 2 shows all of the kinases
with the strongest reduction in levels of human alpha-synuclein.
The columns are labeled similarly to Tables 1A, 1B and 1C.
[0175] Tables 1A-C: Complete list of Kinase candidates that reduce
phosphorylation
TABLE-US-00006 TABLE 1A Serine/Threonine Kinases Up/ # of # of # of
Kinase Gene Name Kinase Name Genbank Down SD SD SD Kinase Family
Group GPRK6 G protein-coupled receptor NM_002082 Down 1.25 1.25
Ser/Thr AGC kinase 6 PDPK1 3-Phosphoinositide NM_002613 Down 2.25
2.75 Ser/Thr AGC dependent protein kinase-1 FLJ11159 RIO kinase 2
(yeast) NM_018343 Down 1.5 1.5 Ser/Thr Atypical *APEG1 Aortic
preferentially NM_005876 Down 1.75 2 2.25 Ser/Thr/Tyr CAMK
expressed gene 1 ARK5 AMP-activated protein NM_014840 Down 1 1 1.5
Ser/Thr CAMK kinase family member 5 *CAMK1 Calcium/calmodulin-
NM_003656 Down 1.5 1.75 2 Ser/Thr CAMK dependent protein kinase I
SSTK Serine/threonine protein NM_032037 Down 1 1 Ser/Thr CAMK
kinase SSTK PHKG2 Phosphorylase kinase, NM_000294 Down 1 2 Ser/Thr
CAMK gamma 2 (testis) CASK Calcium/calmodulin- NM_003688 Down 1.25
1.25 Ser/Thr CAMK dependent serine protein kinase PRKAA2 Protein
kinase, AMP- NM_006252 Down 1 1.75 Ser/Thr CAMK activated, alpha 2
catalytic subunit CDK8 Cyclin-dependent kinase 8 NM_001260 Down 1 1
1.25 Ser/Thr CMGC *CDC2L5 Cell division cycle 2-like 5 NM_003718
Down 1.25 1.5 Ser/Thr/Tyr CMGC ERK8 Extracellular signal- NM_139021
Down 1.5 1.5 Ser/Thr CMGC regulated kinase 8 *CDK4 Cyclin-dependent
kinase 4 NM_000075 Down 1 1 Ser/Thr** CMGC CLK3 CDC-like kinase 3
NM_003992 Down 1 1.5 Ser/Thr/Tyr CMGC PRP4 Pre-mRNA processing
NM_003913 Down 1.25 1.75 Ser/Thr GO factor 4 homolog B (yeast)
CKIIA2 Casein kinase 2, alpha NM_001896 Down 1 1.5 Ser/Thr Other
prime subunit PLKII/SNK Polo like kinase 2 NM_006622 Down 1 1.5
2.25 Ser/Thr Other CKIIA1 Casein kinase 2, alpha NM_001895 Down 1
1.75 2.25 Ser/Thr Other subunit MAP2K1 mitogen-activated protein
NM_002755 Down 1 1.75 Ser/Thr/Tyr STE kinase kinase 1 (MEK1; MKK1)
MAP2K4 mitogen-activated protein NM_003010 Down 1 2 Ser/Thr/Tyr STE
kinase kinase 4 (MEK4; MKK4; JNKK) MAP2K5 mitogen-activated protein
NM_002757 Down 1 1.25 Ser/Thr/Tyr STE kinase kinase 5 (MEK5; MKK5)
TESK2 testis-specific kinase 2 NM_007170 Down 1 1 1.5 Ser/Thr/Tyr
TKL RIPK3 receptor-interacting serine- NM_006871 Down 1 1.25
Ser/Thr/Tyr TKL threonine kinase 3 PRKG2 protein kinase, cGMP-
NM_006259 Down 2.25 Ser/Thr AGC dependent, type II JIK TAO Kinase 3
(MAP3K18) NM_016281 Down 2 Ser/Thr STE PAK6 p21(CDKN1A)-activated
NM_020168 Down 2.5 Ser/Thr STE kinase 6 *CAMK2D Calcium/calmodulin-
NM_001221 Down 1.25 1.5 2 Ser/Thr CAMK dependent protein kinase II-
delta *CDC7L1 CDC7 cell division cycle 7- NM_003503 Down 1.25 2 3
Ser/Thr Other like 1 CDK5 Cyclin-dependent kinase 5 NM_004935 Up 1
1 1.25 Ser/Thr CMGC PRKWNK1 Protein kinase, lysine NM_018979 Up 1
1.5 6.25 Ser/Thr Other deficient 1 CDC42BPB CDC42 binding protein
NM_006035 Up 2.5 3.75 Ser/Thr AGC kinase beta (DMPK-like) PRKCI
protein kinase C, iota NM_002740 Up 1 1.75 Ser/Thr AGC PRKG1
Protein kinase, cGMP- NM_006258 Up 1 1.25 Ser/Thr AGC dependent,
regulatory, Type I SMG1 PI3-kinase-related kinase NM_015092 Up 1.25
1.5 Ser/Thr Atypical SMG1 *BRD3 Bromodomain-containing NM_007371 Up
1.25 1.75 Ser/Thr Atypical protein 3 DAPK1 Death-associated protein
NM_004938 Up 1 1.25 1.25 Ser/Thr CAMK kinase 1 PASK PAS domain
containing NM_015148 Up 1 1 Ser/Thr CAMK serine/threonine kinase
LOC283629 Chromosome 14 open NM_174944 Up 1 1.25 Ser/Thr/Tyr CAMK
reading frame 20; Testis- specific serine kinase 4 CDC2 Cell
division cycle 2, G1 to NM_001786 Up 1.25 2.75 Ser/Thr/Tyr CMGC S
and G2 to M MAPK13 Mitogen-activated protein NM_002754 Up 1 1.25
1.75 Ser/Thr/Tyr CMGC kinase 13 STK35 Serine/threonine kinase 35,
NM_080836 Up 1 1.25 1.5 Ser/Thr Other Clik1 GAK Cyclin G associated
kinase NM_005255 Up 1 1.25 1.75 Ser/Thr Other ANKRD3 ankyrin repeat
domain 3 NM_020639 Up 1 1.75 Ser/Thr/Tyr TKL IRAK3 Interleukin-1
receptor- NM_007199 Up 1.5 1.75 Ser/Thr TKL associated kinase 3
LIMK2 LIM domain kinase 2 NM_005569 Up 1 2 Ser/Thr/Tyr TKL PKMYT1
Protein kinase, membrane- NM_004203 Both 1.25 1.5 1.75 Ser/Thr/Tyr
Other associated, tyrosine/threonine 1 ADRBK2 adrenergic, beta,
receptor NM_005160 Up 2 Ser/Thr AGC kinase 2 (GRK3; BARK2) AKT3
v-akt murine thymoma viral NM_005465 Up 2 Ser/Thr AGC oncogene
homolog 3 (protein kinase B, gamma) CDK10 cyclin-dependent kinase
NM_003674 Up 5.75 Ser/Thr/Tyr CMGC (CDC2-like) 10 EIF2AK3
eukaryotic translation NM_004836 Up 2.5 Ser/Thr Other initiation
factor 2-alpha kinase 3 BIKE BMP2 inducible kinase NM_017593 Up 6
Ser/Thr Other (BMP2K), transcript variant IKBKE inhibitor of kappa
light NM_014002 Up 2 Ser/Thr Other polypeptide gene enhancer in
B-cells, kinase epsilon SDCCAG43 serologically defined colon
NM_006648 Up 4.25 Ser/Thr/Tyr Other cancer antigen 43 FLJ10074
SCY1-like 2 (S. cerevisiae) NM_017988 Up 2.25 Ser/Thr Other
FLJ32685 hypothetical protein NM_152534 Up 3 Ser/Thr/Tyr Other
FLJ32685 NEK11 NIMA (never in mitosis NM_024800 Up 4 Ser/Thr/Tyr
Other gene a)-related kinase 11 TTK TTK protein kinase NM_003318 Up
2.5 Ser/Thr/Tyr Other
TABLE-US-00007 TABLE 1B Tyrosine Kinases Up/ # of # of # of Kinase
Kinase Gene Name Kinase Name Genbank Down SD SD SD Family Group
PDGFRA Platelet-derived growth NM_006206 Down 1.5 2 Tyr TK factor
receptor, alpha SRMS src-related kinase lacking C- NM_080823 Down
1.25 1.75 Tyr TK terminal regulatory tyrosine and N-terminal
myristylation sites PTK6 Protein tyrosine kinase 6 NM_005975 Down
1.25 1.5 Tyr TK ZAP70 zeta-chain (TCR) associated NM_001079 Down
1.5 2 Tyr TK protein kinase 70 kDa ERBB4 v-erb-b2 erythroblastic
NM_005235 Down 1.25 1.5 Tyr TK leukemia viral oncogene homolog 4
(avian) IGF1R insulin-like growth factor 1 NM_000875 Down 1 1.5 Tyr
TK receptor MET met proto-oncogene NM_000245 Down 1 1.5 Tyr TK
(hepatocyte growth factor receptor) MERTK c-mer proto-oncogene
NM_006343 Down 1 1.25 Tyr TK tyrosine kinase JAK2 Janus kinase 2 (a
protein NM_004972 Down 1 1.5 Tyr TK tyrosine kinase) YES1 v-yes-1
Yamaguchi sarcoma NM_005433 Up 1.25 1.25 Tyr TK viral oncogene
homolog 1 ERBB3 v-erb-b2 erythroblastic NM_001982 Up 1 1 Tyr TK
leukemia viral oncogene homolog 3 (avian) EPHA7 EphA7 NM_004440 Up
1 1.25 1.5 Tyr TK BTK Bruton NM_000061 Up 1.25 1.5 Tyr TK
agammaglobulinemia tyrosine kinase EPHB3 EphB3 NM_004443 Up 1 1.75
Tyr TK RET ret proto-oncogene (multiple NM_020975 Up 3 Tyr TK
endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung
disease)
TABLE-US-00008 TABLE 1C No Protein Phosphorylation Activity Up/ #
of # of # of Kinase Kinase Gene Name Kinase Name Genbank Down SD SD
SD Family Group C8FW Tribbles homolog 1 NM_025195 Down 1 1 X CAMK
CHK Choline kinase NM_001277 Down 1 1.5 X GO FLJ13052 NAD kinase
NM_023018 Down 1.25 1.75 X GO FLJ22055 Phosphatidylinositol-4-
NM_024779 Down 1 1 X GO phosphate 5-kinase, type II, gamma CKMT2
Creatine kinase, NM_001825 Down 1.75 2 X GO mitochondrial 2
(sarcomeric) DKFZP586B1621 DKFZP586B1621 protein, NM_015533 Down 1
1.25 X GO function unknown GK Glycerol kinase NM_000167 Down 1.5
1.75 X GO ITPKC Inositol 1,4,5-trisphosphate NM_025194 Down 1 1.25
X GO 3-kinase C NME4 Non-metastatic cells 4, NM_005009 Down 1 1.25
X GO protein expressed in NM23-H6 Non-metastatic cells 6, NM_005793
Down 1.25 1.25 1.75 X GO protein expressed in
(nucleoside-diphosphate kinase) RBSK Ribokinase NM_022128 Down 2.25
X GO ITPKC Inositol 1,4,5-trisphosphate NM_025194 Down 1 1.25 X GO
3-kinase C PMVK Phosphomevalonate NM_006556 Both 1 1.25 2.5 X GO
kinase GS3955 Tribbles homolog 2 NM_021643 Up 1.25 1.25 X CAMK DGKI
diacylglycerol kinase, iota NM_004717 Up 3 X GO HK2 hexokinase 2
NM_000189 Up 2.25 X GO DGKG diacylglycerol kinase, NM_001346 Up
2.25 X GO gamma 90 kDa NBP Coenzyme A synthase NM_025233 Up 2.75 X
GO (COASY), DGKA Diacylglycerol kinase, NM_001345 Up 1.5 1.75 X GO
alpha 80 kDa XYLB Xylulokinase homolog (H. influenzae) NM_005108 Up
1.25 3.5 X GO SPHK2 Sphingosine kinase 2 NM_020126 Up 1.5 2 X GO
PRKRA Protein kinase, interferon- NM_003690 Up 1 1 2.5 X GO
inducible double stranded RNA dependent activator PIP5K2A
Phosphatidylinositol-4- NM_005028 Up 1 1 X GO phosphate 5-kinase,
type II, alpha
TABLE-US-00009 TABLE 2 Kinase whose inhibition modulates synuclein
levels Gene TF Up/ # of # of Kinase Kinase Name Kinase Name Genbank
Plates Down # of SD SD SD Family Group PRKCD protein kinase C,
NM_006254 1, 4, 7 Down 1 1.5 Ser/Thr AGC delta GPRK2L G
protein-coupled NM_005307 3, 6, 9 Down 1.25 1.5 Ser/Thr AGC
receptor kinase 2-like (Drosophila) GPRK5 G protein-coupled
NM_005308 3, 6, 9 Down 1.5 1.5 1.75 Ser/Thr AGC receptor kinase 5
AD034 RIO kinase 1 (yeast) NM_031480 10, 13, 16 Down 1.25 1.5
Ser/Thr Atypical BRDT bromodomain, testis- NM_001726 12, 15, 18
Down 1 1.25 Ser/Thr Atypical specific EEF2K eukaryotic elongation
NM_013302 12, 15, 18 Down 1.5 2.25 Ser/Thr Atypical factor-2 kinase
FASTK Fas-activated NM_006712 12, 15, 18 Down 1.25 1.5 Ser/Thr
Atypical serine/threonine kinase LOC283629 Testis-specific serine
NM_174944 21, 24, 27 Down 1 1 Ser/Thr/ CAMK kinase 4 (TSSK4) Tyr
STK22D serine/threonine NM_032028 21, 24, 27 Down 1.25 1.25 Ser/Thr
CAMK kinase 22D (spermiogenesis associated); TSSK1 ALS2CR7
amyotrophic lateral NM_139158 28, 31, 34 Down 1 1.5 Ser/Thr CMGC
