U.S. patent application number 14/827364 was filed with the patent office on 2016-06-30 for screening methods using fyn in combination with tau protein.
This patent application is currently assigned to Proteome Sciences plc. The applicant listed for this patent is King's College London, Proteome Sciences plc. Invention is credited to Brian Henry Anderton, Helen Byers, Pascal Derkinderen, Christopher Hugh Reynolds, Malcolm Ward, Ritchie Williamson.
Application Number | 20160186233 14/827364 |
Document ID | / |
Family ID | 35064772 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160186233 |
Kind Code |
A1 |
Ward; Malcolm ; et
al. |
June 30, 2016 |
SCREENING METHODS USING FYN IN COMBINATION WITH TAU PROTEIN
Abstract
The present invention provides materials and methods relating to
screening for compounds useful in the treatment of Alzheimer's
disease and related conditions. In particular, screening methods
using tyrosine kinases are provided, as are methods relating to the
role of tyrosine kinases as therapeutic targets.
Inventors: |
Ward; Malcolm; (Surrey,
GB) ; Byers; Helen; (Surrey, GB) ; Anderton;
Brian Henry; (London, GB) ; Derkinderen; Pascal;
(London, GB) ; Reynolds; Christopher Hugh;
(London, GB) ; Williamson; Ritchie; (London,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Proteome Sciences plc
King's College London |
Cobham
London |
|
GB
GB |
|
|
Assignee: |
Proteome Sciences plc
Cobham
GB
King's College London
London
GB
|
Family ID: |
35064772 |
Appl. No.: |
14/827364 |
Filed: |
August 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13683375 |
Nov 21, 2012 |
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14827364 |
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11630720 |
Jun 4, 2007 |
8367360 |
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PCT/GB05/02475 |
Jun 21, 2005 |
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13683375 |
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60580901 |
Jun 21, 2004 |
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Current U.S.
Class: |
424/616 |
Current CPC
Class: |
A61P 25/14 20180101;
G01N 2500/02 20130101; G01N 2440/14 20130101; A61P 21/04 20180101;
G01N 33/6896 20130101; A61P 21/02 20180101; A61P 43/00 20180101;
G01N 2333/9121 20130101; A61P 25/16 20180101; G01N 2333/912
20130101; C12Q 1/485 20130101; A61P 25/28 20180101; A61K 33/40
20130101; A61P 25/00 20180101; A61P 25/02 20180101 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48; A61K 33/40 20060101 A61K033/40 |
Claims
1-51. (canceled)
52. A method of treating or preventing a tauopathy, the method
comprising: (a) screening for a substance which is a candidate
therapeutic agent for treating a tauopathy, said substance being
effective to inhibit the phosphorylation of a tau protein by a
tyrosine kinase, wherein the tau protein comprises at least one
phosphorylation site, comprising: (i) contacting at least one said
substance, the tau protein and the tyrosine kinase under in vitro
conditions in which the tyrosine kinase is capable of
phosphorylating said at least one phosphorylation site of the tau
protein in the absence of the substance; (ii) detecting whether the
substance inhibits the phosphorylation of the tau protein at said
at least one phosphorylation site of the tau protein by the
tyrosine kinase; and (iii) selecting the substance which inhibits
phosphorylation of the tau protein at said at least one
phosphorylation site; and (b) administering the substance selected
by screening step (a) to a patient in a therapeutically or
prophylactically effective amount; wherein the tyrosine kinase is
Fyn and the tau protein is a protein which undergoes
phosphorylation by Fyn and has at least 80% sequence identity with
the amino acid sequence of SEQ ID NO: 1, or a fragment of said tau
protein, said fragment comprising at least 25 amino acids and
including at least one said phosphorylation site; and wherein the
tauopathy is selected from the group consisting of Alzheimer's
Disease (AD), frontotemporal dementia with Parkinsonism linked to
chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP),
Pick's disease, corticobasal degeneration and multiple system
atrophy (MSA).
53. The method of claim 52, wherein the substance obtained from
screening step (a) is effective to inhibit the phosphorylation of
residues Y.sup.18F and/or Y.sup.310F on the tau protein.
54. The method of claim 52, wherein the detecting step further
comprises determining the extent of the inhibition of the
phosphorylation of the tau protein.
55. The method of claim 52, wherein the tau protein is paired
helical filament tau.
56. The method of claim 52, wherein the tau protein has greater
than 90% sequence identity with the tau protein comprising the
amino acid sequence set out in SEQ ID NO: 1.
57. The method of claim 52, wherein the tau protein comprises the
amino acid sequence set out in SEQ ID NO: 1.
58. The method of 52, wherein the tau protein is a fragment of the
tau protein comprising the amino acid sequence set out in SEQ ID
NO: 1.
59. The method of claim 52 wherein the step of detecting the
presence or absence of phosphorylation at one or more sites of the
tau protein employs mass spectroscopy.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/683,375, filed Nov. 21, 2012, which is a divisional of
U.S. patent application Ser. No. 11/630,720, filed Jun. 4, 2007,
now U.S. Pat. No. 8,367,360, issued Feb. 5, 2013, which is the U.S.
National Stage of International Application No. PCT/GB2005/002475,
filed Jun. 21, 2005, which claims the benefit of U.S. Provisional
Application No. 60/580,901, filed Jun. 21, 2004. The entire
disclosure of each of the aforesaid applications is incorporated by
reference in the present application.
FIELD OF THE INVENTION
[0002] The present invention relates to screening methods, and more
particularly to methods which relate to the role of tyrosine
kinases as therapeutic targets for Alzheimer's disease and related
conditions.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is a neurodegenerative disease
characterised by the presence of senile plaques and neurofibrillary
tangles in the brain. The degree of dementia at death correlates
better with neurofibrillary tangle numbers and with neuronal and
synaptic loss than with senile plaque counts. The presence of
neurofibrillary tangles in neurons results in the death of those
neurons, implying that prevention of tangle formation is an
important therapeutic goal. The principal protein that forms the
neurofibrillary tangle is the microtubule-associated protein, tau,
which assembles into filaments that have the appearance of twisting
about each other in pairs and are referred to as paired helical
filaments (PHF). PHF are present in different locations in
degenerating neurons in the Alzheimer brain and when many aggregate
in the neuronal cell body, they produce the neurofibrillary tangle
(Lee et al., 2001).
[0004] Senile plaques have an extracellular central deposit of
amyloid .beta.-peptide (A.beta.), which is surrounded by dystrophic
neurites to form the senile or neuritic plaque. In vitro and in
vivo A.beta. has been shown to be neurotoxic. A.beta. is derived by
proteolytic processing of the larger amyloid precursor protein
(APP). Much attention has been focused on A.beta. production as a
therapeutic target because its production is believed to be an
early event in AD pathogenesis. This is because mutations in the
APP gene, which give rise to autosomal dominant AD, result in
either increased overall production of A.beta. or in a relative
increase in the slightly longer A.beta..sub.42 over A.beta..sub.40,
the former being more amyloidogenic; A.beta..sub.42 has two
additional hydrophobic amino acids at the C-terminus of 40-residue
A.beta..sub.40 thereby endowing the peptide with an increased
tendency to aggregate and form amyloid fibres. Mutations in two
other genes that also cause autosomal dominant AD, presenilin-1 and
presenilin-2 (PS1 & PS2) also result in an increase in the
ratio of A.beta..sub.42 to A.beta..sub.40. The belief that A.beta.
deposition in the brain precedes the appearance of neurofibrillary
tangles has been the basis of the amyloid cascade hypothesis but it
has been uncertain whether tangles are important in pathogenesis or
are only an unimportant epiphenomenon. This has been changed by the
discovery of mutations in the gene for tau in some other related
neurodegenerative diseases.
[0005] The mechanism by which A.beta. kills neurons in the brain
has still to be established. Many studies of A.beta. toxicity have
been conducted in tissue culture using rat brain neuronal cultures.
We have shown that exposure of both foetal rat and human brain
neuronal cultures to aggregated A.beta. induces within 2 to 10
minutes increases in the phosphotyrosine content of several
proteins including tau (Williamson et al., 2002). We have also
shown that this treatment results in activation of the tyrosine
kinases FAK and Fyn, the latter being a member of the src family of
tyrosine kinases. This tyrosine phosphorylation of tau was
prevented by inhibitors that act on the src family of tyrosine
kinases and act on c-Abl.
[0006] It has previously been reported that increased levels of Fyn
are associated with neurons containing abnormally phosphorylated
tau in AD brain (Shirazi and Wood, 1993) and we have demonstrated
using antibodies that recognise phosphotyrosine that PHF-tau from
AD brain contains phosphotyrosine (Williamson et al., 2002). There
are five potential sites for tyrosine phosphorylation in tau, these
are residues 18, 29, 197, 310 and 394, based upon the numbering of
residues in the longest human brain isoforms of tau of 441 amino
acids. We have shown in vitro that Fyn and Lck, both src family
kinases, phosphorylate recombinant human tau and phosphotyrosines
18, 197, 310 and 394 were positively identified in one or more of
their respective tryptic peptides, from sequence information of
fragmented peptides (Scales et al., 2002).
[0007] Neurons in brain slices from transgenic mice in which the
Fyn gene has been disrupted are resistant to A.beta. toxicity
(Lambert et al., 1998). Thus, there is evidence that activation of
Fyn may be involved in A.beta. toxicity.
[0008] It has been reported that A.beta. treatment of microglia in
culture results in activation of several other tyrosine kinases,
namely Syk, Lyn and FAK (McDonald et al., 1997) and, as mentioned
above, we have found that FAK is also activated in primary neurons
exposed to A.beta. (Williamson et al., 2002). Syk has been reported
to phosphorylate .alpha.-synuclein on tyrosine, .alpha.-synuclein
being the principal protein of Lewy bodies which are the
pathological hallmark of Parkinson's disease and are also present
in up to 70% of AD brains (Negro et al., 2002). Finally, we have
found that the protein tyrosine kinase Abl phosphorylates tau in
co-transfected cells and Abl is implicated in activation of the
serine/threonine protein kinase cdk5, which is regarded as a
pathogenically important tau kinase that phosphorylates many
residues in tau that can alternatively be phosphorylated by GSK-3
(Zukerberg et al., 2000). Thus, there is the strong possibility
that tau is a substrate for various tyrosine kinases and that these
need to be considered in the context of the possible pathogenesis
of the tauopathies.
[0009] The presence of intraneuronal deposits of tau in the form of
typical neurofibrillary tangles in AD or other morphologically
distinct tau aggregates in a number of other neurodegenerative
diseases, is the basis for grouping these conditions as
tauopathies. Thus, in addition to AD, the main examples of the
tauopathies are frontotemporal dementia with Parkinsonism linked to
chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP),
Pick's disease, corticobasal degeneration, and multisystem atrophy
(MSA). The intracellular tau deposits (usually neuronal but
sometimes also glial) are filamentous and in a hyperphosphorylated
state compared to the phosphorylation of tau in control human
brain. In the case of AD, this hyperphosphorylated tau is often
referred to as PHF-tau because it is derived from the PHF.
[0010] Other than for AD, deposits of A.beta. in the brain are
either absent or minimal in these other tauopathies. There are some
tauopathy pedigrees with autosomal dominant disease in which the
causative gene has been identified as the tau gene and although
cases with the same mutation may present with apparently different
diseases, they invariably have tau deposits in the brain and are
mostly of the FTDP-17 variety. Thus, the finding of mutations in
the tau gene which result in disease and deposition of tau
aggregates in neurons is compelling evidence for the primary
pathogenic importance of tau deposition in all of these conditions,
including AD, whatever the primary cause of disease. Therefore, the
amyloid cascade hypothesis is borne out by the discovery of tau
mutations and confirms that indeed neurofibrillary tangle formation
may well be subservient to A.beta. deposition in AD, but that in
the other tauopathies lacking A.beta. deposits, then some other
primary event must trigger the tau pathology. Tau abnormalities and
deposition are therefore important therapeutic targets for all
tauopathies, including AD.
[0011] Tau is a phosphoprotein, the function of its phosphorylation
remaining to be unequivocally established. However, increased
phosphorylation of tau on multiple serine and threonine residues
reduces the ability of tau to promote microtubule assembly and to
stabilise assembled microtubules, effects that have been
demonstrated both in vitro and in cells. Many studies have shown
that PHF-tau from AD brain is more heavily phosphorylated on serine
and threonine than tau from control brain. This has been
demonstrated partly by protein sequencing and partly by
demonstrating that certain monoclonal antibodies only label either
PHF-tau or alternatively they label non-phosphorylated tau and not
PHF-tau; the epitopes for many of these antibodies have been mapped
to particular phosphorylated residues present in PHF-tau and absent
from, or present at lower levels in, control brain tau. The
pathological tau from most other cases of other tauopathies seems
to be similarly hyperphosphorylated to PHF-tau.
[0012] These findings strongly imply that similar abnormalities in
regulating phosphorylation of tau are shared by all the tauopathies
including AD. Since phosphorylation of proteins is effected by
protein kinases and dephosphorylation by protein phosphatases,
identifying the protein kinases and phosphatases for tau is
important because these enzymes are potential therapeutic targets
for these diseases.
[0013] As mentioned above, there are five tyrosines in human brain
tau. It has been reported that Fyn phosphorylates tau in
non-neuronal co-transfected cells and that tyrosine 18 is the
preferred phosphorylation site (Lee et al., 1998). We have reported
that PHF-tau isolated from Alzheimer brain is phosphorylated on
tyrosines and others have identified tyrosine 18 as one site of
phosphorylation (Williamson et al., 2002; Lee et al., 2004).