sclerosis 2 (juvenile) chromosome region, candidate 7 CLK4 CDC-like
kinase 4 NM_020666 30, 33, 36 Down 1 1.5 Ser/Thr/ CMGC Tyr CDK5
cyclin-dependent NM_004935 30, 33, 36 Down 1 2 Ser/Thr CMGC kinase
5 CSNK2A2 casein kinase 2, NM_001896 55, 58, 61 Down 1 1.25 1.5
Ser/Thr Other alpha prime polypeptide MAP2K4 mitogen-activated
NM_003010 65, 68, 71 Down 1 2 Ser/Thr/ STE protein kinase kinase 4
Tyr MAP2K1 mitogen-activated NM_002755 65, 68, 71 Down 1 1.75
Ser/Thr/ STE protein kinase kinase 1 Tyr MAP2K5 mitogen-activated
NM_002757 66, 69, 72 Down 1 1.25 Ser/Thr/ STE protein kinase kinase
5 Tyr ANKRD3 ankyrin repeat NM_020639 83, 86, 89 Down 1.25 1.75
Ser/Thr TKL domain 3 (RIPK4) IRAK3 interleukin-1 NM_007199 83, 86,
89 Down 1 1.25 Ser/Thr TKL receptor-associated kinase 3 BMPR2 bone
morphogenetic NM_001204 84, 87, 90 Down 1 2 Ser/Thr TKL protein
receptor, type II (serine/threonine kinase) PRKG2 protein kinase,
NM_006259 1, 4, 7 Down 2 Ser/Thr AGC cGMP-dependent, type II CHEK2
CHK2 checkpoint NM_007194 19, 22, 25 Down 3.25 Ser/Thr/ AGC homolog
(S. pombe) Tyr CDK9 cyclin-dependent NM_001261 28, 31, 34 Down 2
Ser/Thr CMGC kinase 9 (CDC2- related kinase) CDK2 cyclin-dependent
NM_001798 29, 32, 35 Down 2 Ser/Thr CMGC kinase 2 CDKL3
cyclin-dependent NM_016508 29, 32, 35 Down 2.25 Ser/Thr CMGC
kinase-like 3 CDK10 cyclin-dependent NM_003674 29, 32, 35 Down 3
Ser/Thr/ CMGC kinase (CDC2-like) Tyr 10 CDK7 cyclin-dependent
NM_001799 30, 33, 36 Down 2 Ser/Thr CMGC kinase 7 (MO15 homolog,
Xenopus laevis, cdk-activating kinase) PK428 CDC42 binding
NM_003607 40, 46, 52 Down 2 Ser/Thr/ GO protein kinase alpha Tyr
(DMPK-like) FLJ32685 hypothetical protein NM_152534 57, 60, 63 Down
2.75 Ser/Thr/ Other FLJ32685 Tyr NEK11 NIMA (never in NM_024800 57,
60, 63 Down 3.5 Ser/Thr/ Other mitosis gene a)- Tyr related kinase
11 JIK TAO Kinase 3 NM_016281 64, 67, 70 Down 2 Ser/Thr STE
(MAP3K18) PAK6 p21(CDKN1A)- NM_020168 65, 68, 71 Down 2.25 Ser/Thr
STE activated kinase 6 KSR kinase suppressor of NM_013571 83, 86,
89 Down 2.25 Ser/Thr/ TKL ras Tyr AMHR2 anti-Mullerian NM_020547
83, 86, 89 Down 2 Ser/Thr/ TKL hormone receptor, Tyr type II LIMK2
LIM domain kinase 2 NM_005569 84, 87, 90 Down 2 Ser/Thr/ TKL Tyr
BCR breakpoint cluster NM_004327 11, 14, 17 Both 1 1.25 1.5 Ser/Thr
Atypical region ROCK2 Rho-associated, NM_004850 2, 5, 8 Up 1 1
Ser/Thr/ AGC coiled-coil containing Tyr protein kinase 2 SGK2
serum/glucocorticoid NM_170693 2, 5, 8 Up 1.25 1.5 Ser/Thr AGC
regulated kinase 2 SGKL serum/glucocorticoid NM_013257 2, 5, 8 Up
1.25 1.25 Ser/Thr/ AGC regulated kinase-like Tyr pknbeta protein
kinase N3 NM_013355 3, 6, 9 Up 1 1.75 Ser/Thr AGC PRKCH protein
kinase C, eta NM_006255 3, 6, 9 Up 1 1.75 Ser/Thr AGC ROS1 v-ros
UR2 sarcoma NM_002944 10, 13, 16 Up 1 1.5 Ser/Thr/ TK virus
oncogene Tyr homolog 1 (avian) CAMK1 calcium/calmodulin- NM_003656
20, 23, 26 Up 1.25 2 Ser/Thr CAMK dependent protein kinase I CAMK2B
calcium/calmodulin- NM_172078 20, 23, 26 Up 1.5 1.75 Ser/Thr/ CAMK
dependent protein Tyr kinase (CaM kinase) II beta CAMK2D
calcium/calmodulin- NM_001221 21, 24, 27 Up 1.5 2.25 Ser/Thr CAMK
dependent protein kinase (CaM kinase) II delta STK22B
serine/threonine NM_053006 21, 24, 27 Up 1.25 1.25 Ser/Thr/ CAMK
kinase 22B Tyr (spermiogenesis associated) STK29 serine/threonine
NM_003957 21, 24, 27 Up 1 2 Ser/Thr/ CAMK kinase 29 Tyr DYRK1B
dual-specificity NM_004714 28, 31, 34 Up 1 1.25 Ser/Thr/ CMGC
tyrosine-(Y)- Tyr phosphorylation regulated kinase 1B PCTK1 PCTAIRE
protein NM_033018 28, 31, 34 Up 1 1.25 Ser/Thr CMGC kinase 1 SRPK2
SFRS protein kinase 2 NM_182692 30, 33, 36 Up 1 1.25 Ser/Thr/ CMGC
Tyr NEK7 NIMA (never in NM_133494 55, 58, 61 Up 1 1 Ser/Thr/ Other
mitosis gene a)- Tyr related kinase 7 PACE-1 SCY1-like 3 (S.
cerevisiae) NM_020423 56, 59, 62 Up 1 1.5 Ser/Thr Other CNK
cytokine-inducible NM_004073 57, 60, 63 Up 1.25 2 Ser/Thr Other
kinase (polo-like kinase 3- Drosophila) TTBK tau tubulin kinase 2
NM_173500 84, 87, 90 Up 1.25 1.25 Ser/Thr/ CK1 Tyr PRKCZ protein
kinase C, NM_002744 3, 6, 9 Up 2 Ser/Thr AGC zeta TTK TTK protein
kinase NM_003318 64, 67, 70 Up 2.25 Ser/Thr/ Other Tyr ZAP70
zeta-chain (TCR) NM_001079 66, 69, 72 Down 1.25 2 Tyr TK associated
protein kinase 70 kDa FLT3 fms-related tyrosine NM_004119 73, 76,
79 Down 1 1 Tyr TK kinase 3 HCK hemopoietic cell NM_002110 74, 77,
80 Down 1.5 1.75 Tyr TK kinase BMX BMX non-receptor NM_001721 74,
77, 80 Down 1.25 1.5 Tyr TK tyrosine kinase BTK Bruton NM_000061
74, 77, 80 Down 1 1 Tyr TK agammaglobulinemia tyrosine kinase DDR2
discoidin domain NM_006182 75, 78, 81 Down 1 1.25 Tyr TK receptor
family, member 2 CSF1R colony stimulating NM_005211 75, 78, 81 Down
2 Tyr TK factor 1 receptor, formerly McDonough feline sarcoma viral
(v-fms) oncogene homolog LCK lymphocyte-specific NM_005356 39, 45,
51 Both 1 1.25 1.5 Tyr GO protein tyrosine kinase PRKCA protein
kinase C, NM_002737 1, 4, 7 Up 1 1.25 Tyr AGC alpha ROR2 receptor
tyrosine NM_004650 10, 13, 16 Up 1 1 1 Tyr TK kinase-like orphan
receptor 2 PDGFRA platelet-derived NM_006206 11, 14, 17 Up 1 1.25
Tyr TK growth factor receptor, alpha polypeptide SRMS src-related
kinase NM_080823 11, 14, 17 Up 1.25 1.25 Tyr TK lacking C-terminal
regulatory tyrosine and N-terminal myristylation sites TXK TXK
tyrosine kinase NM_003328 12, 15, 18 Up 1 1.5 Tyr TK YES1 v-ros UR2
sarcoma NM_005433 66, 69, 72 Up 1.25 1.25 Tyr TK virus oncogene
homolog 1 (avian) DKFZp61P1010 serine/threonin/tyrosine NM_018243
73, 76, 79 Up 1.25 1.25 Tyr TK kinase 1 (STYK1) EPHA2 EphA3
NM_004431 73, 76, 79 Up 1 1.5 Tyr TK FGR Gardner-Rasheed NM_005248
75, 78, 81 Up 1 1.25 Tyr TK feline sarcoma viral (v-fgr) oncogene
homolog FLT1 fms-related tyrosine NM_002019 75, 78, 81 Up 1.25 1.5
Tyr TK kinase 1 (vascular endothelial growth factor/vascular
permeability factor receptor) EPHB3 EphB3 NM_004443 75, 78, 81 Up 1
1.25 Tyr TK CSS3R colony stimulating NM_005211 38, 44, 50 Up 2.75
Tyr GO factor 1 receptor, formerly McDonough feline sarcoma viral
(v-fms) oncogene homolog GUCY2C guanylate cyclase 2C NM_004963 57,
60, 63 Up 2.25 Tyr Other (heat stable enterotoxin receptor) BCKDK
branched chain NM_005881 10, 13, 16 Down 1.25 1.5 2.25 ? Atypical
ketoacid dehydrogenase kinase BRD4 bromodomain NM_014299 11, 14, 17
Down 1.75 2 ? Atypical containing 4 AK3 adenylate kinase 3
NM_013410 37, 43, 49 Down 1 1.25 X GO FLJ12476 hypothetical protein
NM_022784 39, 45, 51 Down 1 1.25 ? GO FLJ12476 PAPSS2
3-phosphoadenosine NM_004670 42, 48, 54 Down 1 1.5 ? GO
5-phosphosulfate synthase 2 C20orf97 chromosome 20 NM_021158 19,
22, 25 Down 2 X CAMK open reading frame 97 (Tribbles homolog 3)
C8FW Tribbles homolog 1 NM_025195 19, 22, 25 Down 2 X CAMK GS3955
Tribbles homolog 2 NM_021643 20, 23, 26 Down 2 X CAMK FLJ32704
chromosome 9 open NM_157572 37, 43, 49 Down 3.75 X GO reading frame
98 DCK deoxycytidine kinase NM_000788 38, 44, 50 Down 2.25 X GO
KIAA0626 microfibrillar- NM_021647 39, 45, 51 Down 2.5 X GO
associated protein 3- like XYLB Xylulokinase NM_005108 40, 46, 52
Down 3 X GO homolog (H. influenzae) UCK1 uridine-cytidine NM_031432
42, 48, 54 Down 2 X GO kinase 1 GUK1 guanylate kinase 1 NM_000858
39, 45, 51 Both 1 2 2.75 ? GO MGC26954 chromosome 6 open NM_145025
40, 46, 52 Both 1 1 1.25 ? GO
reading frame 199 HK1 hexokinase 1 NM_033498 37, 43, 49 Up 2.25
2.75 X GO CALM3 calmodulin 3 NM_005184 38, 44, 50 Up 2 2.25 X GO
(phosphorylase kinase, delta) RBSK ribokinase NM_022128 40, 46, 52
Up 1.25 1.5 X GO PANK1 pantothenate kinase 1 NM_148978 41, 47, 53
Up 1.5 1.5 X GO P15RS hypothetical protein NM_018170 41, 47, 53 Up
1 1.5 ? GO FLJ10656 PFKFB2 6-phosphofructo-2- NM_006212 42, 48, 54
Up 1 1.25 ? GO kinase/fructose-2,6- biphosphatase 2 CKMT2 Creatine
kinase, NM_001825 38, 44, 50 Up 3.5 X GO mitochondrial 2
(sarcomeric) PGK1 phosphoglycerate NM_000291 40, 46, 52 Up 2.5 X GO
kinase 1
Example 2
Verification of Alpha-Synuclein Phosphorylation Modulation by
Re-Screening and by qRT-PCR
[0176] The kinases that showed either an increase or decrease in
alpha-synuclein phosphorylation from Example 1 were retested to
verify the effect on alpha-synuclein. The confirmation screen was
performed using 10 nM siRNA on the targets identified in Example 1
along with several additional kinases of interest. The higher
concentration of siRNA in Example 1 was used to ensure that
marginal knockdown caused by poorly designed siRNAs could be
observed. By using a much lower siRNA concentration in the
confirmation screens, the chance of effects due to a general
response to the siRNA itself could be much reduced. Some siRNAs
that were later reported by Ambion to be ineffective were also
re-screened (see replacement library screen below). Finally, some
newly identified kinases were screened and those results were added
to the pool of results. The kinases that were identified as
candidates were tested by quantitative RT-PCR (qRT-PCR) to confirm
that they were actually present in the PEAK-Syn cells (see Example
6). The experimental procedures and results for the confirmation
and rescreening were as follows:
Confirmation Screen
[0177] The results for the confirmation screen were grouped into
four categories shown below:
[0178] Completely Confirmed: This category included the kinases for
which all three siRNAs produced identical phenotypes in the 10 nM
screen and in the 100 nM screen.
[0179] Mostly Confirmed: This category included the kinases for
which 2/3 of the siRNAs produced identical phenotypes in the 10nM
and in the 100 nM screen, but one third did not; or, alternatively
one siRNA result was replicated, but for a second siRNA there was a
trend for the same phenotype but with a different siRNA from that
used in the original screen.
[0180] Partly Confirmed: This category included the kinases for
which 1/3 of the siRNAs produced the same phenotype in the 10 nM
screen and in the 100 nM screen.
[0181] Not Confirmed: This category included the kinases for which
either or both of the following occurred: [0182] a) None of the
three siRNAs had any effect on phosphor-alpha-synuclein levels at
10 nM, and/or [0183] b) The siRNAs produced the opposite phenotype
to what was observed in the primary 100 nM screen
[0184] The number of kinases that fell into each category was
tabulated and the results are shown in Table 3. Seven kinases were
completely confirmed, and they are listed in Table 4. Of these
seven, only three were identified as possessing the qualities to be
good candidates for a kinase that directly phosphorylates
alpha-synuclein at ser-129. This is because only three were both
ser/thr kinases and decreased phospho alpha-synuclein levels when
the kinase levels were reduced by the specific siRNA. These
included: APEG1, which is believed to play a role in growth and
differentiation of smooth muscle, PLK2 (SNK), which is expressed in
brain and is believed to play a role in normal cell division, and
CDC7L1, a cell division cycle protein with kinase activity. Of the
three, PLK2 was of the most interest due to its role and
localization in cells, such as activated neurons. Alpha-synuclein
is a synaptic-associated protein thought to be involved in synaptic
plasticity and vesicular transport. Thus, PLK2 was identified as a
very good candidate for a kinase that directly phosphorylates
.alpha.-synuclein.
TABLE-US-00010 TABLE 3 Breakdown of Candidate Hits From 10 nM
Confirmation Screen Number Hit Category of Hits Completely
Confirmed 7 Mostly Confirmed 29 Partly Confirmed 22 No Reactivity
at 10 nM 19 Opposite Reaction to Primary Screen 23 Total Number Of
Hits Re-Screened at 10 nM 100 NOTES: For all subsequent tables ***
denotes where a replacement siRNA has been analyzed and the new
data substituted for that from the ineffective siRNA
Key to shading:
TABLE-US-00011 ##STR00007##
TABLE-US-00012 TABLE 4 Completely Confirmed Hits ##STR00008##
[0185] Table 4 shows the seven candidates whose results were
completely replicated at 10 nM. Only the first three were
identified as having strong potential to be a direct kinase,
because they are ser/thr kinases that reduce phospho-synuclein
levels when the kinase level is reduced.
[0186] There were 29 kinases that fell into the mostly confirmed
category, 12 of which were candidates for a direct kinase. These
are listed in Table 5. There were 17 additional kinases that were
mostly confirmed at 10 .mu.M. Although not likely to be a direct
kinase, these could play a role in the regulation of a direct
kinase and are listed in Table 6. Twenty-two kinases fell into the
partly confirmed category. The ser/thr kinases that decreased
phospho alpha-synuclein (i.e. potentially a direct kinase for
alpha-synuclein) are listed in Table 8, and the remaining
potentially regulatory kinases are listed in Table 8.
TABLE-US-00013 TABLE 5 Potential Direct Serine/Threonine Kinases
that Mostly Confirmed at 10 nM siRNA ##STR00009## ##STR00010##
[0187] The ser/thr kinases shown in Table 5 were identified as
having potential to be a direct kinase that phosphorylates
alpha-synuclein because they significantly reduced
phospho-synuclein levels when the kinase level was reduced. 2/3 of
the siRNAs produced identical results at 10 nM as they did at 100
nM, and as such, were designated as Mostly Confirmed hits.
[0188] The kinases in Table 6 were designated as Mostly Confirmed,
because 2/3 of the siRNAs produced identical results at 10 nM and
100 nM concentration of siRNA. However, because they did not
produce the appropriate phenotype or were the wrong class of kinase
(i.e. tyr or non-protein kinase as opposed to a ser/thr kinase),
they were identified as not likely to be a direct kinase that
phosphorylates ser-129 on alpha-synuclein. Instead, they may be
upstream modulators of alpha-synuclein phosphorylation.
TABLE-US-00014 TABLE 6 Other Kinases That Were Mostly Confirmed at
10 nM siRNA ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015##
TABLE-US-00015 TABLE 7 Potential Direct Serine/Threonine Kinases
that Partly Confirmed at 10 nM siRNA ##STR00016## ##STR00017##
##STR00018##
[0189] The ser/thr kinases in Table 7 were identified as having
potential to be a direct kinase that phosphorylates alpha-synuclein
because they significantly reduced phospho-synuclein levels when
the kinase levels were reduced. Only 1/3 of the siRNAs produced
identical results at 10 nM as they did at 100 nM, and as such, were
designated as Partly Confirmed hits.
[0190] Several candidates had contradictory results and, thus, were
identified as having less potential to be a direct kinase for
alpha-synuclein.
TABLE-US-00016 TABLE 8 Other Kinases That Were Partly Confirmed at
10 nM siRNA ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023##
[0191] The kinases in Table 8 were identified as having less
potential to be direct kinases in the phosphorylation of
alpha-synuclein at ser-129 but could be upstream modulators of
alpha-synuclein phosphorylation. 1/3 of the siRNAs produced
identical results at 10 nM as they did at 100 nM, and as such, were
designated as Partly Confirmed hits. However, several had
contradictory results and, thus, were designated as having less
potential to be direct kinases of alpha-synuclein.
[0192] Forty-two kinases did not have their initial results
confirmed at 10 nM. Of these, 19 fell into category (a) listed
above, and are listed in Table 9. At 10 nM, none of the three
siRNAs at 10 nM produced any change in the phospho-alpha-synuclein
phenotype, indicating that the results for these kinases from the
100 nM screen were possibly due to off-target effects. Twenty-three
kinases (Table 10) produced the opposite effect on
phospho-alpha-synuclein levels at 10 nM than at 100 nM siRNA. There
is a possibility that the results at 10 nM were the true effects
due to the fact that at 100 nM results are sometimes masked by
off-target effects. This can happen at the much higher siRNA
concentration. Alternatively, the true effect may have been seen at
the higher concentration. In any case, these kinases were
designated as less likely to be direct kinases of
alpha-synuclein.
[0193] The nineteen kinases shown in Table 9 had no significant
reactivity at 10 nM compared to controls. Thus, it is possible that
the change in phospho-synuclein levels observed at 100 nM was due
to off-target effects caused by high concentrations of siRNA. GPRK5
and GPRK7 were not candidates in the original 100 nM screen, but
were analyzed at 10 nM siRNA because of additional interest in
their role in alpha-synuclein phosphorylation.
TABLE-US-00017 TABLE 9 Kinases That Had No Reactivity at 10 nM
siRNA ##STR00024##
[0194] The results for the kinases in Table 10 were not confirmed
at 10 nM because they had the opposite effect on phospho-synuclein
levels from that seen at 100 nM. However, it is possible that the
results at 10 nM siRNA were the true results, and that the high
concentration (100 nM) of siRNA was masking the true effects. It is
also possible that the initial effects observed at 100 nM were the
true effects. These were designated as likely to be direct kinases
of alpha-synuclein and set aside to be tested further at a later
date.
TABLE-US-00018 TABLE 10 Kinases Whose Results Were Opposite To The
Primary Screen ##STR00025## ##STR00026## ##STR00027##
Replacement and Up-Dated Library Screens
[0195] Because some siRNAs used in the initial screen were later
identified as being of poor quality, screens were performed at both
concentrations with replacement siRNAs. The data for the
replacement siRNAs was used to replace the data for that specific
siRNA result from the original screen. Statistical data was
tabulated for the three siRNAs for each kinase, and using this,
nine additional kinases were identified as candidates from the
original screen that were missed in the primary screen. These were
retested, and two of the kinases were partially confirmed at 10 nM
siRNA. These were BCKDK and FLJ25965 (KSR2).
[0196] During the process, a number of new kinases were identified
and siRNAs became available. These were tested as in Example 1 as
an AMBION Up-Dates library and new kinase candidates were
identified. Many of the newly identified kinases fell under the GO
(Gene Ontology Consortium) classification. As such, it was
difficult to find detailed information on some of these kinases.
Several of the genes included in this category were not true
kinases, but were kinase binding proteins or adaptor proteins. At
10 nM siRNA, thirteen kinases were confirmed to be candidates for
directly acting on alpha-synuclein. Two of these were likely
candidates for being a direct kinase, see Table 11. The remaining
eleven were designated as possible indirect regulators of
phospho-synuclein levels, see Table 11. Table 11 provides Genbank
accession numbers for the kinase sequences as deposited in Genbank
as of Nov. 1, 2005.
TABLE-US-00019 TABLE 11 Potential Kinase Hits From the Ambion
Updates Library ##STR00028## ##STR00029## ##STR00030##
##STR00031##
[0197] A summary of the results showing the kinase siRNAs that were
identified and verified in Examples 1 and 2 are shown in Tables 12
and 13. From these results, PLK2, APEG1, CDC7L1, MET, IKBKB, CKII,
GRK1, 2, 6 and 7 were identified as kinases that are very likely to
phosphorylate alpha-synuclein directly or indirectly. The kinases
that were identified as having siRNAs that increased
alpha-synuclein phosphorylation (PRKG1, MAPK13, and GAK) could very
well be negative regulators of alpha-synuclein phosphorylation.
[0198] Tables 12 and 13: Summary of Confirmation Studies
TABLE-US-00020 TABLE 12 ##STR00032##
TABLE-US-00021 TABLE 13 GRK Results - Mixture of Mostly, Partially
and Not Confirmed ##STR00033## ##STR00034##
[0199] In the following examples, in vitro kinase assays were
performed on a number of the potential targets identified in
Examples 1 and 2.
Example 3
Identification of Direct Phosphorylation of Alpha-Synuclein In
Vitro
[0200] To determine which of the kinase(s) from the siRNA screen
directly phosphorylated alpha-synuclein, purified kinases were
incubated with alpha-synuclein in in vitro kinase reactions. These
results showed that PLK2, GRK2, 5, 6. and 7 (GPRK2, 5, 6 and 7)
were all capable of phosphorylating alpha-synuclein specifically at
serine 129 and did not phosphorylate serine 87 in vitro, showing
that they could directly phosphorylate alpha-synuclein. MET,
CDC7L1, and IKBKB were shown to be incapable of directly
phosphorylating alpha-synuclein (FIGS. 1A-C).
[0201] Assay conditions for testing recombinant kinase activities
toward recombinant alpha-synuclein at serine 129 were established
and found to be reproducible by immunoblot and ELISA analyses.
Commercially available recombinant kinases were used when possible.
Those that were not available were produced as indicated by
recombinant means.
[0202] In FIGS. 1A-C, recombinant kinases were included in the in
vitro alpha-synuclein (AS) assay by standardizing kinase to
alpha-synuclein substrate at a constant molar ratio (derived from
MW of predicted mature protein) in each reaction (1:200; kinase:
recombinant alpha-synuclein kinase--rAS).--control, +kinase; In
FIG. 1A, a probe for total alpha-synuclein (AS) (mAb Syn-1; 0.1
.mu.g/mL) was used indicating equivalent substrate in each
reaction; In FIG. 1B, a parallel blot was probed for S129
phosphorylation (mAb 11A5 1 .mu.g/mL). Prominent signals came from
GRK6, CKI, CKII and PLK2 (not previously tested by activity
normalization). In FIG. 1C, a parallel blot probed for S87
phosphorylation (pAb, ELADW-110 5 .mu.g/mL). A signal was detected
only with CKI phosphorylation.