[0014] Cultured neurons from transgenic mice in which the tau gene
has been disrupted, such that these animals no longer express the
tau protein, are resistant to exposure to A.beta. and do not die
(Rapoport et al., 2002). This requirement of tau for A.beta. to be
neurotoxic has been confirmed in experiments in which neurons
treated with antisense oligonucleotides to reduce expression of tau
were resistant to the neurotoxic effects of A.beta. exposure (Liu
et al., 2004).
[0015] It remains a considerable problem in the art in identifying
the enzymes responsible for causing phosphorylation of paired
helical filament tau and the sites phosphorylated by those
enzymes.
SUMMARY OF THE INVENTION
[0016] Broadly, the present invention relates to the modulation of
the phosphorylation of tau protein at tyrosine sites through its
interaction with kinases. In particular, the present invention
concerns the identification of tyrosine phosphorylation sites in
tau protein and the kinases that preferentially phosphorylate
subsets of those sites (e.g. tyrosine kinases). This identifies
novel therapeutic targets and interactions that can be employed in
methods of screening for candidate therapeutic agents. In one
aspect, the present invention is based on the identification of the
role played by the protein tyrosine kinase c-Abl in the
phosphorylation of tyrosine 394 in PHF tau. This has not previously
been disclosed in the prior art. Prior to the present invention,
the prior art proposed that tyrosine 394 was phosphorylated by Fyn,
another Src family protein tyrosine kinase. Thus, in contrast to
prior art approaches based on screening for compounds capable of
inhibiting tyrosine 394 phosphorylation by inhibition of Fyn
kinase, in one aspect, the present invention provides methods of
screening for substances useful in the treatment of AD, or another
tauopathy, which are inhibitors of c-Abl.
[0017] Some of the work described herein involved the technically
difficult determination of tau phosphorylation state in PHF tau
from Alzheimer's patients brains, rather than more conventional
approaches employing normal tau or fetal brain tau. Accordingly,
the present invention provides the first disclosure of the presence
of tyrosine phosphorylation in clinical PHF tau, and in some
aspects, the first to link a specific kinase with a specific
phosphorylation event.
[0018] Furthermore, the identification herein of c-Abl as a
candidate for a target to treat AD is based on modifying the
disease associated tyrosine phosphorylation of tau and is further
supported by the observation that c-Abl may be a source of cdk5
activation since cdk5 is proposed as an alternative to GSK-3 in the
production of PHF tau.
[0019] Furthermore, the present invention proposes that fyn, lck
and c-Abl are candidate targets for drugs to treat AD or other
tauopathies because of the the work described herein which links
these kinases to the amyloid cascade theory through their
recruitment to lipid rafts. Without wishing to be bound by any
particular explanation, the present inventors propose that A.beta.
activates these tyrosine kinases through interaction with
cholesterol-rich domains of cell membranes and that this results in
an inappropriate over-association of tau with these regions of
membranes and a subsequent disturbance of intracellular cell
signaling processes. The kinases involved in these signalling
events can therefore be used alone or in any combination as
therapeutic targets for the screening of modulators of the activity
and/or their interaction with tau.
[0020] Accordingly, the present invention provides methods of
screening for candidate compounds useful in the treatment of
Alzheimer's disease or a tauopathy that act by inhibiting specific
phosphorylation of tau protein. These methods can be carried out in
many ways including measurement by mass spectrometry and
immunoassay.
[0021] Considering the requirements described above for the
obligatory expression of both Fyn and tau in order for A.beta. to
be neurotoxic and the previously known fact that Fyn can
phosphorylate tau in cells, the present inventors propose that a
sequence of biochemical events is involved in the processing of
tau. This is that exposure of neurons to A.beta. induces activation
of Fyn and probably other protein tyrosine kinases, which then
phosphorylates tau and this results in a series of further
biochemical changes ending in neuronal cell death, which may
involve hyperphosphorylation of tau on numerous serine and
threonine residues.
[0022] The first stage in identifying the responsible enzymes is to
map all of the phosphorylation sites in PHF-tau and compare the
complement of sites with those in control brain tau. Protein
sequencing studies have in total resulted in the identification of
25 phosphorylation sites in PHF-tau (Hanger et al., 1998;
Morishima-Kawashima et al., 1995); control brain tau has not been
studied as extensively and only a few of these sites have been
identified in tau from adult control human brain or from foetal
control human brain (tau from foetal brain is known to be more
phosphorylated than that from adult brain).
[0023] As described above, the prior art disclosed that of the five
potential tyrosine phosphorylation sites present in human tau
protein at positions 18, 29, 197, 310 and 394, the tyrosine kinases
Lck and Fyn phosphorylate tau at tyrosine positions 18, 310 and 394
and that tyrosine 18 is the preferred phosphorylation site of Fyn.
However, the present invention discloses for the first time that
PHF-tau from AD brain is phosphorylated at Tyr-394, a new result
arising from mass spectrometry experiments. The present invention
also demonstrates that Fyn phosphorylates primarily Tyr-18 in
cells, and that Abl phosphorylates primarily Tyr-394. These
findings mean that the phosphorylation of Tyr-394 may contribute to
AD pathology and that Abl is a potential drug target.
[0024] Accordingly, the work described in the present application
refines the initial indications provided in the prior art and to
investigate how AP may trigger activation of Fyn, how Fyn might
come into contact with tau, and on which particular tyrosine
residues in tau Fyn, and the kinases Syk and Abl, might act.
A.beta. Neurotoxicity--Lipid Rafts
[0025] Fyn is known to be associated with lipid rafts, which are
domains of cell membranes rich in cholesterol and sphingolipids.
Solubilising cells in certain detergents such as Triton X100 at
4.degree. C. enables isolation of lipid rafts since these
cholesterol-rich domains remain insoluble and can be separated from
other cell components by virtue of their low buoyant density. Thus,
lipid rafts are isolated by flotation on sucrose solutions by
ultracentrifugation. Furthermore, it has been reported that binding
of A.beta. to membranes is mediated, at least in part, by
cholesterol and that increasing membrane cholesterol levels is
positively correlated with A.beta. toxicity to neuronal and
endothelial cells (Eckert et al., 2000; Subasinghe et al., 2003;
Wang et al., 2001; Yip et al., 2001). Flotillin, a lipid raft
constituent, accumulates in tangle-bearing neurons in Alzheimer
brain indicating abnormalities in lipid rafts in the diseased brain
(Girardot et al., 2003), and indeed the protein composition of
lipid rafts isolated from Alzheimer brain has been reported to be
abnormal (Ledesma et al., 2003). We have investigated lipid rafts
in the context of A.beta. neurotoxicity.
[0026] The present application describes investigations into the
effects on lipid rafts of exposing neurons to A. To summarise, we
have found by western blotting that lipid rafts isolated from
primary cultures of rat brain cortical neurons contain the marker
protein, flotillin, as well as Fyn, FAK, and small but reproducible
amounts of actin, tubulin and tau. After exposure to 10 .mu.M
A.beta. for 5 min, there is an increase in the phosphotyrosine
content of numerous proteins, as detected with the phosphotyrosine
monoclonal antibody 4G10; there are also increases in the amounts
of Fyn, FAK, tau, tubulin, actin and c-Src kinase, but not
.beta.-catenin, relative to the flotillin content of lipid rafts
compared to untreated neurons.
[0027] We have also found that pre-treatment of neuronal cultures
with the Src family tyrosine kinase inhibitor, PP2, before exposure
to A.beta. and subsequent isolation of lipid rafts, resulted it
blocking of the recruitment of increased quantities of tau and Fyn
to the lipid rafts that was induced by A.
Tyrosine Phosphorylation of Tau
[0028] Fyn has previously been shown to phosphorylate tau in cells
co-transfected with tau and Fyn (Lee et al., 1998). As mentioned
above, we previously found that in vitro Fyn and Lck phosphorylate
human tau on four of the five tyrosines present in human tau (Y18,
Y197, Y310, Y394) (Scales et al., 2002). We have now made a series
of mutant forms of tau in which either each of the five tyrosines
was individually mutated to phenylalanine (F18, F29, F197, F310,
F394) or in which only a single tyrosine remained with the other
four replaced by phenylalanine (Y18-only, Y29-only, Y197-only,
Y310-only, Y394-only).
[0029] Using these mutants, we have found that by treating
non-neuronal cells transfected with these mutant forms of tau with
pervanadate to inhibit tyrosine phosphatases, there is an increase
in endogenous tyrosine phosphorylation of tau, principally on
tyrosine 394 with a contribution from tyrosine 197. In other
experiments in which mutant forms of tau were co-transfected with
Fyn, Syk or Abl tyrosine kinases, we found preferential
phosphorylation of tyrosine 18 and 310 by Fyn, tyrosines 18, 29,
197 and 394 by Syk but Abl phosphorylated preferentially tyrosines
197, 310 and 394. Using rat brain lysate in the presence of
pervanadate to phosphorylate recombinant human tau in vitro, we
have found by mass spectrometry that tyrosines 310 and 394 were
phosphorylated.
[0030] Finally, tyrosine phosphorylation in tau is a physiological
event since we have found by mass spectrometry unequivocal evidence
that tyrosine 394 is phosphorylated in tau isolated from human
foetal brain and in PHF.
Tyrosine Phosphorylation of Tau Generates an SH2 Binding Site for
Fyn
[0031] We have found that in vitro phosphorylation of tau by Lck
generates a binding site for the SH2 domain of Fyn. In summary, the
evidence suggests that more than one tyrosine kinase phosphorylates
tau, with different kinases preferentially phosphorylating
different tyrosine residues, and that A.beta. is capable of
activating at least some of those kinases. The data also
demonstrate that tyrosine phosphorylation of tau generates a
binding site for at least one tyrosine kinase, implying that tau
may be an important cell signalling protein in addition to its role
as a microtubule-associated protein.
[0032] Accordingly, in one aspect, the present invention proposes
that three kinases phosphorylate tau protein at the tyrosine
phosphorylation sites at Tyr18, Tyr29, Tyr197, Tyr310 and Tyr394.
The kinases are Fyn, Syk and Abl. A description and the sequences
of these kinases are provided in:
[0033] Fyn: Semba, K. et al (1986) Proc. Natl. Acad. Sci. USA 83,
5459-5463. See Genbank NM 002037 and that there are two main
isoforms. The present invention is primarily concerned with the
isoform expressed in brain, but the other isoform expressed in
haematopoietic cells, such as T cells, may also find use in' the
method of screening disclosed herein.
[0034] Syk: Law, C. L. et al, J. Biol. Chem. 269, 12310-12319. See
Genbank L28824.
[0035] c-Abl: Fainstein, E., Einat, M., Gokkel, E., Marcelle, C.,
Croce, C. M., Gale, R. P. and Canaani, E. (1989) Oncogene 4,
1477-1481. See Genbank X16416 and M14752. There are several
isoforms involving the N-terminus, but having a similar catalytic
domain.
[0036] In referring to these kinases, the present invention
includes the use of isoforms, splice variants, fragments and
sequence variants, as discussed in more detail below.
[0037] In particular, the kinases and the sites they preferentially
phosphorylate can be used in methods of screening for inhibitors of
phosphorylation or promoters of dephosphorylation. Preferably, the
screening method is for finding substances which are capable of
inhibiting phosphorylation. The screening method may involve
determining whether a candidate substance is capable of binding to
the kinase and/or tau protein, e.g. to inhibit or prevent the
phosphorylation of tau protein at a given site by the kinase in
question. This method may involve contacting the candidate
substance with the kinase and/or tau protein and determining
whether binding occurs, and optionally the affinity of the binding
reaction.
[0038] Alternatively or additionally, the method may comprise
determining whether a candidate substance is capable of inhibiting
a kinase, e.g. to inhibit or prevent the phosphorylation of a
substrate such as a tau protein at a site by one of the kinases, as
disclosed herein. This determination may comprise contacting a
candidate substance with the kinase in question and tau protein or
an alternative substrate (e.g. a fragment of tau comprising the
amino acid sequence around the phosphorylation site), and
determining whether the candidate substance inhibits the kinase
phosphorylating the substrate. The determining step may comprise
determining the extent of the inhibition. In situations where an
initial screen is carried out to identify candidate substances
which are capable of binding to tau protein or a kinase, or are
capable of inhibiting the activity of a kinase, the method may
comprise the further step of determining whether the binding or
inhibiting property of the candidate substance is capable of
inhibiting the phosphorylation of tau protein or a fragment thereof
in the presence of the kinase.
[0039] The screening for candidate substances having these
properties may employ tau protein, or a fragment, active portion or
sequence variant thereof comprising one or more of the relevant
phosphorylation sites. One example of a tau protein that may be
employed in this way is a fragment of tau comprising the amino acid
sequence around the phosphorylation site.
[0040] The sites and the kinases that preferentially phosphorylate
them are tyrosines 18 and 310 by Fyn, tyrosines 18, 29, 197 and 394
by Syk, and tyrosines 197, 310 and 394 by Abl.
[0041] As a consequence of these findings, the new sites and
kinases can be used as the basis of assays and assays methods for
screening for modulators of the phosphorylation of the sites in tau
protein for use or development as therapeutics for the treatment of
tauopathies. As a first step, the candidate substances may be
tested to determine whether they are inhibitors or promoters of the
kinases disclosed herein. Optionally, the method may alternatively
or additionally comprise determining whether a candidate substance
is capable of inhibiting the phosphorylation of tau by a kinase
and/or promoting the dephosphorylation of phosphorylated tau by a
phosphatase (e.g. a tyrosine phosphatase).