[0203] In FIGS. 1D-F, a more focused study was performed with
recombinant kinases from the GPCR-receptor kinase (GRK) family and
PLK2 were included in the in vitro alpha-synuclein (AS) assay by
standardizing kinase to AS substrate at a constant molar ratio
(derived from MW of predicted mature protein) in each reaction
(1:200; kinase: rAS).--control, +kinase; CAM kinases served as
negative controls while CKI and II served as positive controls. In
FIG. 1D, a probe for total AS (mAb Syn-1; 0.1 g/mL) was used
indicating equivalent substrate in each reaction; In FIG. 1E, a
parallel blot probed for S129 phosphorylation (mAb 11A5 1
.mu.g/mL). Prominent signals came from all GRKs except for GRK7. A
specificity between GRK members could be seen with signal and can
be represented as: CKI>GRK6>PLK2>GRK4>GRK5>GRK2. In
FIG. 1F, a parallel blot probed for S87 phosphorylation (pAb,
ELADW-110 5 .mu.g/mL). Signal was detected only with CKI
phosphorylation.
[0204] The assay conditions are defined in Table 14 and were held
constant for all kinases tested. All of the kinases listed were
available as tagged/recombinant protein with the exception of
CDC7L1, PRKG1 and APEG. Those putative targets were expressed in an
in vitro translation system and tested in the in vitro AS assay
without protein concentration or activity measurements.
TABLE-US-00022 TABLE 14 Assay conditions for in vitro kinase
reactions: 1:200 kinase:AS (molarity) total ul Confir- ng kinase;
ul in kin. in ng/ul Units/ Total ul ng co- # Kin mation MW kinase
100ul rxn stock kin ul Dilution kin./rxn kin/rxn factors 1 CKI a'
most 49000 delta 5.1 ng; 6.3 ul 20 814 1000 100000x 3 0.024 (1000x
(0.01 U/ul) dilution) 2 CKII a' part 44,000 alpha 4.6 ng; 4.3 ul 20
1070 500 50000x 3 0.0642 26,000 beta (1000x (0.01 U/ul) dilution) 3
PAK6 part 38,000 4 ng; 1 ul 50 100 0.21 10x (0.021 1.43 14.3 (100x
dilution) U/ul) 4 ARK5 most 78,000 8.1 ng; 8.1 ul 50 100 0.06 0.5
50 (100x dilution) 5 CaMK1 most 68,000 7.1 ng; 7.1 ul 50 100 0.29
10x (0.029 1 10 calmodulin (100x dilution) U/ul) 1uM 6 PHKG2 del
52,000 5.4 ng; 7.7 ul 143 70 0.007 4.3 301 (100x dilution) 7 MAP2K1
del 49,000 5.1 ng; 5.1 ul 20 500 1.69 100x 17.75 90 (100x dilution)
(0.00169 U/ul) 8 GRK6 part 94,000 9.8 ng; 3 ul 34 290 0.008 3.75
1088 (100x dilution) 9 CAMKII del 59,000 6.2 ng; 1.9 ul 31 320 4.93
100x 0.61 195 calmodulin delta (100x dilution) (0.0493 1uM U/ul) 10
Met conf 50,000 5.2 ng; 5.2 ul 50 100 0.022 1.363 136 (100x
dilution) 11 MAPK13 conf 46,000 4.8 ng; 1.1ul 22 450 0.054 0.56 250
(100x dilution) 12 PRKG2 most 117,000 12.2 ng; 2.8 ul 22 440 0.017
1.76 776 (100x dilution) 13 PLK2 conf 106,000 11 ng; 4.1 ul 27 270
0.027 1.1 297 (100x dilution) 14 GRK2 most 82,300 8.6 ng; 1.7 ul 20
500 0.0045 6.7 3350 (100x dilution) 15 GRK4 part 94,000 9.8 ng; 2.5
ul 25 400 0.0012 25 10,000 (100x dilution) 16 GRK5 part 95,200 9.9
ng; 2.1 ul 21 480 0.00018 167 80,160 (100x dilution) 17 GRK7 part
89,700 9.3 ul; 1.9 ul 21 480 0.00067 45 21,600 (100x dilution) 18
CDC7L1 conf 63,800 undetermined; nd nd nd nd nd in vitro
translation 19 PRKG1 conf 76,200 undetermined; nd nd nd nd nd in
vitro translation 20 PDK1 part 59,000 6.1 ng; 3 ul 50 200 0.074 10x
4.05 81 SGK (200x dilution) (0.0074 U/ul) 24 APEG conf 12,600
undetermined; nd nd nd nd nd in vitro translation
[0205] The Standard Conditions were: 40 mM MOPS-NaOH; 1 mM EDTA
MgCl 10 mM pH 8.0, 0.1% BME; 0.01% Brij-35; 5 ug BSA, 100 uM ATP
(5.times.[substrate]), 100 uL volume; 300 ng r-wt-AS (208 nM),
(1:200 kinase: AS or activity normalized 0.03 U/r.times.n, 34C; 17
hrs. Further, those kinases with varying levels of
significance/confirmation from combined screening data were
purchased as recombinant, tagged protein, annotated and
incorporated into a table format for the purposes of establishing
in vitro assays that were comparable based upon normalization to
activity units (determined by the manufacturer from synthetic
substrates) or substrate:enzyme molar ratios determined from MW and
reaction volume. The details of reaction conditions are stipulated
in Table 14. Kin.=kinase. For Confirmation: Most=mostly,
Part=partially, del=deleted, conf=confirmed.
[0206] Kinase activity was initially tested against AS by activity
units as determined from non-native substrates (peptides or
casein). This method was used to get a rough estimate as to
specificity between kinases and whether AS was an in vitro
substrate for the kinase panel. The results of this study are found
in FIGS. 2 and 3. At the time of this experiment, only a portion of
available kinases were obtained and 2/3 kinases from the "most
probable 7 confirmed" were included (PLK2 was not tested). The most
prominent result came from GRK6 (G-protein coupled receptor kinase
6). CKI gave a modest signal and CKII was not detectable. Because
both CK kinases are known to phosphorylate S129 AS, normalization
by activity units was biased against those kinases which had higher
specific activity for tested substrates vs AS. This was likely the
situation for GRK6 which might have preferred AS as a substrate
rather than the peptide substrate which defined its activity
units.
[0207] In the following examples, in vitro kinase assays were
performed on a number of the potential targets identified in
Examples 1 and 2. To correct for the activity bias, kinases were
retested and newly procured kinases were put into an assay that
normalized for molarity. This gave a better measurement of
stoichiometric ratios between enzyme and substrate, thus reporting
the phosphorylation event as a function of AS/kinase interaction.
This was in contrast to the event in which unrelated
substrate/kinase phosphorylation was measured. FIGS. 3A-C
illustrate a more realistic view of AS phosphorylation with roughly
equivalent levels of phospho ser-129 between CKI, GRK6 and PLK2
(one of 7 highly confirmed). With the exception of CKI, none of the
tested kinases were capable of phosphorylating AS at the ser-87
residue. This observation confirmed the specificity/preference of
these kinases for the ser-129 site and/or the low
preference/inaccessibility for the ser-87 site. However, CKI has
been reported to phosphorylate at both sites.
Effect of Acidic Phospholipid on the Assay Results
[0208] The significant levels of activity by GRK6 and PLK2
(polo-like kinase phylogenetically related to the GRK family) in
the in vitro assay combined with the identification of PLK2, GRK2
and GRK1 as decreasers of phosphorylation in the RNAi screen,
prompted a more comprehensive survey of other GRK members. FIGS. 4A
and B indicate the results of GRK 2, 4, 5, 6, 7 and PLK2 compared
in the in vitro assay. This preference could be represented as
CKI>GRK6>PLK2>GRK4>GRK5>GRK2. GRK 7 was not able to
phosphorylate at appreciable levels. All GRKs were unable to
phosphorylate at ser-87 pointing to a specificity for the acidic
sequence flanking amino acid 129. These reactions were quantitated
and confirmed by ELISA measurements. These values more or less
agreed with the immunoblot data with an apparent decrease in PLK2
level vs GRKs. It is likely that most of the AS substrate was
depleted (phosphorylated) based on the assay design (300 ng AS, 210
nM for 17 hr).
[0209] The positive effect of acidic phospholipids on the
phosphorylation of AS has been previously reported Pronin et al.
JBC 275(34): 26515-26522 (2000) and a pronounced effect on GRK 2
and 5 was observed. Because of this report and the many studies
indicating that acidic phospholipids modulate AS conformation, a
mixture of phosphatidylcholine (PC): phosphatidylserine (PS):
phosphatidyl-inositol-phosphate-3 (PIP3) was generated and
incorporated into the established in vitro assay. The lipid mixture
was shown to increase signal for almost all of the kinases tested.
The addition of a lipid environment is likely to imitate the
membrane surface in a cell where AS and GRKs are likely to
associate. Without being bound by the following theory, it is
probably due to a favorable exposure of the C-term of AS upon lipid
binding of the N-term helices of AS. Interestingly, the lipid
effect of ser-87 phosphorylation (as see by the CKI reaction) led
to a decrease in the level of phosphorylation. This may be the
result of epitope masking by lipid interaction if ser-87 is buried
upon helix interaction.
Example 4
Identification of Direct Phosphorylation of Alpha-Synuclein in Cell
Lines
[0210] Because kinases can be more promiscuous in vitro than they
are in cells, an assay was performed in cell lines to confirm the
direct interaction with alpha-synuclein. cDNAs for the kinases that
phosphorylated alpha-synuclein in vitro from Example 3 were
transfected into the PEAK-Syn cell line to see which was capable of
phosphorylating alpha-synuclein ser-129 in cells. The results
showed that GRK6 and, to an even greater extent, PLK2 were able to
mediate alpha-synuclein phosphorylation in cells (FIG. 6).
[0211] cDNA clones for PLK2, GPRK6, APEG1, CDC7 and PRKG1 were
obtained from Origene. The cDNA was transcribed and transfected
into PEAK-Syn cells using Lipofectamine 2000.TM. (Invitrogen). For
each cDNA analyzed, 12 wells of a 96-well plate were transfected,
along with 12 control wells of untransfected cells. Cells were
harvested at 48 hrs post-transfection as per the ELISA screening
protocol, and analyzed by ELISA for total and phospho-synuclein,
and values were normalized for total protein. For those kinase
targets not commercially available as recombinant proteins (namely
APEG, PRKG1 and CDC7LI), an in vitro cell-free reticulocyte system
(Promega) was employed to express protein from human full-length
cDNA clones (Origene). Proper sequence was determined and DNA
prepared. PLK2 and GRK6 cDNA was also included in the study as
positive controls.
[0212] The percentage of phospho-synuclein in untransfected cells
was calculated to be 7.8%. The percentage of phospho-synuclein for
the cells transfected with APEG1, CDC7 and PRKG1 cDNA was only
marginally higher than untransfected cells at 8.9%. These kinases
were considered to have produced a negative result in altering
phospho-synuclein levels, and were considered negative controls for
experimental purposes, as they were subjected to the same rigors of
transfection that the other kinases were exposed to cDNA to PLK2
was transfected into 293-synuclein cells. Cells were harvested 48
hrs following transfection and analyzed by ELISA for total and
phospho-synuclein levels. ELISA values were corrected for total
protein levels. Overexpression of PLK2 resulted in a dramatic
increase in phospho-synuclein levels, increasing phospho-synuclein
expression by 4.3-fold above expression in untransfected cells.
[0213] It is likely that when a direct kinase that phosphorylates
.alpha.-synuclein is introduced into the cell an increase in
phospho-synuclein levels would be observed. This was the case for
both GPRK6 and PLK2 (FIG. 6). The percent phospho-synuclein in
cells transfected with GPRK6 cDNA increased dramatically, from 8.9%
to 18.9%. This increase is significant to 9.25 standard deviations
above the percent phospho-synuclein observed for the negative
kinases. The increase in phospho-synuclein levels for the
PLK2-transfected cells was even more dramatic, increasing the
percent phospho-synuclein almost four-fold to 33.2%. This
represents an extremely significant change, an increase of 22.75
standard deviations above the phospho-synuclein levels observed for
the negative kinases. This dramatic increase was by far the largest
change observed previously in using this assay. This data strongly
indicates GPRK6, and especially PLK2 as very solid contenders as
direct kinases responsible for phosphorylating .alpha.-synuclein.
Thus, as shown in FIG. 6, when GPRK6 cDNA is transfected into
HEK-synuclein cells, the expression of phospho-synuclein increases
2-fold. Introduction of PLK2 cDNA into cells results in an even
more dramatic increase in phospho-synuclein expression, a change of
almost four-fold above control values.
Example 5
Phosphorylation by PLK2 (SNK) GRK6, CKII and IKBKB
[0214] The data in Example 4 was further substantiated for PLK2 by
showing that PLK2 siRNAs reduced alpha-synuclein phosphorylation.
This strengthened the data showing that PLK2 is a likely candidate
as a cellular kinase that directly phosphorylates alpha-synuclein
at Serine 129 (Tables 2 and 12).
[0215] HEK 293 cells stably transfected with alpha-synuclein were
transfected with 10 nM and 100 nM of SmartPool siRNAs (Dharmacon).
SmartPool siRNAs include 4 individual siRNAs to a specific target.
Thus, the actual concentration of each of the four siRNAs
transfected into cells was 2.5 nM and 25 nM respectively. The
results in FIG. 7 show that PLK2 significantly decreased
phospho-synuclein levels, a change of approximately 25%. At 10 nM,
but not at 100 nM of siRNA, GPRK6 significantly increased the
percentage of phospho-synuclein by one standard deviation above the
mean of the control negative kinases (FIG. 7). This is the opposite
effect to what was previously observed in the primary siRNA screen
and may be due to the quality of the siRNA used in the first or
second assays. These results were confirmed by
immunohistochemistry.
[0216] The significant knockdown of phospho-synuclein levels by
different siRNAs from a different source, independently confirms
and solidifies the data, and substantiates the role of PLK2 as a
direct kinase that phosphorylates .alpha.-synuclein. These
experiments were then performed on two other kinases identified in
the screens to be of interest, casein kinase two (CKII) and
IKBKB.
[0217] The individual CKII catalytic subunits were hits in the
primary siRNA screen (see Example 1 and Table 1B) and confirmed at
the 10 mM siRNA screen. It was of interest to determine if the
individual CKII subunits .alpha..sup.1 and .alpha.', when
cotransfected with PLK2 or each other, had additive effects on
alpha-synuclein phosphorylation. Transfections were performed using
the individual CKII subunits A (.alpha..sup.1) and B (.alpha.'),
cotransfected with PLK2 or each other. Overexpression of these
catalytic subunits increased phospho-synuclein levels by 1.75 and 1
standard deviations respectively (the effect was not additive).
When each of the individual subunits was co-transfected with PLK2,
the levels of phospho-synuclein increased over that of PLK2 alone
(18.6% phospho-synuclein) by 1.25 standard deviations each to 22.8%
phospho-synuclein. However when both subunits were co-transfected
with PLK2 phospho-synuclein levels were not significantly increased
above that for PLK2 alone (21.4% phospho-synuclein).
[0218] IKBKB siRNA knockdown resulted in a significant decrease in
alpha-synuclein phosphorylation so this gene was tested for
capacity to phosphorylate alpha synuclein. Transfections and ELISA
analysis were performed as per standard procedure. Previous in
vitro experiments demonstrated that IKBKB was not a direct
synuclein kinase as it did not phosphorylate synuclein in a direct
kinase assay (see Example 3), but may be an upstream regulator of
synuclein phosphorylation. Thus, IKBKB was over-expressed in
HEK-syn cells to identify the effect on phosphorylation of
synuclein. Following introduction of IKBKB cDNA into cells,
synuclein phosphorylation increased from 8.3% in the negative
(empty vector) control to 21.5%, a 2.6-fold increase. This
represented an increase in synuclein phosphorylation that was
significant to almost 53 standard deviations. The PLK2 positive
control increased synuclein phosphorylation to 65.8%, an almost
8-fold increase in phosphorylation (significant to 230 standard
deviations). Although the effect on synuclein phosphorylation was
much more modest for IKBKA, a related kinase, (1.2 fold) than for
IKBKB, it was still significant to 1.4 standard deviations.
Example 6
Synphilin as an Alternative Therapeutic Target
[0219] Synphilin is a synuclein-associated protein that has been
shown to bind alpha-synuclein. To determine if the presence of
synphilin can enhance the phosphorylation of alpha-synuclein, it
was over-expressed in HEK cells with and without alpha-synuclein
and PLK2. Transfections were performed according to standard
protocol, followed by alpha-synuclein ELISA and analysis. Cells
were also harvested for Western blot analysis. Transfected cell
lysates were analyzed for total synuclein using 1H7 antibody and
phospho-serine 129 synuclein using 11A5 antibody (See WO 05047860).
The total amount of DNA transfected into cells remained constant at
0.16 .mu.g/well of a 96-well plate. The type of DNA introduced into
cells varied, with empty vector being used to make up the full
quota of DNA. Varying concentrations of alpha-synuclein, PLK2, and
synphilin cDNA were introduced into naive HEK cells. Cells
transfected with all three showed a slight increase in total
synuclein. For phospho-synuclein, the levels in untransfected cells
were below the limit of quantitation. Introducing alpha-synuclein
alone yielded 5.2% phospho-synuclein, which was marginally less
than co-transfection of synuclein with synphilin (5.4%
phospho-synuclein). Co-transfection of PLK2 and synuclein yielded
levels similar those observed for transfecting PLK2 into HEK-syn
stable cells, 60% phospho-synuclein. Strikingly, concurrent
over-expression of all three cDNA's (PLK2, synuclein and synphilin)
resulted in 83.3% phospho-synuclein in the HEK cells. Thus,
synphilin increased synuclein phosphorylation in PLK2,
alpha-synuclein over-expressed HEK cells.
[0220] Increased phosphorylation of alpha synuclein in the presence
of synphilin can be explained by synphilin binding to the PLK2
polo-box thereby facilitating phosphorylation of synuclein by PLK2.
Synuclein itself is unlikely to bind the polo-box domain.
Example 7
PLK2 Activity: Phosphorylation of Alpha Synuclein and Familial
Mutants of Alpha Synuclein
[0221] To analyze PLK2 phosphorylation of a number of known
familial mutants of alpha synuclein, in vitro studies were
performed and the phosphorylation of the alpha synuclein and
mutants analyzed. The familial mutants (FPD) were A30P, A53T, and
E46K.
[0222] All in vitro reactions were performed using the following
conditions, 10 mM MgCl2, 100 .mu.M ATP, 27 mM HEPES, 250 ng/ml
PLK2, 1/50 dilution of Protease Inhibitor solution (1 tablet in 1
ml of reaction buffer), 40 mM Nitrophenylphosphate, 1 mg/ml of 95%
Type II-S Phosphatidylcholine from soybean, and 10, 100, or 1000 nM
alpha synuclein (AS). The reaction was incubated at 37.degree. C.
The activity was analyzed by autoradiography.
[0223] PLK2 was found to be more active against wild-type alpha
synuclein than beta synuclein. Further, the mutant alpha-synucleins
were phosphorylated more at a given concentration (especially at
lower concentrations) than WT. A trend of PLK2 activity was
identified with PLK2 activity being highest with FPD mutants,
followed by wild-type alpha synuclein, and minimally against beta
synuclein. This order is consistent with a mechanism by which
phosphorylation of alpha synuclein drives Lewy body formation and
subsequent pathology.
Example 8
Confirmation of the Presence of Kinases in HEK-Synuclein and
SY5Y-Synuclein Cells
[0224] qRT-PCR was performed to determine if the kinases of
interest were expressed in HEK-synuclein and SY5Y-synuclein cells.
In Table 15 all samples were normalized to GAPDH expression. In
addition, two of the negative kinases were analyzed in each
experiment as a reference. Of the 24 potential direct kinase
candidates tested, 20 were detected in the HEK293-synuclein cells,
including PLK2. Thus, the remaining completely confirmed kinases
were detected in the cells (FIG. 9). Four of the potential direct
kinases tested, GPRK1, GPRK7, ERK8 and RIPK3, were not detected.
GPRK6 was barely detectable.
[0225] The qRT-PCR was performed as follows: the mRNA levels were
normalized to GAPDH mRNA expression levels. Total RNA was purified
from a cell pellet using the QIAGEN RNeasy Kit and protocol.
Primer-probe sets for 24 of the potential direct kinases and the
four indirect completely confirmed kinases were ordered from
Applied Biosystems (TaqMan Gene Expression Assays), along with
reverse transcriptase, RNase inhibitors and standard PCR reagents.
A one-step RT-PCR/qRT-PCR reaction as performed an ABI7500
Real-Time PCR machine for each primer-probe set using 20 ng or 200
ng total RNA using the following cycling conditions: 48.degree.
C./30 mins (RT-PCR step), 95.degree. C./10 mins (denature), then 40
cycles of 95.degree. C./15 secs, 60.degree. C./1 min. For each
primer-probe set, an RT-negative reaction and PCR-negative reaction
was performed. The RT-negative controls for background
amplification of DNA (not RNA) that is contaminating the purified
RNA. The PCR-negative control was to ensure all of the PCR reagents
were free of contaminating RNA and DNA, and should have had no
signal.
[0226] All three of the completely confirmed potential direct
kinases, along with the four indirect completely confirmed kinases
were easily detected in SY5Y-synuclein cells, indicating this cell
line may be a viable option for a neuronally-derived cell line for
further experimental analysis of kinases.
TABLE-US-00023 TABLE 15 qRT-PCR demonstration of the presence of
kinases in HEK-synuclein and SY5Y-synuclein cells Sample Name
Relative Expression (to GAPDH) SYN APEG1 2.23 SYN SNK/PLK2 3.57 SYN
CDC7L1 1758.34 SYN PRKG1 7.36 SYN MAPK13 8.97 SYN GAK 2.17 SYN MET
891.44 SY5Y-SYN APEG1 1698.45 SY5Y-SYN 208.66 SNK/PLK2 SY5Y-SYN
CDC7L1 24.42 SY5Y-SYN PRKG1 42.22 SY5Y-SYN MAPK13 45.73 SY5Y-SYN
GAK 17.63 SY5Y-SYN MET 86.22
Example 9
Identification of Increased Phosphorylation of Alpha-Synuclein in
293 Cells and Neuronally-Derived Cell Lines
[0227] PLK2 and GRK were overepressed in 293 cells stably
transfected with alpha synuclein. ELISA and Western blot were
performed to identify increase in phospho-synuclein with PLK and
GRK kinases and an increase in phosphorylation was demonstrated. A
second method was used to confirm the increase using the same
biotinylated antibodies used in the ELISA for immunostaining (11A5)
in 293 cells. This method also demonstrated an increase in
phospho-synuclein in cells transfected with PLK2 and to a lesser
extent GRK. The increase was detected in a small population of
cells that brightly stain for 11A5, not a general increase in all
cells. The amount of total synuclein (measured using the 5C12
antibody) did not appear to change. This was a significant increase
in phosphorylation in the 293 cells. Thus, it was of interest to
see if the results could be repeated in neuroblastoma cells.
[0228] To identify that the dramatic upregulation of
phospho-synuclein observed with PLK2 and GPRK6 occurs in
neuronally-derived cells, the same experiment was performed in
human neuroblastoma cells (SY5Y cells). Immunostaining results
showed that PLK2 caused an increase in the phospho-synuclein in a
small population of cells, in a very similar pattern to the 293
cell experiments. Quantitation was performed by
immunohistochemistry using the ArrayScan.TM. in two ways. First all
cells were counted and did not show any difference. Then just the
bright cells were counted and this analysis showed about a 5-10
fold increase in the number of 11A5 positive cells that were PLK
transfected, with a slight increase with GRK6 as well.
[0229] The cDNA transfection experiment is repeated in HCC cells
and immunohistochemistry is performed with a variety of
alpha-synuclein antibodies on the cells that have been transfected
with PLK2 and GPRK6. Cells may be treated with additional reagents
to mimic the pathology of Parkinson's disease; such reagents could
include, for example, rotenone, paraquat, hydrogen peroxide, or
ferric chloride. In this way, inclusion formation and/or
alpha-synuclein aggregation is observed in these cells. Antibodies
used to look for inclusions/aggregation include LB509, SYN-1, 11A5
and ELADW-110.
[0230] Next, cDNA for PLK2 and GPRK6 siRNA is transfected in
primary neuronal cultures in preparation for introducing targets
into a mouse model. The method is performed as in Example 4.
qRT-PCR is performed (as in Example 2) using SYSY-synuclein RNA.
SY5Y-synuclein cells are derived from neuroblastoma cells and have
been stably transfected with a WT-synuclein vector.
Example 10
Distribution of Lentivirus-Expressed Alpha-Synuclein in Human
Cortical Culture (HCC)--a Cellular Model for Lewy Body Disease
[0231] Of interest was the identification of a cellular model for
Lewy body disease and/or for PD pathology. Thus,
lentivirus-mediated expression of alpha-synuclein in human cortical
cultures was used to establish a model of alpha-synuclein
deposition in vivo. Experiments were performed on donors, and HCC
cells overexpressing wild-type and variant alpha-synuclein to
fractionate the cells and localize wild-type alpha-synuclein and
variant alpha-synuclein within the cells. In one experiment
aggregation of alpha synuclein in a manner matching LB disease was
observed in the HCC cells. Further, in one experiment when PLK2 was
expressed, the phosphorylation of alpha-synuclein as well as the
aggregation increased. In other experiments this was not
observed.
[0232] Further experiments were performed to determine whether
extending culture might increase the accumulation of overexpressed
synuclein, and would stress the cells, which also might favor
synuclein deposition or toxicity. Accordingly, HCC were transduced
with viral vectors expressing WT, A53T, S129A or both A53T/S129A
alpha-synuclein mutants. Following transfection cells were grown in
vitro for 9, 16 or 23 days before collecting and fractionating.
ELISA results were normalized to protein concentration and showed
an accumulation of synuclein in the soluble fraction with
increasing time. Somewhat greater accumulation was observed with
the S129A mutant.
[0233] When further experiments were performed with WT,
119-truncated, and E46K AS, the results were as follows. The higher
the expression of wild-type the larger the portion of alpha
synuclein recovered in the soluble fraction. E46K synuclein showed
a 50-100% increase in the amount of phosphorylated alpha-synuclein.