[0042] Accordingly, in a further aspect, the present invention
provides the use of (a) a kinase which is capable of
phosphorylating tau protein at the one or more of the sites
disclosed herein and (b) a substrate of the kinase, wherein the
kinase and substrate are used for identifying candidate substances
which are capable of inhibiting phosphorylation of the substrate by
a kinase.
[0043] In the present invention, the tau protein comprising the
phosphorylation sites may be substantially full length and/or wild
type tau or PHF tau protein, or may be a fragment, active portion
or sequence variant thereof. In other embodiments, the present
invention may employ a corresponding nucleic acid molecule encoding
the tau protein. Where a tau protein which is a fragment, active
portion or sequence variant is employed, the phosphorylation
site(s) may be present with surrounding amino acids from the tau
protein sequence. Preferably, the present invention employs PHF tau
protein. In the present invention the numbering of tau and PHF tau
is according to the sequence disclosed FIG. 1 of Goedert et al
(1989) Neuron 3, 519-526: Multiple isoforms of human
microtubule-associated protein Tau: sequences and localisation in
neurofibrillary tangles of Alzheimer's Disease Goedert M,
Spillantini M G, Rutherford D, Jakes R and Crowther R A.
[0044] Alternatively or additionally, any of the above defined tau
proteins may possess phosphorylation at one or more of the
phosphorylation sites. This enables the effects of cooperative
phosphorylation of the protein to be studied, that is, where the
phosphorylation of one site is dependent in changes to the tau
protein caused by one or more preceding or simultaneous
phosphorylation steps.
[0045] Thus, in some embodiments of the present invention, the tau
protein may include one or more of the known tau phosphorylation
sites.
[0046] In a further aspect, the present invention provides a method
of screening for substances which are capable of inhibiting
phosphorylation at one or more of the site(s) of a substrate by a
kinase, the method comprising: [0047] (a) contacting at least one
candidate substance, a kinase which is capable of phosphorylating
tau protein at the one or more of the sites disclosed herein and a
substrate of the kinase; [0048] (b) determining whether, and
optionally the extent to which, the candidate substance inhibits
the phosphorylation of the substrate by the kinase; and, [0049] (c)
selecting the candidate substance which inhibits phosphorylation of
the substrate.
[0050] The method disclosed herein may be employed for identifying
candidate substances useful in treating or developing lead
compounds for treating tauopathies.
[0051] In all aspects of the invention, the substrate may be a tau
protein, or comprise a fragment of tau protein, which includes one
or more of the phosphorylation site(s) acted on by the kinase. For
example, in the case of c-Abl, the substrate may be a fragment of
tau protein based on the amino acid sequence surrounding Tyr 394.
However, in other embodiments, other non-tau based substrates of
the kinase may be employed, for example where a substrate of the
kinase is readily available. In this case, the method may comprise
the further step of confirming whether a candidate substance
selected in an initial screen has the property of inhibiting the
phosphorylation of the tau protein under conditions in which the
kinase is capable of phosphorylating the site(s) of the tau protein
in the absence of the candidate substance.
[0052] In this embodiment, the method may additionally involve
including a phosphatase inhibitor in step (a) to inhibit
phosphatases that may be present in the system from
dephosphorylating the tau protein.
[0053] In a further aspect, the present invention provides a method
of screening for substances which are capable of promoting
dephosphorylation at one or more of the site(s) of a substrate by a
phosphatase, the method comprising: [0054] (a) contacting at least
one candidate substance, a phosphatase which is capable of
dephosphorylating tau protein at the one or more of the sites
disclosed herein and a substrate of the phosphatase; [0055] (b)
determining whether, and optionally the extent to which, the
candidate substance promotes the dephosphorylation of the substrate
by the phosphatase; and, [0056] (c) selecting the candidate
substance which promotes the dephosphorylation of the
substrate.
[0057] In this embodiment, the method may additionally involve
including a kinase inhibitor in step (a) to inhibit kinases that
may be present in the system from phosphorylating the tau
protein.
[0058] Examples of screening techniques suitable for use according
to the present invention will be well known to the skilled person.
By way of example, a cell based screening assay may be carried out
by co-transfecting cells with nucleic acid encoding tau and
encoding one or more of Fyn, Syk or Abl tyrosine kinases, and
determining the effect that candidate compounds have on tau
phosphorylation, in particular at tyrosines 18 and 310 by Fyn,
tyrosines 18, 29, 197 and 394 by Syk and tyrosines 197, 310 and 394
by Abl. Preferred methods of screening may involve the use of mass
spectroscopy to determine the phosphorylation at sites of tau, and
this is described in detail below. Conveniently, the methods of
screening may be carried out in a multiplex assay format in which a
solid phase is employed on which a plurality of substrates are
immobilised (e.g. in an array), the substrates corresponding to
phosphorylation sites of tau. By way of example, the substrates may
comprise fragments of tau protein. This is described in more detail
below. The present invention therefore provides a kit or solid
phase adapted for carrying out a multiplex screening assay
according to the present invention.
[0059] In some embodiments, the method may comprise, having
identified a candidate substance according to one of the methods
disclosed herein, the further step(s) of optimising the candidate
substance to improve one or more of its properties and/or
formulating it as a pharmaceutical.
[0060] The methods and uses disclosed herein employ one of more
kinases selected from Fyn, Syk or Abl. However, the screening
method may comprise investigating the effect of one or more further
enzymes on phosphorylation sites of tau. Examples of suitable
further enzymes for use in any aspect of the invention are provided
below in the section on multiplex assays.
[0061] In a further aspect, the invention provides for the use of a
modulator of tau protein phosphorylation obtainable by the methods
described herein in the treatment of a tauopathy. Preferably, the
modulator is an inhibitor of tau protein phosphorylation.
[0062] In a related aspect, the invention provides for the use of a
c-Abl, Syk or Fyn inhibitor in the preparation of a medicament for
the treatment of a tauopathy.
[0063] In the present invention, preferably the step of detecting
the presence and extent of phosphorylation and dephosphorylation in
the tau protein can be carried out using mass spectroscopy as
described in detail below.
[0064] Alternatively, or additionally, site specific recognition
agents which are capable of distinguishing between a site which is
phosphorylated and one which is not may be used. Examples of such
agents known in the art are site specific antibodies such as
monoclonal antibody AT100.
[0065] In a further aspect, the present invention provides a
substance obtainable from one of the methods disclosed herein which
is capable of inhibiting the phosphorylation or promoting the
dephosphorylation of a tau protein at one or more of the above
defined sites.
BRIEF DESCRIPTION OF THE FIGURES
[0066] FIG. 1 shows the amino acid sequence of the human tau
isoform used for the numbering given in this application (SEQ ID
NO: 1). The phosphorylation sites Y18, Y29, Y197, Y310 and Y394 are
indicated in bold.
[0067] FIG. 2 shows the amino acid sequence of the p150 isoform of
human c-Abl (SEQ ID NO: 2).
[0068] FIG. 3 shows the amino acid sequence of human Syk (SEQ ID
NO: 3).
[0069] FIG. 4 shows the amino acid sequence of isoform 1 of human
Fyn (SEQ ID NO: 4).
[0070] Embodiments of the present invention will now be discussed
in more detail by way of example and not limitation.
DETAILED DESCRIPTION
Tau Proteins
[0071] The assays and assay methods disclosed herein can employ
wild-type or full length tau proteins, kinases or phosphatases or
fragments, active portions or derivatives thereof. In the case of
tau proteins, the materials used in the assays may be
unphosphorylated or partially phosphorylated as discussed
above.
[0072] In the present invention, derivatives of the tau proteins,
kinases (especially Fyn, Syk and Abl) or phosphatases have an amino
acid sequence which differs by one or more amino acid residues from
the wild-type amino acid sequence, by one or more of addition,
insertion, deletion and substitution of one or more amino acids.
Thus, variants, derivatives, alleles, mutants and homologues, e.g.
from other organisms, are included. Thus, a derivative of tau
protein or kinase may include 1, 2, 3, 4, 5, greater than 5, or
greater than 10 amino acid alterations such as substitutions with
respect to the wild-type sequence.
[0073] Preferably, a fragment or derivative of a protein used in
the assays disclosed herein shares sequence identity with the
corresponding portion of the relevant wild-type sequence of the
protein, and preferably has at least about 60%, or 70%, or 75%, or
80%, or 85%, 90% or 95% sequence identity. Preferred fragments
comprise at least 5, at least 10, at least 15, at least 20 or at
least 25 amino acids which correspond to or share sequence identity
with tau protein. Optionally, the fragments may comprise a fragment
of tau protein linked or conjugated to other moieties, for example
expression tags, purification tags, groups to enable the fragment
to be immobilised or otherwise manipulated, or labels. As is
well-understood, identity at the amino acid level is generally in
terms of amino acid identity which may be defined and determined by
the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215:
403-10, which is in standard use in the art.
[0074] Identity may be over the full-length of the relevant peptide
or over a contiguous sequence of about 5, 10, 15, 20, 25, 30, 35,
50, 75, 100 or more amino acids, compared with the relevant
wild-type amino acid sequence. Alternatively, nucleic acid encoding
a fragment or derivative may hybridise to the corresponding wild
type nucleic acid under stringent conditions, for example as
disclosed in textbooks such as Ausubel, Short Protocols in
Molecular Biology, 1992 or Sambrook et al, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbour Laboratory Press, 1989,
using a hybridization solution comprising: 5.times.SSC,
5.times.Denhardt's reagent, 0.5-1.0% SDS, 100 pg/ml denatured,
fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to
50% formamide. Hybridization is carried out at 37-42.degree. C. for
at least six hours. Following hybridization, filters are washed as
follows: (1) 5 minutes at room temperature in 2.times.SSC and 1%
SDS; (2) 15 minutes at room temperature in 2.times.SSC and 0.1%
SDS; (3) 30 minutes-1 hour at 37.degree. C. in 1.times.SSC and 1%
SDS; (4) 2 hours at 42-65.degree. C. in 1.times.SSC and 1% SDS,
changing the solution every 30 minutes.
[0075] One common formula for calculating the stringency conditions
required to achieve hybridization between nucleic acid molecules of
a specified sequence homology is (Sambrook et al., 1989):
T.sub.m,=81.5.degree. C.+16.6 Log [Na+]+0.41(% G+C)-0.63(%
formamide)-600/#bp in duplex.
[0076] As an illustration of the above formula, using [Na+]=[0.368]
and 50% formamide, with GC content of 42% and an average probe size
of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA
duplex decreases by 1-1.5.degree. C. with every 1% decrease in
homology. Thus, targets with greater than about 75% sequence
identity would be observed using a hybridization temperature of
42.degree. C. Such a sequence would be considered substantially
homologous to the nucleic acid sequence of the present
invention.
Methods of Screening for Inhibitors and Enhancers
[0077] It is well known that pharmaceutical research leading to the
identification of a new drug may involve the screening of very
large numbers of candidate substances, both before and even after a
lead compound has been found. This is one factor which makes
pharmaceutical research very expensive and time-consuming. Means
for assisting in the screening process can have considerable
commercial importance and utility.
[0078] As detailed above, methods of screening for a substance
which are inhibitors of phosphorylation of tau protein or promoters
of dephosphorylation of tau protein can be carried out by
contacting one or more test substances with the tau protein and
kinase or phosphatase (as defined herein) in a suitable reaction
medium, and determining the presence or extent of phosphorylation
of dephosphorylation in the presence and absence of the candidate
substance. A difference in activity in the presence and absence of
the candidate substance is indicative of a modulating effect.
[0079] Preliminary assays in vitro may be followed by, or run in
parallel with, in vivo assays. Of course, the person skilled in the
art will design any appropriate control experiments with which to
compare results obtained in test assays.
[0080] Performance of an assay method according to the present
invention may be followed by isolation and/or manufacture and/or
use of a compound, substance or molecule which tests positive for
ability to modulate interaction between one of the phosphorylation
sites of tau protein (as defined herein) and a kinase (as disclosed
herein) or a phosphatase.
[0081] The precise format of an assay of the invention may be
varied by those of skill in the art using routine skill and
knowledge. For example, interaction between substances may be
studied in vitro by labelling one with a detectable label and
bringing it into contact with the other which has been immobilised
on a solid support. Suitable detectable labels, especially for
peptidyl substances include .sup.35S-methionine which may be
incorporated into recombinantly produced peptides and polypeptides.
Recombinantly produced peptides and polypeptides may also be
expressed as a fusion protein containing an epitope which can be
labelled with an antibody.
[0082] The protein which is immobilized on a solid support may be
immobilized using an antibody against that protein bound to a solid
support or via other technologies which are known per se. A
preferred in vitro interaction may utilise a fusion protein
including glutathione-S-transferase (GST). This may be immobilized
on glutathione agarose beads. In an in vitro assay format of the
type described above a test compound can be assayed by determining
its ability to diminish the amount of labelled peptide or
polypeptide which binds to the immobilized GST-fusion polypeptide.
This may be determined by fractionating the glutathione-agarose
beads by SDS-polyacrylamide gel electrophoresis. Alternatively, the
beads may be rinsed to remove unbound protein and the amount of
protein which has bound can be determined by counting the amount of
label present in, for example, a suitable scintillation
counter.
[0083] The amount of a candidate substance which may be added to an
assay of the invention will normally be determined by trial and
error depending upon the type of compound used. Typically, from
about 0.001 nM to 1 mM or more concentrations of putative inhibitor
compound may be used, for example from 0.01 nM to 100 .mu.M, e.g.
0.1 to 50 .mu.M, such as about 10 .mu.M. Greater concentrations may
be used when a peptide is the test substance. Even a molecule which
has a weak effect may be a useful lead compound for further
investigation and development.