However, the E46K mutation did not markedly affect relative amounts
of synuclein recovered in the membrane-bound or insoluble
fractions. Expression of 119-truncated alpha synuclein led to a
slight increase in the relative amount accumulating in the
insoluble fraction (about 3 fold higher relative to WT). The
increase is expected in view of the published results suggesting
that truncated synuclein forms fibrils much more readily in vitro
than does full-length (Murray et al. 2003 Biochemistry 42:8530).
The 119 truncation resulted in an increased association with
membranes consistent with the N-terminal domain being responsible
for association with lipid bilayers. The increased association with
membranes may mitigate the increased tendency of the soluble
protein to aggregate. The response of the insoluble fraction to
increases in levels of soluble synuclein on overexpression and to
truncation, a change favoring aggregation, suggest that it provides
a way to identify factors affecting aggregation in the
intraneuronal milieu.
[0234] The increased alpha-synuclein in the soluble compartment
might be shifting the alpha-synuclein to a potentially more
vulnerable compartment, leading to changes which could result in
increased deposition. Since the kinases proposed to phosphorylate
alpha-synuclein at Ser129 are soluble, it seems likely that the
soluble alpha-synuclein is more accessible to phosphorylation as
well.
[0235] Additional experiments are performed to identify inhibitors
of the phosphorylation and/or aggregation in this cellular model by
expressing the inhibitors in the cells and identifying a reduction
in the phosphorylation and/or aggregation.
Example 11
Analysis of Endogenous Kinase Activity in Alpha-Synuclein Knock-Out
Mice
[0236] The utilization of an alpha-synuclein knockout
(alpha-synuclein KO) mouse brain for the identification of a
putative alpha-synuclein kinase has the advantage over the siRNA
screen in the following ways: 1) the use of brain material provides
relevant and possibly higher levels of brain-specific kinase
activity which the HEK cell line may not provide; 2) cofactors may
be present in brain (lipid, protein, etc.) which may not be present
in cells and 3) absence of any endogenous alpha-synuclein which
could be detected as a phosphorylated AS. The inclusion of 25-50
.mu.g of extracts (soluble and detergent soluble) with recombinant
alpha-synuclein (rAS) was assessed with 250 .mu.M ATP to determine
if appreciable kinase activity was present in crude material. FIG.
8A shows total alpha-synuclein in each reaction indicating
equivalent loadings of rAS. In FIG. 8B, the levels of
phospho-ser-129 alpha-synuclein were investigated. The rAS in both
TBS (sucrose soluble) and TX (Triton-S 100 soluble) extracts was
phosphorylated, with roughly twice the level of signal from the TBS
material than the TX (although reactions were not normalized for
protein). Phosphorylation levels were increased by addition of CKI
but were not significantly affected by the addition of
phospholipids from soybean. An identical blot was probed for ser-87
phosphorylation in FIG. 9B. This pAb presents cross-reactivity with
rAS at 100 ng, thus levels above background indicate true
phosphorylation at the ser-87 site. In both TBS and TX reactions
there is no significant phosphorylation at this site whereas the
CKI spike achieved phosphorylation at appreciable levels. These
experiments suggest that measurable and real kinase activit(ies)
are present in the soluble and membrane fractions of KO mouse brain
and are specific to the ser-129 site compared to ser-87. The
potential exists for phosphorylation at other serine or threonine
sites in alpha-synuclein but antibodies are not yet available to
detect such modifications. Thus, measurable levels of ser-129
specific kinase activity/activities are present in alpha-synuclein
KO mouse brain extracts and could serve as starting material for
purification of a kinase from the brain.
[0237] In FIGS. 8A, 8B, 9A and 9B, cortices of alpha-synuclein KO
mouse brain were Dounce homogenized to obtain 200 mM sucrose
soluble and 0.1% Triton X-100 soluble extracts with protease and
phosphatase inhibitors present. 20 .mu.l of sample (100 .mu.l total
volume of reaction) was incubated with 2.4 .mu.g of wt-rAS in the
presence or absence of 1000 units of casein kinase I(CKI) as a
positive control, and/or 200 .mu.g of phosphatidycholine (PC;
soybean lecithin) to increase kinase activiti(es). Reactions were
loaded on SDS-PAGE (130 ng total AS) and immunoblotted with Syn-1
(total Syn; 0.1 ug/ml), 11A5 (phospho ser-129; 1 .mu.g/ml) or
ELADW110 (phospho ser-87; 2 .mu.g/mL).
[0238] The above data shows that PLK2 other direct and/or indirect
kinases (such as GRK6), and modulators such as synphilin are novel
targets for therapeutic intervention in DLB and PD. PLK2 is a
preferred target because it can directly phosphorylate
alpha-synuclein specifically at ser-129.
Example 12
Effect of Overexpression of PLK Family Members on Synuclein
Phosphorylation
[0239] Overexpression of PLK family members PLK1, 2 and 3, but not
PLK4, increased synuclein phosphorylation above the level of the
endogenous kinase in HEK-293 cells.
Methods
[0240] HEK-293 naive cells were transfected with an expression
vector encoding PLK1, PLK2, PLK3, or PLK4 under the control of a
CMV promoter, or empty vector, together with an expression vector
encoding WT-synuclein. All vectors had the sequence coding for the
protein of interest. Transfection was accomplished using
Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) with 0.08 .mu.g
vector. Cells were washed and harvested 48 hours post-transfection.
A Micro BCA (Pierce) total protein assay and total synuclein and
phospho-synuclein ELISAs were performed on each plate of treated
cells.
Results
[0241] Results are summarized in Table 16.
TABLE-US-00024 TABLE 16 Proportion of Fold Change total synuclein
that in Percent is phoshorylated at ser-129 Synuclein Endogenous
Kinase 2.0% Phosphorylation Endogenous Kinase + 1.6% 0.8 synphilin
Over-Expressed PLK1 4.1% 2.1 Over-Expressed PLK2 42.3% 21.2
Over-Expressed PLK3 79.2% 39.6 Over-Expressed PLK4 1.9% 1.0
PLK2
[0242] Over-expression of PLK2 in the 293 cells yielded similar
results to those observed in previous experiments, resulting in a
21-fold increase in synuclein phosphorylation, from 2% to
42.3%.
PLK3
[0243] The over-expression of PLK3 generated an even more striking
39-fold increase in synuclein phosphorylation, from 2% to 79.2%
phospho-synuclein. However, knockdown of PLK3 does not decrease the
percentage of phosphorylated synuclein in the 293 cells (see
Example 13, infra). PLK3 is structurally most similar to PLK2 of
all the PLK family members, so even if PLK3 is not the synuclein
kinase, it may be able to perform the same physiological tasks as
PLK2. It has been proposed that in the absence of PLK2 expression,
PLK3 can functionally compensate for the absent PLK2 (Smith et al.,
2006, "Epigenetic inactivation implies a tumor suppressor function
in hematologic malignancies for Polo-like kinase 2 but not
Polo-like kinase 3.Cell Cycle." Cell Cycle 5:1262-4). In addition,
over-expression experiments can yield off-target and
non-physiological effects and results should be substantiated
through additional experimentation such as siRNA knockdown or in
vitro transcription/translation experiments.
PLK1
[0244] Over-expression of PLK1 resulted in a two-fold increase in
the percent of synuclein phosphorylation (from 2% to 4.1%). While
not as robust an increase in synuclein phosphorylation as the
21-fold increase with PLK2 or 39-fold increase with PLK3
over-expression, it is still significant. However, as noted above,
overexpression of proteins can yield results that do not accurately
represent the physiological state within cells and tissues.
Over-expression of genes can produce off-target effects and can
result in erroneous localizations within cells that do not
characterize the true physiological state, and results of
over-expression experiments should be substantiated with additional
experimentation such as siRNA knockdown or in vitro
transcription/translation experiments.
PLK4
[0245] Over-expression of PLK4 did not change the percentage of
synuclein phosphorylation in 293 cells. The structure of PLK4 is
much different from those of the other PLKs, and has only a single
polo box domain rather than the two that the other three family
members have. The results do not exclude the possibility that in
other tissue types, PLK4 may be able to phosphorylate
synuclein.
In Vitro Biochemical Assays
[0246] In vitro biochemical assays using each of the four PLK
family members as the kinase to phosphorylate synuclein were
conducted. The results (not shown) mirror the cell-based
overexpression assays described above, with PLK1 being able to
phosphorylate synuclein moderately, PLK2 and PLK3 having extremely
robust phosphorylation of synuclein, and PLK4 exhibiting no ability
to phosphorylate synuclein.
Example 13
siRNA Knockdown of PLK Family Members
[0247] HEK-293 naive cells were transfected with 40 nM, 100 nM and
200 nM of Dharmacon On Target Plus Smart Pool siRNAs (Darmacon,
Lafayette, Colo.) designed to knock down the expression of each of
the four PLK family members. The transfection was performed using
Lipofectamine 2000 (Invitrogen Carlsbad, Calif.). Cells were washed
and harvested 48 hours post-transfection. A Micro BCA (Pierce)
total protein assay and total synuclein and phospho-synuclein
ELISAs were performed on each plate of treated cells.
PLK2
[0248] The results of the siRNA knockdown experiment are summarized
in FIG. 10. In agreement with what we have observed previously for
PLK2 siRNA inhibition (see Example 5) knockdown resulted in a 25%
decrease in the percentage of phosphorylated synuclein in the 293
cells.
PLK3
[0249] The knockdown of PLK3 also had no effect on synuclein
phosphorylation, which is not in agreement with the PLK3
overexpression data. This may be due to the fact that
overexpression can be somewhat promiscuous, and not indicative of
the true physiological state within cells. It may also be that PLK3
is not the synuclein kinase in 293 cells, but it could still be the
synuclein kinase in neurons or other cells due to the potential for
isoform switching between cell types. Thus, although PLK3 knockdown
does not reduce synuclein phosphorylation as we would expect from
inhibition of the synuclein kinase, it does not completely
eliminate PLK3 as being the correct PLK family member as the
synuclein kinase in neurons.
PLK1
[0250] Cells treated with PLK1 siRNA showed a 40% decrease in the
total protein levels compared to the negative siRNA control,
indicating that treatment of cells with PLK1 siRNA inhibits
proliferation of cells. This has been noted in the literature, and
confirms the role of PLK1 in mitosis. Taking into account this
change in total protein levels, knockdown of PLK1 transcript
results in a 60-90% increase in the percentage of
phospho-synuclein. This indicates that PLK1 is negative regulator
of synuclein phosphorylation. It appears that when PLK1 is present
in cell, it regulates PLK2 or it's upstream pathway and accordingly
the level of PLK2-mediated phosphorylation of synuclein. This
increase in phospho-synuclein with PLK1 knockdown has been observed
in two independent experiments, and is intriguing as a potential
regulator of PLK2-driven synuclein phosphorylation.
PLK4
[0251] Knockdown of PLK4 with siRNA had no effect on synuclein
phosphorylation, in accord with the PLK4 overexpression data above
(Example 11).
Example 14
Treatment of Primary Neuronal Cultures with Kinase Inhibitors
[0252] The effect on levels of serine-129 synuclein was tested for
kinase inhibitors with various specificities in rat and mouse
primary cortical cell culture was tested.
[0253] Table 17 shows the inhibitors used in the experiment:
TABLE-US-00025 TABLE 17 Inhibitor Primary Specificity 1 ELN-481080
PLK1 2 ELN-481574-2 (BI 2536) PLK 1, 2, 3 3 ELN-481530 JNK3 4 DMAT
(2-Dimethylamino-4,5,6,7- Casein kinase 2
tetrabromo-1H-benzimidazole) 5 Scytonemin PLK1, PKC.beta.1,
PKC.beta.2, Cdk1/B, Myt1, and Chk1 6 K252A Generic kinase inhibitor
7 Wortmannin Generic kinase inhibitor 8 N-benzoyl staurosporine
Generic kinase inhibitor 9 TBB (4,5,6,7-Tetrabromo-2-
ATP/GTP-competitive azabenzimidazole) inhibitor of casein kinase
2
Preparation of Mouse Neuronal Cell Cultures
[0254] Mouse cortical cultures from fetal (i) Swiss-Webster, (ii)
C56BL/6 WT and (iii) C56BL/6 E46K-Synuclein Transgenic mice were
prepared and maintained in B27/DMEM/1% Penicillin-Streptomycin at
37.degree. C./10% CO.sub.2 for three to fourteen days and then
treated with kinase inhibitors.
[0255] Cultures were exposed to inhibitors for two hours in
B27/DMEM/1% Penicillin-Streptomycin. Cells were immediately washed
in 100 .mu.L of PBS plus Mg.sup.2+ and Ca.sup.2+, and harvested in
ice-cold CEB minus EGTA plus protease inhibitors, frozen on dry ice
and stored at -80.degree. C. until processed. A Micro BCA (Pierce)
total protein assay, and total synuclein and phospho-synuclein
ELISAs were performed on each plate of treated cells using standard
methods.
Preparation of Rat Neuronal Cell Cultures
[0256] Rat Ventral Mesencephalon (RVM) cultures were prepared from
E15 Wistar rats. The RVM cultures were cultured for 2 days and
transduced with 0.75 MOI E46K-Synuclein lentivirus alone, or 0.75
MOI E46K-Synuclein lentivirus plus 0.75 MOI ca-PLK2 lentivirus. The
mid-brain region, ventral mesencephalon, from embryonic day 15
Wistar rats were dissected and pooled for processing for culture as
described previously in Steven et al., 2001, Genetics 10:1317-24
with the medium supplement replaced with B27 (Invitrogen) and 1%
FBS (HyClone). Transduced cultures were maintained in neuronal
media with B27/1% FBS and 37.degree. C./5% CO.sub.2 until treated
with inhibitors. On DIV 17 (which corresponds to 15 days after
viral transduction of the cultures) cells were treated with
inhibitors for two hours. Cells were then harvested as detailed
above in neuronal media with B27/1% FBS. A Micro BCA (Pierce) total
protein assay, and total synuclein and phospho-synuclein ELISAs
were performed on each plate of treated cells using standard
methods.
Results
[0257] The effects of inhibitors on phosphorylation of
alpha-synuclein are summarized in Tables 18 and 19. Table 18 shows
that the extent of inhibition of synuclein phosphorylation by
selected inhibitors is similar for the endogenous kinase in mouse
and rat primary cultures, as well as a constitutively active PLK2
variant (caPLK2). In caPLK2 the polo-box has been deleted,
activating the Thr to Asp mutation in the kinase activation loop.
Table 19 shows the rank order of potency for selected inhibitors is
similar between cell types and across species (mouse, rat and
human)
[0258] The results of treatment of several primary neuronal
cultures with PLK and other inhibitors were very similar to those
observed in 293 cells in both the EC.sub.50 values (Table 18) and
the rank order of potency (Table 19). The EC.sub.50 values for the
potent PLK inhibitor BI 2536 (ELN-481574-2) in all cellular
paradigms were in the nanomolar range, with most EC.sub.50 values
being 100 nM or less, and has an IC.sub.50 of 27 nM in the in vitro
biochemical assay. This PLK inhibitor is a very potent inhibitor of
synuclein phosphorylation in primary neuronal cultures in mouse and
rat, substantiating the role of a PLK family member as the
synuclein kinase.
[0259] The second most potent inhibitor in most of the cellular
paradigms tested was the generic kinase inhibitor K252A (EC.sub.50
values of 3-7 .mu.M). In the in vitro biochemical reaction, K252A
does not inhibit PLK2-driven synuclein phosphorylation
(IC.sub.50>10 .mu.M), indicating that its inhibition of
synuclein phosphorylation is through an upstream or downstream
regulator of PLK2.
[0260] The third most potent inhibitor of synuclein phosphorylation
in primary neuronal cells is ELN-481080, a PLK1 inhibitor that also
has inhibitory activity on PLK2, and to a lesser degree, PLK3 and
PLK4 (see Table 21). The IC.sub.50 of ELN-481080 was 7.7 .mu.M, and
the EC.sub.50 values in most cellular paradigms was 12-38
.mu.M.
[0261] The fourth most potent inhibitor of synuclein
phosphorylation in primary neuronal cells is the casein kinase 2
inhibitor DMAT
(2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole). The
EC.sub.50 values for inhibition of synuclein phosphorylation in
each of the cell types were 16-33 .mu.M. In the biochemical assay
DMAT was the second most potent inhibitor of synuclein
phosphorylation with an IC.sub.50 of 2.07 .mu.M. That DMAT can
directly act on PLK2 to inhibit synuclein phosphorylation suggests
that reports by others using to DMAT to show that the CKII is the
synuclein kinase may be due to the inhibitory effect the DMAT has
on PLK2 and not on casein kinase 2. To confirm that the inhibitory
effect DMAT is due to inhibition of PLK2, MCC were treated with
TBB, a very specific CKII inhibitor. TBB had no effect on synuclein
phosphorylation, even at concentrations of 100 .mu.M (data not
shown). Thus, we are confident that the inhibitory effect of DMAT
on synuclein phosphorylation is due to its direct activity on PLK2
and not CKII.
[0262] The other four inhibitors tested, Scytonemin, Wortmannin and
N-benzoyl staurosporine did not inhibit 50% of synuclein
phosphorylation in any of the cells or in the in vitro assay.
ELN-481530, the JNK3 inhibitor, also did not inhibit PLK2-driven
phosphorylation in vitro, or in most cell types tested. However, in
RVM stably transduced with E46K-synuclein (but not in conjunction
with caPLK2), concentrations of 1 .mu.M or greater of ELN-481530
and above inhibited 50% of synuclein phosphorylation, with the
inhibition reaching a plateau of 50%. In addition, 293 cells
over-expressing human WT-synuclein and WT-PLK2 reached the same
plateau of 50% inhibition at 10 .mu.M ELN-481530. While further
work needs to be done to elucidate the role that JNK may play in
synuclein phosphorylation, it seems reasonable to suggest the JNK
may be a regulator of PLK2 and synuclein phosphorylation in certain
cell types.
TABLE-US-00026 TABLE 18 The EC.sub.50/IC.sub.50 values for selected
inhibitors are similar between cell types and across species
(mouse, rat and human) RVM E46K- Over- Straight E46K-Syn TG
Swiss-Webster RVM E46K- Synuclein Endogenous Expressed Inhibitor
Biochemical WT-MCC MCC MCC Synuclein caPLK2 Kinase (293) PLK2
(293)* ELN-481080 7.7 .mu.M 17.3 .mu.M 12.3 .mu.M 14.8 .mu.M ~100
.mu.M 25.2 .mu.M ~100 .mu.M 38 .mu.M ELN-481574-2 0.027 .mu.M
<0.1 .mu.M <0.1 .mu.M 0.092 .mu.M 0.598 .mu.M 0.078 .mu.M
0.054 .mu.M 0.07 .mu.M DMAT 2.07 .mu.M 18.7 .mu.M 16.6 .mu.M 21.6
.mu.M 20 .mu.M 28.5 .mu.M 33 .mu.M 18.6 .mu.M K252A >10 .mu.M 3
.mu.M 3.1 .mu.M 2.9 .mu.M 3.2 .mu.M 3.5 .mu.M 3.6 .mu.M 7.1 .mu.M
ELN-481530 >100 .mu.M >100 .mu.M >100 .mu.M NT ~1 .mu.M
>100 .mu.M >100 .mu.M ~10 .mu.M Scytonemin >100 .mu.M
>100 .mu.M >100 .mu.M NT >100 .mu.M >100 .mu.M >100
.mu.M >100 .mu.M Wortmannin >100 .mu.M >100 .mu.M >100
.mu.M NT >100 .mu.M >100 .mu.M >100 .mu.M >100 .mu.M
N-Benzoyl >100 .mu.M >100 .mu.M >100 .mu.M NT >100
.mu.M >100 .mu.M >100 .mu.M >100 .mu.M Staurosporine *See
Example 14 NT - Inhibitor not tested in these cells
TABLE-US-00027 TABLE 19 The rank order of potency for selected
inhibitors is similar for different cell types and species (mouse,
rat and human) RVM E46K- Over- Straight E46K-Syn TG Swiss-Webster
RVM E46K- Synuclein Endogenous Expressed Inhibitor Biochemical
WT-MCC MCC MCC Synuclein caPLK2 Kinase (293) PLK2 (293)* ELN-481080
3 3 3 3 5 3 4 5 ELN-481574-2 1 1 1 1 1 1 1 1 DMAT 2 4 4 4 4 4 3 4
K252A 4 2 2 2 3 2 2 2 ELN-481530 NA NA NA NA 2 NA NA 3 Scytonemin
NA NA NA NA NA NA NA NA Wortmannin NA NA NA NA NA NA NA NA
N-Benzoyl NA NA NA NA NA NA NA NA Staurosporine *See Example 14
NA--Not Applicable due to an EC.sub.50 not being reached
Example 15
Specificity of ELN-481574
[0263] PLK inhibitor ELN-481574-2 (BI 2536) exhibited high potency
for reducing alpha-synuclein phosphorylation in a variety of cells,
including primary neuronal cells (see Example 14). The inhibitor
was screened against a panel of 260 kinases (almost half the
kinome) and found that at 10 uM, ELN-481574 was very potent in
inhibiting PLK2 and PLK3, although the compound also inhibited
several CaMKs and casein kinases. The ten kinases that were
inhibited the most by ELN-481574 were subjected to a nine-point
dose-response (0.003 uM to 3 uM) of the compound to determine the
IC.sub.50. The ELN-481574 compound has 16-fold selectivity for PLK2
(IC.sub.50 11 nM) and 13-fold selectivity for PLK3 (IC.sub.50 14
nM) over the next closest kinase IC.sub.50 (CaMKII.delta. 182 nM).
See Table 20 for a summary. This confirms that this inhibitor is
indeed potent and highly selective for at least two members of the
PLK family.
TABLE-US-00028 TABLE 20 Summary of IC.sub.50 Values for
ELN-481574-2 Kinase IC50 (nM) PLK2 11 PLK3 14 CaMKII.delta. 182 FAK
239 EGFR(L858R) 264 Fes 341 MLCK 643 PKC.mu. 1,126 CK1.gamma.3
1,231 CaMKII.beta. 1,496
Example 16
Effect of Kinase Inhibitors in HEK-293 Cells Overexpressing PLK
Family Members
[0264] HEK-293 cells as described in Example 12, which overexpress
synuclein in conjunction with empty vector or one of the PLK family
members, were exposed to inhibitors. EC.sub.50 values (Table 21)
and a rank order of potency (Table 22) was similar to that seen in
primary neuronal cells (see Example 13). The PLK inhibitor
ELN-481574-2 was the most potent inhibitor of synuclein
phosphorylation by the endogenous kinase and by each of the
over-expressed PLKs. The EC.sub.50 for the endogenous kinase was 99
nM, and was 36 nM, 80 nM, 438 nM and 192 nM for each of the PLK1,
2, 3 and 4. The EC.sub.50 for PLK3 is somewhat higher than it is
for the endogenous kinase or the other PLKs.
[0265] The second most potent inhibitor of synuclein
phosphorylation was DMAT, with EC.sub.50 values of .about.14-74
.mu.M for the over-expressed PLKs and 56 .mu.M for the endogenous
kinase. The PLK1 inhibitor ELN-481080 shows selectivity for PLK1
(16.7 .mu.M) over PLK2 (47.8 .mu.M), and while an EC.sub.50 was not
reached for inhibition of synuclein phosphorylation by
over-expressed PLK3 or PLK4, ELN-481080 did inhibit 30-40% of
synuclein phosphorylation at concentrations of 30-100 .mu.M. It
should be noted that while the EC.sub.50 for ELN-481080 activity on
the endogenous kinase was >1100M in this experiment, in previous
experiments, the EC.sub.50 has been .about.100 .mu.M (Table 18),
which is still within the three-fold range of variation of the
EC.sub.50 of PLK2.
[0266] In this experiment, treatment of overexpressed PLK family
members with the JNK3 inhibitor ELN-481530 did not inhibit
synuclein phosphorylation by 50%. However, it did inhibit 30-45% of
synuclein phosphorylation by cells overexpressing PLK2 or PLK3 at
concentrations of 1 .mu.M and above. In the presence of endogenous
kinase or over-expressed PLK1 or PLK4, inhibition was lower,
reaching a maximum of 15-20%.
TABLE-US-00029 TABLE 21 The EC.sub.50 values for ELN-481574-2 is
similar for the four PLK family members EC.sub.50 of Selected
Inhibitors Based on the Percent Inhibition of Synuclein
Phosphorylation Inhibitor Synuclein + Vector Synuclein + PLK1
Synuclein + PLK2 Synuclein + PLK3 Synuclein + PLK4 ELN-481080
>100 .mu.M 16.7 .mu.M 47.8 .mu.M >100 .mu.M >100 .mu.M
ELN-481574-2 0.099 .mu.M 0.036 .mu.M 0.08 .mu.M 0.438 .mu.M 0.192
.mu.M DMAT 55.7 .mu.M 27.2 .mu.M 13.9 .mu.M 37.3 .mu.M 74.3 .mu.M
ELN-481530 >100 .mu.M >100 .mu.M >100 .mu.M >100 .mu.M
>100 .mu.M
TABLE-US-00030 TABLE 22 The rank order of potency of inhibitors
tested is very similar between PLK family members Rank Order of
Potency of Inhibitors Based on Percent Inhibition of Synuclein
Phosphorylation Inhibitor Synuclein + Vector Synuclein + PLK1
Synuclein + PLK2 Synuclein + PLK3 Synuclein + PLK4 ELN-481080 NA 2
3 3 NA ELN-481574-2 1 1 1 1 1 DMAT 2 3 2 2 2 ELN-481530 NA NA NA NA
NA NA--Not Applicable due to an EC.sub.50 not being reached
Example 17
Effect of Staurosporin in HEK-293 Cells Overexpressing PLK2
[0267] HEK-293 cells as described in Example 12, which overexpress
synuclein or synuclein and PLK2. The EC.sub.50 value for the
endogenous kinase (synuclein only) was 4.35 .mu.M, and 11.16 .mu.M
for PLK2 over-expressing cells.