[0084] Combinatorial library technology provides an efficient way
of testing a potentially vast number of different substances for
ability to modulate activity of a polypeptide. Such libraries and
their use are known in the art. Compounds which may be used may be
natural or synthetic chemical compounds used in drug screening
programmes. Extracts of plants which contain several characterised
or uncharacterised components may also be used.
[0085] Antibodies directed to the site of interaction in either
protein form a further class of putative inhibitor compounds.
Candidate inhibitor antibodies may be characterised and their
binding regions determined to provide single chain antibodies and
fragments thereof which are responsible for disrupting the
interaction. Antibodies may also be employed as site specific
recognition agents for determining whether phosphorylation of a
site in tau protein has occurred during as assay.
[0086] Antibodies may be obtained using techniques which are
standard in the art. Methods of producing antibodies include
immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or
monkey) with the protein or a fragment thereof. Antibodies may be
obtained from immunised animals using any of a variety of
techniques known in the art, and screened, preferably using binding
of antibody to antigen of interest. For instance, Western blotting
techniques or immunoprecipitation may be used (Armitage et al.,
1992, Nature 357: 80-82). Isolation of antibodies and/or
antibody-producing cells from an animal may be accompanied by a
step of sacrificing the animal.
[0087] As an alternative or supplement to immunising a mammal with
a peptide, an antibody specific for a protein may be obtained from
a recombinantly produced library of expressed immunoglobulin
variable domains, e.g. using lambda bacteriophage or filamentous
bacteriophage which display functional immunoglobulin binding
domains on their surfaces; for instance see WO 92/01047. The
library may be naive, that is constructed from sequences obtained
from an organism which has not been immunised with any of the
proteins (or fragments), or may be one constructed using sequences
obtained from an organism which has been exposed to the antigen of
interest.
[0088] Antibodies according to the present invention may be
modified in a number of ways. Indeed the term "antibody" should be
construed as covering any binding substance having a binding domain
with the required specificity.
[0089] Thus the invention covers antibody fragments, derivatives,
functional equivalents and homologues of antibodies, including
synthetic molecules and molecules whose shape mimicks that of an
antibody enabling it to bind an antigen or epitope.
[0090] Example antibody fragments, capable of binding an antigen or
other binding partner are the Fab fragment consisting of the VL,
VH, CI and CH1 domains; the Fd fragment consisting of the VH and
CH1 domains; the Fv fragment consisting of the VL and VH domains of
a single arm of an antibody; the dAb fragment which consists of a
VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent
fragment including two Fab fragments linked by a disulphide bridge
at the hinge region. Single chain Fv fragments are also
included.
[0091] A hybridoma producing a monoclonal antibody according to the
present invention may be subject to genetic mutation or other
changes. It will further be understood by those skilled in the art
that a monoclonal antibody can be subjected to the techniques of
recombinant DNA technology to produce other antibodies or chimeric
molecules which retain the specificity of the original antibody.
Such techniques may involve introducing DNA encoding the
immunoglobulin variable region, or the complementarity determining
regions (CDRs), of an antibody to the constant regions, or constant
regions plus framework regions, of a different immunoglobulin. See,
for instance, EP 0 184 187 A, GB 2 188 638 A or EP 0 239 400 A.
Cloning and expression of chimeric antibodies are described in EP 0
120 694 A and EP 0 125 023 A.
[0092] Hybridomas capable of producing antibody with desired
binding characteristics are within the scope of the present
invention, as are host cells, eukaryotic or prokaryotic, containing
nucleic acid encoding antibodies (including antibody fragments) and
capable of their expression. The invention also provides methods of
production of the antibodies including growing a cell capable of
producing the antibody under conditions in which the antibody is
produced, and preferably secreted.
[0093] The reactivities of antibodies on a sample may be determined
by any appropriate means. Tagging with individual reporter
molecules is one possibility. The reporter molecules may directly
or indirectly generate detectable, and preferably measurable,
signals. The linkage of reporter molecules may be directly or
indirectly, covalently, e.g. via a peptide bond or non-covalently.
Linkage via a peptide bond may be as a result of recombinant
expression of a gene fusion encoding antibody and reporter
molecule. The mode of determining binding is not a feature of the
present invention and those skilled in the art are able to choose a
suitable mode according to their preference and general
knowledge.
[0094] Other candidate inhibitor compounds may be based on
modelling the 3-dimensional structure of a polypeptide or peptide
fragment and using rational drug design to provide potential
inhibitor compounds with particular molecular shape, size and
charge characteristics.
Mass Spectroscopy
[0095] An LC/MS/MS based strategy was used to discover new
phosphorylation sites within tau protein isolated from AD brain. So
called PHF-tau was initially extracted from a heat-stable
preparation of human AD brain material and subsequently further
purified by ion exchange chromatography. Having been separated
using SDS-PAGE, phospho-peptide mapping was then undertaken.
Coomassie stained bands are excised, reduced, alkylated and
enzymatically digested using a suite of proteases such as trypsin,
chymotrypsin and endoproteinase Asp-N. Resulting peptide mixtures
are then analysed by LC/MS/MS using a Q-TOF micro instrument with
peptide separation achieved using a 75 micron ID PepMap reversed
phase column with peptides eluted using a gradient of acetonitrile
at a flowrate of 200 nl/min.
[0096] Database searching against bespoke index files is performed
utilising the Mascot algorithm (Matrix Science). All MS/MS spectra
relating to phosphopeptides are then subsequently visually verified
to check the result.
[0097] Tandem MS/MS of peptides may be used to provide sequence
information by virtue of the fragment ions produced. Fragmentation
occurs generally across the peptide bond leading to a ladder of
sequence ions that are diagnostic of the amino acid sequence. The
difference between consecutive ions in a series indicates the mass
of the amino acid at that position in the peptide. The most common
ion types are b and y ions. The C-terminal containing fragments are
designated y-ions and the N-terminal containing fragments are
designated b-ions (Roepstorff, P., Fohlman, J. J. Biomed. Mass
Spectrom. 1984, 11, 601). Peptides created by trypsin proteolysis
and ionised by electrospray generally form ions that are doubly
charged. This stems from the presence of basic groups within the
peptide, namely, the alpha amino group at the N-terminus and the
side chain of the C-terminal lysine or arginine. MS/MS spectra of
such peptides generally yield a prominent y-type ion series in the
high mass end of the spectrum (Bonner, R., Shushan, B. Rapid
Commun. Mass Spectrom. 1995, 9, 1067-1076). Ideally, for de novo
sequencing purposes, a complete set of complementary b and y ions
will ensure a high confidence level for the proposed peptide
sequence. Moreover, if fragment ions representing the complete
sequence of the peptide are present, the site of attachment of the
phosphate group can be deduced from the position and pattern of
these fragment ions. Therefore, it is possible in most instances to
discover the exact site of phosphorylation in each phosphopeptide.
In some instances we have even found MS/MS spectra to be
heterogeneous. Here two (or more) distinct phosphopeptides are
represented in the same spectrum. This is because each
phosphopeptide form has the same molecule weight and the same
number of phosphate groups, but these are attached to different
amino acids within the peptide. Therefore, both forms give rise to
precursor ions of the same m/z ratio, which are then selected
simultaneously by the mass spectrometer during the MS/MS
experiment. In such cases, we refer to the phosphopeptides
concerned as "regiomers"
Multiplex Assays for Screening Compounds
[0098] In drug development it is desirable to develop rapid high
throughput assays with simple read out to show whether a compound
has an effect on the proposed target. In the case of compounds
inhibiting an enzyme function, such as a kinase, it is possible to
develop an artificial substrate for the target enzyme that is
modified by the enzyme in a way that the level of modification can
be readily detected. In the presence of an inhibitory compound, the
substrate is not modified and this can also be readily
detected.
[0099] In the case of inhibitors of tau phosphorylation, it is
necessary to monitor the effect of inhibiting specific protein
kinases on the phosphorylation status of a large number of sites.
In one aspect, it is possible to prepare artificial substrates
corresponding to each of the phosphorylation sites on tau and
assess each compound for their ability to inhibit the
phosphorylation of each site independent of the other sites. In
such a system, each compound would be added to multiple wells each
well containing the proposed kinase target, one of the
phosphorylation site-specific artificial substrates and a reporter
system to show phosphorylation, such as a monoclonal antibody that
binds specifically to the substrate in either the phosphorylated or
unphosphorylated form, and which antibody is labelled with a
fluorescent marker, an enzyme that converts a colour less substrate
into a coloured product, or an enzyme that promotes the production
of a luminescent signal. In such an assay, it is desirable that the
artificial substrate for the target is immobilised on a solid
surface such that as part of the assay procedure any unreacted
antibody is removed from the system by washing before the result is
read. Such assays may be run in microtitre wells of varying formats
of typically 96, or more typically 384, or even more typically 1536
wells, or alternatively may be run on a microarray based on a solid
support such as glass.
[0100] Alternatively, the effect of different kinase inhibitors on
the global phosphorylation status of tau may be designed. In such
an assay, full length recombinant tau protein carrying no
phosphorylations, or one or more desirable phosphorylations may be
used as the substrate. Alternatively, a mixture of equal amounts of
all of the artificial substrates representing single
phosphorylation sites may be used. Each screening assay will
determine the effect of compounds on the inhibition of one, two or
more protein kinases with known activity for the phosphorylation of
tau. As with the more simple assays described above substrate,
target kinase and compound are added to a well of a microtitre
plate and incubated with appropriate buffers and other constituents
that permit the phosphorylation of substrate in the absence of an
inhibitory compound. The phosphorylation status of the substrate
may then be determined using a mixture of antibodies or other
molecules with specificity for individual phosphorylation sites on
tau, wherein such antibodies or other molecules are each labelled
with a unique reporter such as a fluorescent dye or compounds with
unique spectral properties in infra-red, visible or ultraviolet
spectra. After removal of antibodies that remain unbound to the
phosphorylated substrate(s), levels of each specific reporter are
determined using an appropriate reading device, and the levels of
phosphorylation at each specific site in tau is revealed by
comparison with a control where no kinase inhibitor was added.
[0101] In a preferred embodiment of such a multiplex screening
assay, the substrate is dephosphorylated recombinant tau protein
and the kinase is selected from CK1, CK2, GSK-3a, GSK-3b, PKA,
CDK5, ERK1/2, SAPK1g, SAPK2a, SAPK2b, SAPK3, SAPK4, stress
activated protein kinase family kinases (SAPKs) such as p38MAPK and
JNK, MARK family kinases such as 110K, cdc2, cdk2, PKC, PKN, TTK,
PKB, DYRK, PK, CaMKII, PKD, or a mixture of one of more these
kinases. Reporter systems are preferably labelled antibodies,
typically monoclonal antibodies, for example those that can be
obtained from rabbits or mice using techniques well known in the
art. Labels are preferably fluorescent or colorimetric compounds
that are covalently attached to antibodies, more preferably
fluorescent or colorimetric nanoparticles and are most preferably
nanoparticles with unique Raman spectra.
Development of Mimetic Substances
[0102] Once candidate substance have been found in the assays and
screens according to the present invention, they may be used to
design mimetic compounds for development as drugs. The designing of
mimetics to a known pharmaceutically active compound is a known
approach to the development of pharmaceuticals based on a "lead"
compound. This might be desirable where the active compound is
difficult or expensive to synthesise or where it is unsuitable for
a particular method of administration, e.g. peptides are unsuitable
active agents for oral compositions as they tend to be quickly
degraded by proteases in the alimentary canal. Mimetic design,
synthesis and testing is generally used to avoid randomly screening
large number of molecules for a target property.
[0103] There are several steps commonly taken in the design of a
mimetic from a compound having a given target property. Firstly,
the particular parts of the compound that are critical and/or
important in determining the target property are determined. In the
case of a peptide, this can be done by systematically varying the
amino acid residues in the peptide, e.g. by substituting each
residue in turn. These parts or residues constituting the active
region of the compound are known as its "pharmacophore".
[0104] Once the pharmacophore has been found, its structure is
modelled according to its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data from a
range of sources, eg spectroscopic techniques, X-ray diffraction
data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the
bonding between atoms) and other techniques can be used in this
modelling process.
[0105] In a variant of this approach, the three-dimensional
structure of the ligand and its binding partner are modelled. This
can be especially useful where the ligand and/or binding partner
change conformation on binding, allowing the model to take account
of this in the design of the mimetic.
[0106] A template molecule is then selected onto which chemical
groups which mimic the pharmacophore can be grafted. The template
molecule and the chemical groups grafted on to it can conveniently
be selected so that the mimetic is easy to synthesise, is likely to
be pharmacologically acceptable, and does not degrade in vivo,
while retaining the biological activity of the lead compound. The
mimetic or mimetics found by this approach can then be screened to
see whether they have the target property, or to what extent they
exhibit it. Further optimisation or modification can then be
carried out to arrive at one or more final mimetics for in vivo or
clinical testing.
Inhibitors
[0107] The term "inhibitor" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes the expression or kinase activity of a kinase.
Preferably, a kinase inhibitor is a specific or near-specific
inhibitor which inhibits the expression or activity a desired
kinase without affecting other kinases.
[0108] Kinase inhibitors include antibodies, dominant negative
forms and small molecule inhibitors.
[0109] Small molecule inhibitors of Abl activity include
phenylaminopyrimidines such as imatinib or imatinib mesylate
(Glivec/Gleevec.TM.,
4-[)4-methyl-1-piperazinyl)methyl]-N-(4-methyl-3-[4-(3-pyridinyl)2-pyrimi-
dinyl[amino]-phenyl]benzamide methanesulfonate; Novartis);
BMS-354825
[n-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-me-
thylpyrimidin-4-ylamino)thiazole-5-carbozamide]; PD 173955 (Parke
Davis); pyridopyrimidines such as PD166326 (Parke Davis); ON 012380
(Onconova).
[0110] Small molecule inhibitors of Syk activity include picetannol
(3,4,3',5'-tetrahydroxy-trans-stilbene); 574711
(3-(1-Methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfo-
namide, Calbiochem); ER-27319; and BAY61-3606.
[0111] Small molecule inhibitors of Fyn activity include PP1
(4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine).
[0112] Inhibitors of kinase expression include antisense RNA or
siRNA as described below, triple-helix nucleic acids or
ribozymes.
[0113] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra; Lee et
al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:
456 (1988); Dervan et al., Science, 251:1360 (1991).
[0114] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4:469-471
(1994), and PCT publication No. WO 97/33551 (published Sep. 18,
1997).
Antisense
[0115] Expression of nucleic acid sequences that are complementary
in sequence to a coding sequence of a gene (`antisense` nucleic
acids) can inhibit production of the protein product from the gene.
It is not known exactly how this occurs, but it is thought that the
antisense nucleic acid sequences hybridise to cellular mRNA,
forming a double stranded molecule. The cell does not translate the
mRNA in this double-stranded form, so translation is inhibited.
Antisense nucleic acids may have other effects, including
inhibition of transcription and splicing inhibition.
[0116] The term `antisense` nucleic acid indicates a nucleic acid
sequence which is sufficiently complementary to the RNA molecule
for which the antisense nucleic acid is specific to cause molecular
hybridisation between the antisense nucleic acid and the mRNA such
that translation of the mRNA is inhibited. Such hybridisation must
occur under in vivo conditions, that is, inside the cell.
[0117] Oligomers of about fifteen nucleotides or greater and
molecules that hybridise to the AUG initiation codon are
particularly efficient, since they are easy to synthesize and are
likely to pose fewer problems than larger molecules when
introducing them into cells.
RNA Interference
[0118] RNA interference (RNAi) is a process of sequence-specific,
post-transcriptional gene silencing in animals and plants,
initiated by double-stranded RNA (dsRNA) that is homologous in
sequence to the silenced gene. RNAi is mediated by short
double-stranded RNA molecules (small interfering RNAs or siRNAs).
siRNAs may be introduced into a cell as short RNA oligonucleotides
of 10-15 bp, or as longer dsRNAs which are subsequently cleaved to
produce siRNAs. The RNA may be introduced into the cell as RNA, or
may be transcribed from a DNA or RNA vector.
[0119] Methods relating to the use of RNAi to silence genes in C.
elegans, Drosophila, plants, and mammals are known in the art (Fire
A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15,
358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15,
485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2,
110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001);
Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M.,
et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell
101, 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366
(2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001);
WO0129058; WO9932619, and Elbashir S M, et al., 2001 Nature
411:494-498).
[0120] In some embodiments, the siRNA has an overhang at one or
both ends of one or more deoxythymidine bases. The overhang is not
to be interpreted as part of the siRNA sequence. Where present, it
serves to increase the stability of the siRNA within cells by
reducing its susceptibility to degradation by nucleases.
[0121] siRNA molecules may be synthesized using standard solid or
solution phase synthesis techniques which are known in the art.
Linkages between nucleotides may be phosphodiester bonds or
alternatives, for example, linking groups of the formula P(O)S,
(thioate); P(S)S, (dithioate); P(O)NR'2; P(O)R'; P(O)OR6; CO; or
CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl
(1-9C) is joined to adjacent nucleotides through --O-- or
--S--.
[0122] Alternatively, siRNA molecules or longer dsRNA molecules may
be made recombinantly by transcription of a nucleic acid sequence,
preferably contained within a vector as described below.
[0123] Another alternative is the expression of a short hairpin RNA
molecule (shRNA) in the cell. shRNAs are more stable than synthetic
siRNAs. A shRNA consists of short inverted repeats separated by a
small loop sequence. One inverted repeat is complimentary to the
gene target. The shRNA is then processed into an siRNA which
degrades the target gene mRNA and suppresses expression. shRNAs can
produced within a cell by transfecting the cell with a DNA
construct encoding the shRNA sequence under control of a RNA
polymerase III promoter, such as the human H1 or 7SK promoter.
Alternatively, the shRNA may be synthesised exogenously and
introduced directly into the cell. Preferably, the shRNA sequence
is between 40 and 100 bases in length, more preferably between 40
and 70 bases in length. The stem of the hairpin is preferably
between 19 and 30 base pairs in length. The stem may contain G-U
pairings to stabilise the hairpin structure.
[0124] Modified nucleotide bases can be used in addition to the
naturally occurring bases, and may confer advantageous properties
on siRNA molecules containing them.
[0125] For example, modified bases may increase the stability of
the siRNA molecule, thereby reducing the amount required for
silencing. The provision of modified bases may also provide siRNA
molecules which are more, or less, stable than unmodified
siRNA.
[0126] The term `modified nucleotide base` encompasses nucleotides
with a covalently modified base and/or sugar. For example, modified
nucleotides include nucleotides having sugars which are covalently
attached to low molecular weight organic groups other than a
hydroxyl group at the 3' position and other than a phosphate group
at the 5' position. Thus modified nucleotides may also include 2'
substituted sugars such as 2'-O-methyl-; 2-O-alkyl; 2-O-allyl;
2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-halo or 2; azido-ribose,
carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such
as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,
and sedoheptulose.
[0127] Modified nucleotides are known in the art and include
alkylated purines and pyrimidines, acylated purines and
pyrimidines, and other heterocycles. These classes of pyrimidines
and purines are known in the art and include pseudoisocytosine,
N4,N4-ethanocytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl
uracil, dihydrouracil, inosine, N6-isopentyl-adenine,
1-methyladenine, 1-methylpseudouracil, 1-methylguanine,
2,2-dimethylguanine, 2methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-methyladenine,
7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy amino
methyl-2-thiouracil, -D-mannosylqueosine,
5-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl
ester, psuedouracil, 2-thiocytosine, 5-methyl-2 thiouracil,
2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic
acid methylester, uracil 5-oxyacetic acid, queosine,
2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil,
5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine,
and 2,6,diaminopurine, methylpsuedouracil, 1-methylguanine,
1-methylcytosine.
Pharmaceutical Compositions
[0128] Following identification of a substance which modulates or
affects phosphorylation or dephosphorylation of tau protein, the
substance may be investigated further. Furthermore, it may be
manufactured and/or used in preparation, i.e. manufacture or
formulation, of a composition such as a medicament, pharmaceutical
composition or drug. These may be administered to individuals.
[0129] Thus, the present invention extends in various aspects not
only to a substance identified using the screening assays and assay
methods disclosed herein, but also a pharmaceutical composition,
medicament, drug or other composition comprising such a substance,
a method comprising administration of such a composition to a
patient, e.g. to treat tauopathies, use of such a substance in
manufacture of a composition for administration for the treatment
of tauopathies, and a method of making a pharmaceutical composition
comprising admixing such a substance with a pharmaceutically
acceptable excipient, vehicle or carrier, and optionally other
ingredients.
[0130] The substances identified as kinase inhibitors or
phosphatase promoters in the assays and assay methods of the
present invention, or compounds or substances arising from further
development or optimisation, may be formulated in pharmaceutical
compositions. These compositions may be employed for the treatment
of tauopathies, that is conditions which are characterised by
neurofibrillary tangles or aggregates of tau protein. Tauopathies
are a recognised class of conditions known to those skilled in the
art and include Alzheimer's disease (AD), frontotemproal dementia
with Parkinsonism linked to chromosome 17 (FTDP-17), progressive
supranuclear palsy (PSP), Pick's disease, corticobasal degeneration
and multiple system atrophy (MSA). The intracellular tau deposits
are usually neuronal or glial and are filamentous and generally in
a hyperphosphorylated state as compared to the level of
phosphorylation in tau from control human brain. In the case of AD,
this hyperphosphorylated tau is often referred to as paired helical
filament tau (PHF) tau because it is derived from the PHF.
[0131] These compositions may comprise, in addition to one of the
above substances, a pharmaceutically acceptable excipient, carrier,
buffer, stabiliser or other materials well known to those skilled
in the art. Such materials should be non-toxic and should not
interfere with the efficacy of the active ingredient. The precise
nature of the carrier or other material may depend on the route of
administration, e.g. oral, intravenous, cutaneous or subcutaneous,
nasal, intramuscular, intraperitoneal routes.
[0132] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may include a
solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally include a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0133] For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient will be
in the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability. Those
of relevant skill in the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as Sodium
Chloride Injection, Ringer's Injection, Lactated Ringer's
Injection. Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0134] Whether it is a polypeptide, antibody, peptide, nucleic acid
molecule, small molecule or other pharmaceutically useful compound
according to the present invention that is to be given to an
individual, administration is preferably in a "prophylactically
effective amount" or a "therapeutically effective amount" (as the
case may be, although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual. The actual
amount administered, and rate and time-course of administration,
will depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage etc, is within
the responsibility of general practitioners and other medical
doctors, and typically takes account of the disorder to be treated,
the condition of the individual patient, the site of delivery, the
method of administration and other factors known to practitioners.
Examples of the techniques and protocols mentioned above can be
found in Remington's Pharmaceutical Sciences, 20th Edition, 2000,
pub. Lippincott, Williams & Wilkins. A composition may be
administered alone or in combination with other treatments, either
simultaneously or sequentially, dependent upon the condition to be
treated.
Experimental
[0135] Purification of PHF-Tau from Alzheimer Brain
[0136] Paired helical filament (PHF) tau was purified from
Alzheimer brain as described in Hanger et al, 1998. Briefly, brain
tissue was homogenised and insoluble PHF-tau was recovered by
differential centrifugation. Following solubilisation in guanidine
and dialysis against a re-naturing buffer, PHF-tau was purified by
anion-exchange and reversed-phase chromatography.
Preparation of Mutant Forms of Human Tau
[0137] To generate the five tau constructs each with a single
tyrosine replaced by phenylalanine, a QuikChange.RTM. XL
site-directed mutagenesis kit (Stratagene, Amsterdam, The
Netherlands) was used. Primers (custom-synthesised by
Sigma-Genosis) were as follows: to convert Tyr-18 to Phe (giving
tau construct Y18F), forward primer 5'-CAC GCT GGG ACG TTC GGG TTG
GGG GAC-3' (Primer A; SEQ ID NO: 5), and reverse primer 5'-GTC CCC
CAA CCC GAA CGT CCC AGC GTG-3' (SEQ ID NO: 6); to convert Tyr-29 to
Phe (giving Y29F), forward primer 5'-GAT CAG GGG GGC TTC ACC ATV
CAC CAA G-3' (Primer B; SEQ ID NO: 7), and reverse primer 5'-C TTG
GTG CAT GGT GAA GCC CCC CTG ATC-3' (SEQ ID NO: 8); to convert
Tyr-197 to Phe (giving Y197F), 5'-GAT CGC AGC GGC TTC AGC AGC CCC
GG-3' (Primer C; SEQ ID NO: 9), and reverse primer 5'-CC GGG GCT
GCT GAA GCC GCT GCG ATC-3' (SEQ ID NO: 10); to convert Tyr-310 to
Phe (giving Y310F), forward primer 5'-GGC AGT GTG CAA ATA GTC TTC
AAA CCA GTT GAC CTG AG-3' (Primer D; SEQ ID NO: 11), and reverse
primer 5'-CT CAG GTC AAC TGG TTT GAA GAC TAT TTG CAC ACT GCC-3'
(SEQ ID NO: 12); and to convert Tyr-394 to Phe (giving Y394F),
forward primer 5'-GCG GAG ATC GTG TTC AAG TCG CCA GTG G-3' (Primer
E; SEQ ID NO: 13), and reverse primer 5'-C CAC TGG CGA CTT GAA CAC
GAT CTC CGC-3' (SEQ ID NO: 14). The sequence of the full insert was
determined for each construct (Lark Technologies).
[0138] To change all five tyrosines to phenylalanines a
QuikChange.RTM. multi site-directed mutagenesis kit (Stratagene)
was used, with the five primers A to E (above). Colonies were
sequenced, and as well as identifying constructs where all five
tyrosines had been replaced by phenylalanine (TauYallF), constructs
with a single tyrosine remaining were found that contained four
phenylalanines and just Tyr-18, Tyr-29 or Tyr-197. Mutants
containing just Tyr-310 or just Tyr-394 were generated from the
all-Phe construct by single site-directed mutagenesis as above
using the following primers: for Tyr-310 only, forward primer
5'-GGC AGT GTG CAA ATA GTC TAC AAA CCA GTT GAC CTG AG-3' (SEQ ID
NO: 15), and reverse primer 5'-CT CAG GTC AAC TGG TTT GTA GAC TAT
TTG CAC ACT GCC-3' (SEQ ID NO: 16); and for Tyr-394 only, forward
primer 5'-GCG GAG ATC GTG TAC AAG TCG CCA GTG G-3' (SEQ ID NO: 17),
and reverse primer 5'-C CAC TGG CGA CTT GTA CAC GAT CTC CGC-3' (SEQ
ID NO: 18). The five constructs with one remaining tyrosine were
termed Y18 only, Y29 only, etc, and their tau coding sequences
verified (Lark Technologies).