[0268] The above examples are illustrative only and do not define
the invention; other variants will be readily apparent to those of
ordinary skill in the art. The scope of the invention is
encompassed by the claims of any patent(s) issuing herefrom. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the issued claims along with their
full scope of equivalents. All publications, references (including
accession numbers), and patent documents cited in this application
are incorporated by reference in their entirety for all purposes to
the same extent as if each individual publication or patent
document were so individually denoted.
Sequence CWU 1
1
2212795DNAHomo sapiens 1gcacaagtgg accggggtgt tgggtgctag tcggcaccag
aggcaagggt gcgaggacca 60cggccggctc ggacgtgtga ccgcgcctag ggggtggcag
cgggcagtgc ggggcggcaa 120ggcgaccatg gagcttttgc ggactatcac
ctaccagcca gccgccagca ccaaaatgtg 180cgagcaggcg ctgggcaagg
gttgcggagc ggactcgaag aagaagcggc cgccgcagcc 240ccccgaggaa
tcgcagccac ctcagtccca ggcgcaagtg cccccggcgg cccctcacca
300ccatcaccac cattcgcact cggggccgga gatctcgcgg attatcgtcg
accccacgac 360tgggaagcgc tactgccggg gcaaagtgct gggaaagggt
ggctttgcaa aatgttacga 420gatgacagat ttgacaaata acaaagtcta
cgccgcaaaa attattcctc acagcagagt 480agctaaacct catcaaaggg
aaaagattga caaagaaata gagcttcaca gaattcttca 540tcataagcat
gtagtgcagt tttaccacta cttcgaggac aaagaaaaca tttacattct
600cttggaatac tgcagtagaa ggtcaatggc tcatattttg aaagcaagaa
aggtgttgac 660agagccagaa gttcgatact acctcaggca gattgtgtct
ggactgaaat accttcatga 720acaagaaatc ttgcacagag atctcaaact
agggaacttt tttattaatg aagccatgga 780actaaaagtt ggggacttcg
gtctggcagc caggctagaa cccttggaac acagaaggag 840aacgatatgt
ggtaccccaa attatctctc tcctgaagtc ctcaacaaac aaggacatgg
900ctgtgaatca gacatttggg ccctgggctg tgtaatgtat acaatgttac
tagggaggcc 960cccatttgaa actacaaatc tcaaagaaac ttataggtgc
ataagggaag caaggtatac 1020aatgccgtcc tcattgctgg ctcctgccaa
gcacttaatt gctagtatgt tgtccaaaaa 1080cccagaggat cgtcccagtt
tggatgacat cattcgacat gacttttttt tgcagggctt 1140cactccggac
agactgtctt ctagctgttg tcatacagtt ccagatttcc acttatcaag
1200cccagctaag aatttcttta agaaagcagc tgctgctctt tttggtggca
aaaaagacaa 1260agcaagatat attgacacac ataatagagt gtctaaagaa
gatgaagaca tctacaagct 1320taggcatgat ttgaaaaaga cttcaataac
tcagcaaccc agcaaacaca ggacagatga 1380ggagctccag ccacctacca
ccacagttgc caggtctgga acacccgcag tagaaaacaa 1440gcagcagatt
ggggatgcta ttcggatgat agtcagaggg actcttggca gctgtagcag
1500cagcagtgaa tgccttgaag acagtaccat gggaagtgtt gcagacacag
tggcaagggt 1560tcttcgggga tgtctggaaa acatgccgga agctgattgc
attcccaaag agcagctgag 1620cacatcattt cagtgggtca ccaaatgggt
tgattactct aacaaatatg gctttgggta 1680ccagctctca gaccacaccg
tcggtgtcct tttcaacaat ggtgctcaca tgagcctcct 1740tccagacaaa
aaaacagttc actattacgc agagcttggc caatgctcag ttttcccagc
1800aacagatgct cctgagcaat ttattagtca agtgacggtg ctgaaatact
tttctcatta 1860catggaggag aacctcatgg atggtggaga tctgcctagt
gttactgata ttcgaagacc 1920tcggctctac ctccttcagt ggctaaaatc
tgataaggcc ctaatgatgc tctttaatga 1980tggcaccttt caggtgaatt
tctaccatga tcatacaaaa atcatcatct gtagccaaaa 2040tgaagaatac
cttctcacct acatcaatga ggataggata tctacaactt tcaggctgac
2100aactctgctg atgtctggct gttcatcaga attaaaaaat cgaatggaat
atgccctgaa 2160catgctctta caaagatgta actgaaagac ttttcgaatg
gaccctatgg gactcctctt 2220ttccactgtg agatctacag ggaagccaaa
agaatgatct agagtatgtt gaagaagatg 2280gacatgtggt ggtacgaaaa
caattcccct gtggcctgct ggactggttg gaaccagaac 2340aggctaaggc
atacagttct tgactttgga caatccaaga gtgaaccaga atgcagtttt
2400ccttgagata cctgttttaa aaggtttttc agacaatttt gcagaaaggt
gcattgattc 2460ttaaattctc tctgttgaga gcatttcagc cagaggactt
tggaactgtg aatatacttc 2520ctgaagggga gggagaaggg aggaagctcc
catgttgttt aaaggctgta attggagcag 2580cttttggctg cgtaactgtg
aactatggcc atatataatt ttttttcatt aatttttgaa 2640gatacttgtg
gctggaaaag tgcattcctt gttaataaac tttttattta ttacagccca
2700aagagcagta tttattatca aaatgtcttt ttttttatgt tgaccatttt
aaaccgttgg 2760caataaagag tatgaaaacg cagaaaaaaa aaaaa
27952685PRTHomo sapiens 2Met Glu Leu Leu Arg Thr Ile Thr Tyr Gln
Pro Ala Ala Ser Thr Lys1 5 10 15Met Cys Glu Gln Ala Leu Gly Lys Gly
Cys Gly Ala Asp Ser Lys Lys20 25 30Lys Arg Pro Pro Gln Pro Pro Glu
Glu Ser Gln Pro Pro Gln Ser Gln35 40 45Ala Gln Val Pro Pro Ala Ala
Pro His His His His His His Ser His50 55 60Ser Gly Pro Glu Ile Ser
Arg Ile Ile Val Asp Pro Thr Thr Gly Lys65 70 75 80Arg Tyr Cys Arg
Gly Lys Val Leu Gly Lys Gly Gly Phe Ala Lys Cys85 90 95Tyr Glu Met
Thr Asp Leu Thr Asn Asn Lys Val Tyr Ala Ala Lys Ile100 105 110Ile
Pro His Ser Arg Val Ala Lys Pro His Gln Arg Glu Lys Ile Asp115 120
125Lys Glu Ile Glu Leu His Arg Ile Leu His His Lys His Val Val
Gln130 135 140Phe Tyr His Tyr Phe Glu Asp Lys Glu Asn Ile Tyr Ile
Leu Leu Glu145 150 155 160Tyr Cys Ser Arg Arg Ser Met Ala His Ile
Leu Lys Ala Arg Lys Val165 170 175Leu Thr Glu Pro Glu Val Arg Tyr
Tyr Leu Arg Gln Ile Val Ser Gly180 185 190Leu Lys Tyr Leu His Glu
Gln Glu Ile Leu His Arg Asp Leu Lys Leu195 200 205Gly Asn Phe Phe
Ile Asn Glu Ala Met Glu Leu Lys Val Gly Asp Phe210 215 220Gly Leu
Ala Ala Arg Leu Glu Pro Leu Glu His Arg Arg Arg Thr Ile225 230 235
240Cys Gly Thr Pro Asn Tyr Leu Ser Pro Glu Val Leu Asn Lys Gln
Gly245 250 255His Gly Cys Glu Ser Asp Ile Trp Ala Leu Gly Cys Val
Met Tyr Thr260 265 270Met Leu Leu Gly Arg Pro Pro Phe Glu Thr Thr
Asn Leu Lys Glu Thr275 280 285Tyr Arg Cys Ile Arg Glu Ala Arg Tyr
Thr Met Pro Ser Ser Leu Leu290 295 300Ala Pro Ala Lys His Leu Ile
Ala Ser Met Leu Ser Lys Asn Pro Glu305 310 315 320Asp Arg Pro Ser
Leu Asp Asp Ile Ile Arg His Asp Phe Phe Leu Gln325 330 335Gly Phe
Thr Pro Asp Arg Leu Ser Ser Ser Cys Cys His Thr Val Pro340 345
350Asp Phe His Leu Ser Ser Pro Ala Lys Asn Phe Phe Lys Lys Ala
Ala355 360 365Ala Ala Leu Phe Gly Gly Lys Lys Asp Lys Ala Arg Tyr
Ile Asp Thr370 375 380His Asn Arg Val Ser Lys Glu Asp Glu Asp Ile
Tyr Lys Leu Arg His385 390 395 400Asp Leu Lys Lys Thr Ser Ile Thr
Gln Gln Pro Ser Lys His Arg Thr405 410 415Asp Glu Glu Leu Gln Pro
Pro Thr Thr Thr Val Ala Arg Ser Gly Thr420 425 430Pro Ala Val Glu
Asn Lys Gln Gln Ile Gly Asp Ala Ile Arg Met Ile435 440 445Val Arg
Gly Thr Leu Gly Ser Cys Ser Ser Ser Ser Glu Cys Leu Glu450 455
460Asp Ser Thr Met Gly Ser Val Ala Asp Thr Val Ala Arg Val Leu
Arg465 470 475 480Gly Cys Leu Glu Asn Met Pro Glu Ala Asp Cys Ile
Pro Lys Glu Gln485 490 495Leu Ser Thr Ser Phe Gln Trp Val Thr Lys
Trp Val Asp Tyr Ser Asn500 505 510Lys Tyr Gly Phe Gly Tyr Gln Leu
Ser Asp His Thr Val Gly Val Leu515 520 525Phe Asn Asn Gly Ala His
Met Ser Leu Leu Pro Asp Lys Lys Thr Val530 535 540His Tyr Tyr Ala
Glu Leu Gly Gln Cys Ser Val Phe Pro Ala Thr Asp545 550 555 560Ala
Pro Glu Gln Phe Ile Ser Gln Val Thr Val Leu Lys Tyr Phe Ser565 570
575His Tyr Met Glu Glu Asn Leu Met Asp Gly Gly Asp Leu Pro Ser
Val580 585 590Thr Asp Ile Arg Arg Pro Arg Leu Tyr Leu Leu Gln Trp
Leu Lys Ser595 600 605Asp Lys Ala Leu Met Met Leu Phe Asn Asp Gly
Thr Phe Gln Val Asn610 615 620Phe Tyr His Asp His Thr Lys Ile Ile
Ile Cys Ser Gln Asn Glu Glu625 630 635 640Tyr Leu Leu Thr Tyr Ile
Asn Glu Asp Arg Ile Ser Thr Thr Phe Arg645 650 655Leu Thr Thr Leu
Leu Met Ser Gly Cys Ser Ser Glu Leu Lys Asn Arg660 665 670Met Glu
Tyr Ala Leu Asn Met Leu Leu Gln Arg Cys Asn675 680
685319DNAArtificialSynthetic siRNA sequence 3ccggagatct cgcggatta
19419DNAArtificialSynthetic siRNA sequence 4ggggcaaagt gctgggaaa
19519DNAArtificialSynthetic siRNA sequence 5tcacagcaga gtagctaaa
19619DNAArtificialSynthetic siRNA sequence 6gggaaaagat tgacaaaga
19719DNAArtificialSynthetic siRNA sequence 7gattgtgtct ggactgaaa
19819DNAArtificialSynthetic siRNA sequence 8gcacagagat ctcaaacta
19919DNAArtificialSynthetic siRNA sequence 9acacagaagg agaacgata
191019DNAArtificialSynthetic siRNA sequence 10aggagaacga tatgtggta
191119DNAArtificialSynthetic siRNA sequence 11cataagggaa gcaaggtat
191219DNAArtificialSynthetic siRNA sequence 12gctagtatgt tgtccaaaa
191319DNAArtificialSynthetic siRNA sequence 13gaagacatct acaagctta
191419DNAArtificialSynthetic siRNA sequence 14catcaatgag gataggata
191519DNAArtificialSynthetic siRNA sequence 15gacatgtggt ggtacgaaa
191619DNAArtificialSynthetic siRNA sequence 16cagaacaggc taaggcata
191719DNAArtificialSynthetic siRNA sequence 17gtgcattcct tgttaataa
191821DNAArtificialSynthetic siRNA sequence 18gguauacaau gccguccuct
t 211921DNAArtificialSynthetic siRNA sequence 19ggacuuugga
acugugaaut t 212021DNAArtificialSynthetic siRNA sequence
20gggaaaagau ugacaaagat t 212125PRTArtificialSynthetic motif
characterizing one class of zinc finger proteins 21Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa His
Xaa Xaa Xaa Xaa Xaa His20 2522140PRTHomo sapiens 22Met Asp Val Phe
Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val1 5 10 15Ala Ala Ala
Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys20 25 30Thr Lys
Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val35 40 45Val
His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr50 55
60Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys65
70 75 80Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val
Lys85 90 95Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu
Gly Ile100 105 110Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala
Tyr Glu Met Pro115 120 125Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro
Glu Ala130 135 140
* * * * *
References