Preparation and Purification of Recombinant Human Tau
[0139] A plasmid expressing the largest tau isoform (2N4R) was used
to prepare and purify recombinant human tau as described previously
(Mulot et al., 1994). Briefly, a bacterial cell lysate expressing
2N4R tau was heated and centrifuged to remove heat-labile proteins.
The supernatant was fractionated with ammonium sulphate and
precipitated material was solubilised and dialysed into buffer
prior to cation-exchange chromatography. Proteins were eluted with
NaCl and fractions containing tau were pooled and dialysed against
Mes buffer pH 6.25, 5 mM DTT, and stored frozen.
In Vitro Phosphorylation of Recombinant Tau by Rat Brain Lysate
[0140] A rat brain extract containing active protein kinases was
prepared by homogenising a rat brain in ice-cold buffer (2 ml
buffer per g brain) containing 25 mM Tris-HCl pH7.5, 5 mM EGTA, 2
mM dithiothreitol (DTT), 2 .mu.M okadaic acid, 1 mM sodium
orthovanadate and protease inhibitors. The homogenate was
centrifuged at 100,000 g for 1 hr, and incubated on ice for 30 min
with 2 mM ATP and 10 .mu.M okadaic acid. This extract (rat brain
supernatant, RBS) contained 7 mg/ml protein (Bradford).
[0141] Recombinant 2N4R tau protein (100 .mu.g/ml) was
phosphorylated by incubating with RBS (1.8 mg/ml protein) in 50 mM
Tris-HCl buffer pH 7.5 with 5 mM MgCl.sub.2, 3 mM ATP, 5 mM EGTA, 1
mM sodium orthovanadate, 10 .mu.M okadaic acid, 1 mM DTT and
protease inhibitors at 37.degree. C. for 24 hr. The reaction
mixture was then heated at 100.degree. C. for 5 min, incubated on
ice for 10 min, and centrifuged for 10 min at 16000 g. The
supernatant containing the tau protein was aspirated off and
analysed by Western blotting and mass spectrometry.
In Vitro Phosphorylation of Recombinant Tau by Tyrosine Protein
Kinases
[0142] Recombinant human tau (1 .mu.g) was incubated with 50 ng of
either Abl or Syk (Upstate) in 30 .mu.l of kinase buffer (HEPES 50
mM pH 7.4, 10 mM MnCl.sub.2 in the presence of 1 mM ATP) for 30
minutes at 30.degree. C. 30 .mu.l of SDS-PAGE sample buffer was
added to stop the reaction.
In Vitro Phosphorylation of Recombinant Tau by Lck and Subsequent
Binding of SH2 Domain of Fyn
[0143] Recombinant 2N4R tau protein (440 .mu.g/ml) was incubated at
30.degree. with purified recombinant Lck (20-100 .mu.g/ml), 40 mM
.beta.-glycerophosphate buffer pH 7.5, 3 mM ATP, 25 mM MgCl.sub.2,
5 mM MnCl.sub.2, 1 mM DTT, 100 .mu.M EDTA, 1 mM sodium
orthovanadate and protease inhibitors. After 6 hr the tubes were
heated at 100.degree. for 5 min, cooled for 10 min on ice, and
centrifuged for 10 min at 16000 g. The supernatants were checked
for tyrosine phosphorylation by Western blotting, using 4G10
anti-phosphotyrosine antibody (Upstate, Inc) and anti-tau antibody
(Dako).
[0144] Interaction of tau with the SH2 domain of Fyn was
investigated by incubating tyrosine-phosphorylated or
non-phosphorylated tau (5 .mu.g/ml) with glutathione-Sepharose.RTM.
beads containing 2-5 .mu.g of GST-Fyn-SH2 fusion protein or GST as
control. After mixing for 60 min at 4.degree. the beads were washed
.times.3 and analysed by Western blotting for tau and for
phosphotyrosine as above.
In-Gel Proteolytic Digestion of Tau
[0145] PHF-tau or in vitro phosphorylated tau proteins were
separated on 10% (wt/vol) polyacrylamide gels and stained with
colloidal Coomassie Blue G. Protein bands corresponding to tau were
excised, carbamidomethylated, and digested with proteolytic enzymes
(trypsin or Asp-N). Peptides were extracted from gel pieces by a
series of acetonitrile and aqueous washes, dried and resuspended in
50 mM ammonium bicarbonate.
Amyloid Beta Treatment of Neurons and Isolation of Lipid Rafts
[0146] Rat and human primary cortical neuronal cultures were
treated with fibrillar A.beta..sub.25-35 or A.beta..sub.1-42 for
1-30 min. Lipid rafts were prepared from control untreated and
A.beta.-treated neuronal cultures by scraping the cells from one 80
cm.sup.2 flask into 2 ml 1% Triton X-100 in 25 mM Mes, pH 6.5
containing protease inhibitors. Cells were disrupted by Dounce
homgenization. The homogenate was mixed with 2 ml of 90% sucrose
(w/v) in 25 mM Mes, 150 mM NaCl pH 6.5 and placed in a 12 ml
centrifuge tube. A 5-35% step sucrose gradient was formed by
overlaying the homogenate mix with 4 ml of a 35% (w/v) sucrose
solution followed by 4 ml of a 5% (w/v) sucrose solution. This was
then centrifuged at 39,000 rpm for 18 hr in a Beckman SW41 rotor. 1
ml fractions were collected from the top of each gradient. The
lipid raft fraction partitioned at the interface between the 5%
(w/v) sucrose layer and the 35% sucrose (w/v) layer, fractions 4
and 5. Lipid raft fractions were concentrated by mixing the raft
fractions (4 and 5) with 10 ml dd H.sub.2O and centrifuging at
39,000 rpm for 2 h in a Beckman SW41 rotor. The supernatant was
aspirated and the remaining pellet was resuspended in 100 .mu.l of
2.times. sample buffer. Western blots of lipid raft proteins were
probed for antibodies to flotillin and protein loading corrected by
scanning densitometry. Tau was detected in lipid rafts by probing
western blots of lipid raft proteins using a polyclonal anti-tau
antibody (DAKO).
Phosphorylation of Tyrosine Residues in Tau in Cultured Cells
Treated with Pervanadate
[0147] In a first set of experiments, COS-7 cells were transiently
transfected with V5 tagged human tau longest isoform or with V5
tagged mutants of tau where one tyrosine has been replaced by one
phenylalanine (named Y18F, Y29F, Y197F, Y310F and Y394F)
constructs. In a second set of experiments, COS-7 cells were
transiently transfected with the V5 tagged tau (441) construct or
with V5 tagged mutants of tau where only one tyrosine is remaining,
the four other tyrosines being replaced by phenyalanine (named
Y18-only, Y29-only, Y197-only, Y310-only and Y394-only according to
the remaining tyrosine). In order to increase tau tyrosine
phosphorylation, cells were treated with the tyrosine phosphatase
inhibitor pervanadate for 20 minutes. Cells were harvested in NETF
buffer (100 mM NaCl, 2 mM EGTA, 50 mM Tris-Cl pH 7.4 and 50 mM NaF)
containing 1% NP-40, 2 mM orthovanadate and protease inhibitors.
Samples were precleared with 40 .mu.l of protein G-Sepharose beads,
and immunoprecipitations were carried out with monoclonal anti-V5
antibodies preadsorbed on protein G-Sepharose beads. Cells were
harvested in NETF lysis buffer containing 1% NP-40 and tau was
immunoprecipitated using an anti-V5 antibody. Resulting
immunoprecipitates were separated in duplicate by SDS-PAGE and
transferred co nitrocellulose. Immunoblots were performed on
duplicate membranes using 4G10 phosphotyrosine antibody or TP70
antibody (total tau antibody). Bound antibodies were visualized by
enhanced chemiluminescence detection. Quantification was achieved
by scanning the autoradiograms with GS710 Calibrated Imaging
Densitometer (Bio-Rad) and measurement of relative optical density
with Quantity One 4.0.3 software (Bio-Rad).
Phosphorylation of Tyrosine Residues in Tau in Cultured Cells by
Co-Expression of Tyrosine Kinases, Fyn, Src, Abl, Syk
[0148] Fyn cDNA was a gift from D. Markby (Sugen, San Francisco),
Src cDNA was from upstate (Src cDNA allelic pack), Abl and
Abl.DELTA.XB cDNA (a constitutively active form of Abl, with
deletion of most of the SH3 domain) were from Richard A. Van Etten
(Molecular Oncology Research Institute, Boston), Syk cDNA was a
gift from H. Band (Brigham and Women's Hospital, Boston). CHO cells
were used for the co-transfection experiments. CHO cells were
transiently transfected with the V5 tagged human tau longest
isoform and with V5 tagged mutants of tau where one tyrosine has
been replaced by one phenylalalanine (named Y18F, Y29F, Y197F,
Y310F and Y394F) constructs. In every experiment, cells were
co-transfected with the empty vector or with the protein tyrosine
kinase expression vector (Fyn, Src, Abl or Abl.DELTA.XB).
Harvesting of cells, immunoprecipitation and Western analysis were
performed as described in the section "Phosphorylation of tyrosine
residues in tau in cultured cells treated with pervanadate".
Results
New Sites of Tyrosine Phosphorylation Found in PHF-Tau
[0149] Current literature reports 25 known phosphorylation sites
(all are serine or threonine) identified by direct means in PHF-tau
(Hanger et al, 1998)(Morishima-Kawashima et al., 1995). There are a
further 2-3 sites that have been identified by antibody reactivity
only. On the basis of antibody labelling, it has been reported that
tyrosine 18 is phosphorylated in a proportion of PHF-tau in AD
brain. We have found an additional 12 phosphorylation sites in
PHF-tau, one of which is a tyrosine residue (tyr394), bringing the
total number of sites to 37. We have also found that tyr394 is
phosphorylated in tau isolated from human foetal brain.
New Sites of Tyrosine Phosphorylation on Recombinant Tau Generated
by Rat Brain Lysate
[0150] Mass spectrometry of proteolytic digests of tau that had
been phosphorylated with rat brain supernatant demonstrated
phosphorylation on tyrosines 310 and 394, in addition to many
serines and threonines.
[0151] A.beta. treatment of neurons and lipid raft composition
A.beta..sub.25-35 and A.beta..sub.1-42 treatment of primary rat
neuronal cultures resulted in a rapid increase in the tyrosine
phosphorylation of neuronal protein components of lipid rafts. No
increase in the phosphoserine or phosphothreonine content of lipid
raft proteins was observed after AP-treatment. The increase in
tyrosine phosphorylation was concomitant with an increased
partitioning of Fyn, tau, and tubulin into lipid rafts.
[0152] Focal adhesion kinase (FAK) levels transiently increased in
lipid rafts in response to AP while levels of the classic lipid
raft protein flotillin remained unchanged. Inhibition of tyrosine
phosphorylation with the tyrosine kinase inhibitor PP2 abrogated
the AP-induced increase in tyrosine phosphorylation of lipid raft
proteins and partitioning of tau into lipid rafts.
Phosphorylation of Tyrosine Residues in Tau in Cultured Cells
Treated with Pervanadate
[0153] The first set of five mutants where one tyrosine residue was
exchanged with phenylalanine (Y18F, Y29F, Y197F, Y310F and Y394F)
were transfected into COS-7 cells and cells were treated for 20
minutes with pervanadate. Western analysis performed on
immunoprecipitated tau, using 4G10 antiphosphotyrosine antibody
shows that the Y394F mutant construct is the only single tyrosine
mutation that results in a significant effect, reducing tyrosine
phosphorylation to approximately 10% of the wild-type control.
Tyrosine phosphorylation of the Y18F, Y29F, Y310F constructs were
not significantly different from the wild-type control. Concerning
the Y197F mutant construct, it should be pointed out that a
decrease in tyrosine phosphorylation was observed in two of the
five experiments that were done with this construct. To confirm
these results, we transfected into COS-7 cells the second set of
mutants in which only one tyrosine residue remains as the sole
tyrosine with the other four replaced by phenylalanine (Y18-only,
Y29-only, Y197-only, Y310-only and Y394-only). Analysis using
phosphotyrosine antibodies showed that no tyrosine phosphorylation
could be elicited by pervanadate in Y18-only, Y29-only, Y310-only
mutant constructs, whereas pervanadate induces an increase in
tyrosine phosphorylation of the Y394-only similar to the one
observed in wild-type tau. In two of the four experiments made with
the Y197-only mutant construct, a faint but clear-cut
phosphotyrosine immunoreactivity was detectable. Taken together,
these results suggest that the majority of tyrosine phosphorylation
of tau in pervanadate-treated COS-7 cells occurs on tyrosine
394.
Phosphorylation of Tyrosine Residues in Tau in Cultured Cells Over
Expressing Fyn
[0154] Wild type tau and the first set of five mutants where one
tyrosine residue was exchanged with phenylalanine (Y18F, Y29F,
Y197F, Y310F and Y394F) were co-transfected with the empty vector
or with a Fyn-expression vector into CHO cells. Western analysis
performed on immunoprecipitated tau, using 4G10 phosphotyrosine
antibody, shows that the Y18F and Y310F mutant constructs are the
two single tyrosine mutations that results in a significant effect,
each mutation reducing tyrosine phosphorylation to approximately
50% of the wild-type control. Taken together, the results suggest
that tyrosine 18 and 310 are the main sites phosphorylated by
Fyn.
Phosphorylation of Tyrosine Residues in Tau in Cultured Cells
Over-Expressing Abl
[0155] Wild type tau and the first set of five mutants where one
tyrosine residue was exchanged with phenylalanine (Y18F, Y29F,
Y197F, Y310F and Y394F) were co-transfected with the empty vector
or with Ab.DELTA.XB expression vector into CHO cells. Western
analysis performed on immunoprecipitated tau, using 4G10
phosphotyrosine antibodies, shows that the Y394F is the tyrosine
mutation with the strongest effect reducing tyrosine
phosphorylation to approximately 25% of the wild-type control.
Y197F and Y310F mutant constructs also have a significant effect
reducing tyrosine phosphorylation to approximately 70% of the
wild-type control each. In contrast, Y18F and Y29F mutant
constructs were not different from the wild type control. Taken
together, the results suggest that Abl primarily phosphorylates tau
on tyrosine 394 and that tyrosines 197 and 310 are also
phosphorylated by this kinase. In contrast, Abl does not
phosphorylate tyrosines 18 and 29.
Phosphorylation of Tyrosine Residues in Tau in Cultured Neurons
Over-Expressing Syk
[0156] Wild type tau and the first set of five mutants where one
tyrosine residue was exchanged with phenylalanine (Y18F, Y29F,
Y197F, Y310F and Y394F) were co-transfected with the empty vector
or with a Syk expression vector into CHO cells.
[0157] Western analysis performed on immunoprecipitated tau, using
4G10 phosphotyrosine antibodies, shows that single mutants of tau
(i.e. with one tyrosine mutated to phenylalanine, the other four
tyrosines still being present) showed no significant decreases in
tau tyrosine phosphorylation. Mutants with only Y18, Y29, Y197 or
Y394 could each be phosphorylated to 20-25% of the level found with
wild-type, indicating that Syk can phosphorylate tau at each of
these sites.
SH2 Domain of Fyn Binding to Tyrosine-Phosphorylated Tau
[0158] Co-sedimentation experiments using GST-SH2 proteins bound to
glutathione beads demonstrated that tyrosine phosphorylated tau,
but not control non-phosphorylated tau, could bind to the SH2
domain of Fyn (isoform B).
Use of STI 571 to Determine Whether Phosphorylation of Tau in Cells
is Catalysed by an Abl-Like Kinase
[0159] The chemical compound STI 571, also known as Imatinib
mesylate, Gleevec.RTM., Glivec, formerly CGP 57148B, chemical name
4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyri-
midinyl]amino]-phenyl]benzamide methanesulphonate, is a known
inhibitor of tyrosine protein kinases and an effective
antileukaemic agent. It is selective for Abl but also inhibits a
small number of other tyrosine protein kinases including the
platelet-derived growth factor receptor and c-Kit.
[0160] An experiment to confirm whether an Abl-like kinase
phosphorylates tau in cells could be carried out as follows. COS-7,
CHO or SH-SY5Y cells are transfected with a suitable tau construct,
e.g. a plasmid containing Tau2N4R-V5-His (wild-type) and after 48
hours treated with STI-571 followed 1 hour later with 100
micromolar pervanadate or control.
[0161] After a further one hour cells are harvested and cell
lysates are immunoprecipitated with anti-V5 antibody and analysed
by Western blotting with the antiphosphotyrosine antibody 4G10. It
would be expected that, as already shown with the compound PP2, STI
571 will inhibit the tyrosine-phosphorylation of tau.
Hypothesis
[0162] Our hypothesis is that AR is neurotoxic to neurons by a
mechanism that obligatorily requires the involvement of tau and
certain protein tyrosine kinases; likely candidate tyrosine kinases
include Fyn and Abl but others may be required. We envisage a
mechanism in which exposure of neurons to A.beta. induces
activation of one or more of these tyrosine kinases, which then
phosphorylate tau and this generates binding sites for other cell
signaling proteins, including for example an SH2 binding site for
Fyn. Tyrosine phosphorylated tau then binds to lipid raft
components of cell membranes in amounts that are pathological and
this triggers unknown but detrimental cell signalling processes
that result in neurodegeneration and cell death.
REFERENCES
[0163] The references cited herein are all expressly incorporated
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disorganization leads to reduced plasmin activity in Alzheimer's
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interacts with src-family non-receptor tyrosine kinases. J. Cell
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Litersky, J. M., Bhaskar, K., Fang, S. M., Do, L. H., Andreadis,
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P., Zhou, Y. X., Deng, C., and Li, X. J. (2003). Lack of
huntingtin-associated protein-1 causes neuronal death resembling
hypothalamic degeneration in Huntington's disease. J. Neurosci. 23,
6956-6964. [0173] Liu, T., Perry, G., Chan, H. W., Verdile, G.,
Martins, R. N., Smith, M. A., and Atwood, C. S. (2004).
Amyloid-beta-induced toxicity of primary neurons is dependent upon
differentiation-associated increases in tau and cyclin-dependent
kinase 5 expression. J. Neurochem. 88, 554-563. McDonald, D. R.,
Brunden, K. R., and Landreth, G. E. (1997). Amyloid fibrils
activate tyrosine kinase-dependent signaling and superoxide
production in microglia. J. Neurosci. 17, 2284-2294. [0174]
Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M.,
Yoshida, H., Titani, K., and Ihara, Y. (1995). Proline-directed and
non-proline-directed phosphorylation of PHF-tau. J. Biol. Chem.
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Anderton, B. H., and Hanger, D. P. (1994). PHF-tau from Alzheimer's
brain comprises four species on SDS-PAGE which can be mimicked by
in vitro phosphorylation of human brain tau by glycogen synthase
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M., Donella-Deana, A., Massimino, M. L., and Pinna, L. A. (2002).
Multiple phosphorylation of alpha-synuclein by protein tyrosine
kinase Syk prevents eosin-induced aggregation. FASEB J. 16,
210-212. [0177] Rapoport, M., Dawson, H. N., Binder, L. I., Vitek,
M. P., and Ferreira, A. (2002). Tau is essential to
beta-amyloid-induced neurotoxicity. Proc. Natl. Acad. Sci. U. S. A
99, 6364-6369. [0178] Scales, T. M. E., Williamson, R., Anderton,
B. H., and Reynolds, C. H. Tyrosine phosphorylation of specific
sites on tau by Src family kinases. Neurobiology of Aging 23,
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Wood, J. G. (1993). The protein tyrosine kinase, fyn, in
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I., and Small, D. H. (2003). Cholesterol is necessary both for the
toxic effect of Abeta peptides on vascular smooth muscle cells and
for Abeta binding to vascular smooth muscle cell membranes. J.
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T. A. (2001). Reduction in cholesterol and sialic acid content
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Clark, B. R., Gibb, G., Reynolds, C. H., Kellie, S., Bird, I. N.,
Varndell, I. M., Sheppard, P. W., Everall, I., and Anderton, B. H.
(2002). Rapid tyrosine phosphorylation of neuronal proteins
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Patrick, G. N., Nikolic, M., Humbert, S., Wu, C. L., Lanier, L. M.,
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(2000). Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine
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26, 633-646.
Sequence CWU 1
1
181441PRTHomo sapiens 1Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met
Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp
Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr
Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu
Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys
Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp
Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90
95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro
100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg
Met Val 115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys
Lys Ala Lys Gly 130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro
Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn
Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175 Pro Ala Pro Lys Thr
Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190 Asp Arg Ser
Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205 Arg
Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215
220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys
225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu
Lys Asn Val 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys
His Gln Pro Gly Gly 260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys
Leu Asp Leu Ser Asn Val Gln 275 280 285 Ser Lys Cys Gly Ser Lys Asp
Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300 Ser Val Gln Ile Val
Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys
Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335
Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340
345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly
Asn 355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn
Ala Lys Ala 370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys
Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu
Ser Asn Val Ser Ser Thr Gly Ser 405 410 415 Ile Asp Met Val Asp Ser
Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430 Ser Ala Ser Leu
Ala Lys Gln Gly Leu 435 440 2 1130PRTHomo sapiens 2Met Leu Glu Ile
Cys Leu Lys Leu Val Gly Cys Lys Ser Lys Lys Gly 1 5 10 15 Leu Ser
Ser Ser Ser Ser Cys Tyr Leu Glu Glu Ala Leu Gln Arg Pro 20 25 30
Val Ala Ser Asp Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala Arg Trp 35
40 45 Asn Ser Lys Glu Asn Leu Leu Ala Gly Pro Ser Glu Asn Asp Pro
Asn 50 55 60 Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser Gly Asp
Asn Thr Leu 65 70 75 80 Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu
Gly Tyr Asn His Asn 85 90 95 Gly Glu Trp Cys Glu Ala Gln Thr Lys
Asn Gly Gln Gly Trp Val Pro 100 105 110 Ser Asn Tyr Ile Thr Pro Val
Asn Ser Leu Glu Lys His Ser Trp Tyr 115 120 125 His Gly Pro Val Ser
Arg Asn Ala Ala Glu Tyr Leu Leu Ser Ser Gly 130 135 140 Ile Asn Gly
Ser Phe Leu Val Arg Glu Ser Glu Ser Ser Pro Gly Gln 145 150 155 160
Arg Ser Ile Ser Leu Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg Ile 165
170 175 Asn Thr Ala Ser Asp Gly Lys Leu Tyr Val Ser Ser Glu Ser Arg
Phe 180 185 190 Asn Thr Leu Ala Glu Leu Val His His His Ser Thr Val
Ala Asp Gly 195 200 205 Leu Ile Thr Thr Leu His Tyr Pro Ala Pro Lys
Arg Asn Lys Pro Thr 210 215 220 Val Tyr Gly Val Ser Pro Asn Tyr Asp
Lys Trp Glu Met Glu Arg Thr 225 230 235 240 Asp Ile Thr Met Lys His
Lys Leu Gly Gly Gly Gln Tyr Gly Glu Val 245 250 255 Tyr Glu Gly Val
Trp Lys Lys Tyr Ser Leu Thr Val Ala Val Lys Thr 260 265 270 Leu Lys
Glu Asp Thr Met Glu Val Glu Glu Phe Leu Lys Glu Ala Ala 275 280 285
Val Met Lys Glu Ile Lys His Pro Asn Leu Val Gln Leu Leu Gly Val 290
295 300 Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Phe Met Thr
Tyr 305 310 315 320 Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg
Gln Glu Val Asn 325 330 335 Ala Val Val Leu Leu Tyr Met Ala Thr Gln
Ile Ser Ser Ala Met Glu 340 345 350 Tyr Leu Glu Lys Lys Asn Phe Ile
His Arg Asp Leu Ala Ala Arg Asn 355 360 365 Cys Leu Val Gly Glu Asn
His Leu Val Lys Val Ala Asp Phe Gly Leu 370 375 380 Ser Arg Leu Met
Thr Gly Asp Thr Tyr Thr Ala His Ala Gly Ala Lys 385 390 395 400 Phe
Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe 405 410
415 Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val Leu Leu Trp Glu Ile
420 425 430 Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile Asp Leu Ser
Gln Val 435 440 445 Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg
Pro Glu Gly Cys 450 455 460 Pro Glu Lys Val Tyr Glu Leu Met Arg Ala
Cys Trp Gln Trp Asn Pro 465 470 475 480 Ser Asp Arg Pro Ser Phe Ala
Glu Ile His Gln Ala Phe Glu Thr Met 485 490 495 Phe Gln Glu Ser Ser
Ile Ser Asp Glu Val Glu Lys Glu Leu Gly Lys 500 505 510 Gln Gly Val
Arg Gly Ala Val Ser Thr Leu Leu Gln Ala Pro Glu Leu 515 520 525 Pro
Thr Lys Thr Arg Thr Ser Arg Arg Ala Ala Glu His Arg Asp Thr 530 535
540 Thr Asp Val Pro Glu Met Pro His Ser Lys Gly Gln Gly Glu Ser Asp
545 550 555 560 Pro Leu Asp His Glu Pro Ala Val Ser Pro Leu Leu Pro
Arg Lys Glu 565 570 575 Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu Asp
Glu Arg Leu Leu Pro 580 585 590 Lys Asp Lys Lys Thr Asn Leu Phe Ser
Ala Leu Ile Lys Lys Lys Lys 595 600 605 Lys Thr Ala Pro Thr Pro Pro
Lys Arg Ser Ser Ser Phe Arg Glu Met 610 615 620 Asp Gly Gln Pro Glu
Arg Arg Gly Ala Gly Glu Glu Glu Gly Arg Asp 625 630 635 640 Ile Ser
Asn Gly Ala Leu Ala Phe Thr Pro Leu Asp Thr Ala Asp Pro 645 650 655
Ala Lys Ser Pro Lys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly Ala 660
665 670 Leu Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser Pro His Leu Trp
Lys 675 680 685 Lys Ser Ser Thr Leu Thr Ser Ser Arg Leu Ala Thr Gly
Glu Glu Glu 690 695 700 Gly Gly Gly Ser Ser Ser Lys Arg Phe Leu Arg
Ser Cys Ser Ala Ser 705 710 715 720 Cys Val Pro His Gly Ala Lys Asp
Thr Glu Trp Arg Ser Val Thr Leu 725 730 735 Pro Arg Asp Leu Gln Ser
Thr Gly Arg Gln Phe Asp Ser Ser Thr Phe 740 745 750 Gly Gly His Lys
Ser Glu Lys Pro Ala Leu Pro Arg Lys Arg Ala Gly 755 760 765 Glu Asn
Arg Ser Asp Gln Val Thr Arg Gly Thr Val Thr Pro Pro Pro 770 775 780
Arg Leu Val Lys Lys Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp 785
790 795 800 Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro Asn Leu Thr
Pro Lys 805 810 815 Pro Leu Arg Arg Gln Val Thr Val Ala Pro Ala Ser
Gly Leu Pro His 820 825 830 Lys Glu Glu Ala Glu Lys Gly Ser Ala Leu
Gly Thr Pro Ala Ala Ala 835 840 845 Glu Pro Val Thr Pro Thr Ser Lys
Ala Gly Ser Gly Ala Pro Gly Gly 850 855 860 Thr Ser Lys Gly Pro Ala
Glu Glu Ser Arg Val Arg Arg His Lys His 865 870 875 880 Ser Ser Glu
Ser Pro Gly Arg Asp Lys Gly Lys Leu Ser Arg Leu Lys 885 890 895 Pro
Ala Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly 900 905
910 Lys Pro Ser Gln Ser Pro Ser Gln Glu Ala Ala Gly Glu Ala Val Leu
915 920 925 Gly Ala Lys Thr Lys Ala Thr Ser Leu Val Asp Ala Val Asn
Ser Asp 930 935 940 Ala Ala Lys Pro Ser Gln Pro Gly Glu Gly Leu Lys
Lys Pro Val Leu 945 950 955 960 Pro Ala Thr Pro Lys Pro Gln Ser Ala
Lys Pro Ser Gly Thr Pro Ile 965 970 975 Ser Pro Ala Pro Val Pro Ser
Thr Leu Pro Ser Ala Ser Ser Ala Leu 980 985 990 Ala Gly Asp Gln Pro
Ser Ser Thr Ala Phe Ile Pro Leu Ile Ser Thr 995 1000 1005 Arg Val
Ser Leu Arg Lys Thr Arg Gln Pro Pro Glu Arg Ile Ala 1010 1015 1020
Ser Gly Ala Ile Thr Lys Gly Val Val Leu Asp Ser Thr Glu Ala 1025
1030 1035 Leu Cys Leu Ala Ile Ser Arg Asn Ser Glu Gln Met Ala Ser
His 1040 1045 1050 Ser Ala Val Leu Glu Ala Gly Lys Asn Leu Tyr Thr
Phe Cys Val 1055 1060 1065 Ser Tyr Val Asp Ser Ile Gln Gln Met Arg
Asn Lys Phe Ala Phe 1070 1075 1080 Arg Glu Ala Ile Asn Lys Leu Glu
Asn Asn Leu Arg Glu Leu Gln 1085 1090 1095 Ile Cys Pro Ala Thr Ala
Gly Ser Gly Pro Ala Ala Thr Gln Asp 1100 1105 1110 Phe Ser Lys Leu
Leu Ser Ser Val Lys Glu Ile Ser Asp Ile Val 1115 1120 1125 Gln Arg
1130 3635PRTHomo sapiens 3Met Ala Ser Ser Gly Met Ala Asp Ser Ala
Asn His Leu Pro Phe Phe 1 5 10 15 Phe Gly Asn Ile Thr Arg Glu Glu
Ala Glu Asp Tyr Leu Val Gln Gly 20 25 30 Gly Met Ser Asp Gly Leu
Tyr Leu Leu Arg Gln Ser Arg Asn Tyr Leu 35 40 45 Gly Gly Phe Ala
Leu Ser Val Ala His Gly Arg Lys Ala His His Tyr 50 55 60 Thr Ile
Glu Arg Glu Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Arg 65 70 75 80
Thr His Ala Ser Pro Ala Asp Leu Cys His Tyr His Ser Gln Glu Ser 85
90 95 Asp Gly Leu Val Cys Leu Leu Lys Lys Pro Phe Asn Arg Pro Gln
Gly 100 105 110 Val Gln Pro Lys Thr Gly Pro Phe Glu Asp Leu Lys Glu
Asn Leu Ile 115 120 125 Arg Glu Tyr Val Lys Gln Thr Trp Asn Leu Gln
Gly Gln Ala Leu Glu 130 135 140 Gln Ala Ile Ile Ser Gln Lys Pro Gln
Leu Glu Lys Leu Ile Ala Thr 145 150 155 160 Thr Ala His Glu Lys Met
Pro Trp Phe His Gly Lys Ile Ser Arg Glu 165 170 175 Glu Ser Glu Gln
Ile Val Leu Ile Gly Ser Lys Thr Asn Gly Lys Phe 180 185 190 Leu Ile
Arg Ala Arg Asp Asn Asn Gly Ser Tyr Ala Leu Cys Leu Leu 195 200 205
His Glu Gly Lys Val Leu His Tyr Arg Ile Asp Lys Asp Lys Thr Gly 210
215 220 Lys Leu Ser Ile Pro Glu Gly Lys Lys Phe Asp Thr Leu Trp Gln
Leu 225 230 235 240 Val Glu His Tyr Ser Tyr Lys Ala Asp Gly Leu Leu
Arg Val Leu Thr 245 250 255 Val Pro Cys Gln Lys Ile Gly Thr Gln Gly
Asn Val Asn Phe Gly Gly 260 265 270 Arg Pro Gln Leu Pro Gly Ser His
Pro Ala Thr Trp Ser Ala Gly Gly 275 280 285 Ile Ile Ser Arg Ile Lys
Ser Tyr Ser Phe Pro Lys Pro Gly His Arg 290 295 300 Lys Ser Ser Pro
Ala Gln Gly Asn Arg Gln Glu Ser Thr Val Ser Phe 305 310 315 320 Asn
Pro Tyr Glu Pro Glu Leu Ala Pro Trp Ala Ala Asp Lys Gly Pro 325 330
335 Gln Arg Glu Ala Leu Pro Met Asp Thr Glu Val Tyr Glu Ser Pro Tyr
340 345 350 Ala Asp Pro Glu Glu Ile Arg Pro Lys Glu Val Tyr Leu Asp
Arg Lys 355 360 365 Leu Leu Thr Leu Glu Asp Lys Glu Leu Gly Ser Gly
Asn Phe Gly Thr 370 375 380 Val Lys Lys Gly Tyr Tyr Gln Met Lys Lys
Val Val Lys Thr Val Ala 385 390 395 400 Val Lys Ile Leu Lys Asn Glu
Ala Asn Asp Pro Ala Leu Lys Asp Glu 405 410 415 Leu Leu Ala Glu Ala
Asn Val Met Gln Gln Leu Asp Asn Pro Tyr Ile 420 425 430 Val Arg Met
Ile Gly Ile Cys Glu Ala Glu Ser Trp Met Leu Val Met 435 440 445 Glu
Met Ala Glu Leu Gly Pro Leu Asn Lys Tyr Leu Gln Gln Asn Arg 450 455
460 His Val Lys Asp Lys Asn Ile Ile Glu Leu Val His Gln Val Ser Met
465 470 475 480 Gly Met Lys Tyr Leu Glu Glu Ser Asn Phe Val His Arg
Asp Leu Ala 485 490 495 Ala Arg Asn Val Leu Leu Val Thr Gln His Tyr
Ala Lys Ile Ser Asp 500 505 510 Phe Gly Leu Ser Lys Ala Leu Arg Ala
Asp Glu Asn Tyr Tyr Lys Ala 515 520 525 Gln Thr His Gly Lys Trp Pro
Val Lys Trp Tyr Ala Pro Glu Cys Ile 530 535 540 Asn Tyr Tyr Lys Phe
Ser Ser Lys Ser Asp Val Trp Ser Phe Gly Val 545 550 555 560 Leu Met
Trp Glu Ala Phe Ser Tyr Gly Gln Lys Pro Tyr Arg Gly Met 565 570 575
Lys Gly Ser Glu Val Thr Ala Met Leu Glu Lys Gly Glu Arg Met Gly 580
585 590 Cys Pro Ala Gly Cys Pro Arg Glu Met Tyr Asp Leu Met Asn Leu
Cys 595 600 605 Trp Thr Tyr Asp Val Glu Asn Arg Pro Gly Phe Ala Ala
Val Glu Leu 610 615 620 Arg Leu Arg Asn Tyr Tyr Tyr Asp Val Val Asn
625 630 635 4537PRTHomo sapiens 4Met Gly Cys Val Gln Cys Lys Asp
Lys Glu Ala Thr Lys Leu Thr Glu 1 5 10 15 Glu Arg Asp Gly Ser Leu
Asn Gln Ser Ser Gly Tyr Arg Tyr Gly Thr 20 25 30 Asp Pro Thr Pro
Gln His Tyr Pro Ser Phe Gly Val Thr Ser Ile Pro 35 40 45 Asn Tyr
Asn Asn Phe His Ala Ala Gly Gly Gln Gly Leu Thr Val Phe 50 55 60
Gly Gly Val Asn Ser Ser Ser His Thr Gly Thr Leu Arg Thr Arg Gly 65
70 75 80 Gly Thr Gly
Val Thr Leu Phe Val Ala Leu Tyr Asp Tyr Glu Ala Arg 85 90 95 Thr
Glu Asp Asp Leu Ser Phe His Lys Gly Glu Lys Phe Gln Ile Leu 100 105
110 Asn Ser Ser Glu Gly Asp Trp Trp Glu Ala Arg Ser Leu Thr Thr Gly
115 120 125 Glu Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Val Asp
Ser Ile 130 135 140 Gln Ala Glu Glu Trp Tyr Phe Gly Lys Leu Gly Arg
Lys Asp Ala Glu 145 150 155 160 Arg Gln Leu Leu Ser Phe Gly Asn Pro
Arg Gly Thr Phe Leu Ile Arg 165 170 175 Glu Ser Glu Thr Thr Lys Gly
Ala Tyr Ser Leu Ser Ile Arg Asp Trp 180 185 190 Asp Asp Met Lys Gly
Asp His Val Lys His Tyr Lys Ile Arg Lys Leu 195 200 205 Asp Asn Gly
Gly Tyr Tyr Ile Thr Thr Arg Ala Gln Phe Glu Thr Leu 210 215 220 Gln
Gln Leu Val Gln His Tyr Ser Glu Arg Ala Ala Gly Leu Cys Cys 225 230
235 240 Arg Leu Val Val Pro Cys His Lys Gly Met Pro Arg Leu Thr Asp
Leu 245 250 255 Ser Val Lys Thr Lys Asp Val Trp Glu Ile Pro Arg Glu
Ser Leu Gln 260 265 270 Leu Ile Lys Arg Leu Gly Asn Gly Gln Phe Gly
Glu Val Trp Met Gly 275 280 285 Thr Trp Asn Gly Asn Thr Lys Val Ala
Ile Lys Thr Leu Lys Pro Gly 290 295 300 Thr Met Ser Pro Glu Ser Phe
Leu Glu Glu Ala Gln Ile Met Lys Lys 305 310 315 320 Leu Lys His Asp
Lys Leu Val Gln Leu Tyr Ala Val Val Ser Glu Glu 325 330 335 Pro Ile
Tyr Ile Val Thr Glu Tyr Met Asn Lys Gly Ser Leu Leu Asp 340 345 350
Phe Leu Lys Asp Gly Glu Gly Thr Ala Leu Lys Leu Pro Asn Leu Val 355
360 365 Asp Met Ala Ala Gln Val Ala Ala Gly Met Ala Tyr Ile Glu Arg
Met 370 375 380 Asn Tyr Ile His Arg Asp Leu Arg Ser Ala Asn Ile Leu
Val Gly Asn 385 390 395 400 Gly Leu Ile Cys Lys Ile Ala Asp Phe Gly
Leu Ala Arg Leu Ile Glu 405 410 415 Asp Asn Glu Tyr Thr Ala Arg Gln
Gly Ala Lys Phe Pro Ile Lys Trp 420 425 430 Thr Ala Pro Glu Ala Ala
Leu Tyr Gly Arg Phe Thr Ile Lys Ser Asp 435 440 445 Val Trp Ser Phe
Gly Ile Leu Leu Thr Glu Leu Val Thr Lys Gly Arg 450 455 460 Val Pro
Tyr Pro Gly Met Asn Asn Arg Glu Val Leu Glu Gln Val Glu 465 470 475
480 Arg Gly Tyr Arg Met Pro Cys Pro Gln Asp Cys Pro Ile Ser Leu His
485 490 495 Glu Leu Met Ile His Cys Trp Lys Lys Asp Pro Glu Glu Arg
Pro Thr 500 505 510 Phe Glu Tyr Leu Gln Ser Phe Leu Glu Asp Tyr Phe
Thr Ala Thr Glu 515 520 525 Pro Gln Tyr Gln Pro Gly Glu Asn Leu 530
535 527DNAArtificial SequencePrimer 5cacgctggga cgttcgggtt gggggac
27627DNAArtificial SequencePrimer 6gtcccccaac ccgaacgtcc cagcgtg
27728DNAArtificial SequencePrimer 7gatcaggggg gcttcaccat gcaccaag
28828DNAArtificial SequencePrimer 8cttggtgcat ggtgaagccc ccctgatc
28926DNAArtificial SequencePrimer 9gatcgcagcg gcttcagcag ccccgg
261026DNAArtificial SequencePrimer 10ccggggctgc tgaagccgct gcgatc
261138DNAArtificial SequencePrimer 11ggcagtgtgc aaatagtctt
caaaccagtt gacctgag 381238DNAArtificial SequencePrimer 12ctcaggtcaa
ctggtttgaa gactatttgc acactgcc 381328DNAArtificial SequencePrimer
13gcggagatcg tgttcaagtc gccagtgg 281428DNAArtificial SequencePrimer
14ccactggcga cttgaacacg atctccgc 281538DNAArtificial SequencePrimer
15ggcagtgtgc aaatagtcta caaaccagtt gacctgag 381638DNAArtificial
SequencePrimer 16ctcaggtcaa ctggtttgta gactatttgc acactgcc
381728DNAArtificial SequencePrimer 17gcggagatcg tgtacaagtc gccagtgg
281828DNAArtificial SequencePrimer 18ccactggcga cttgtacacg atctccgc
28
* * * * *