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MOLECULAR INTERACTIONS IN NEURONS
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/426,212, filed November 14, 2002, and U.S. Provisional Application No.
60/426,213,
filed November 14, 2002. This application is also (a) a continuation-in-part
of PCT
Application No. US02/24655, filed August 2, 2002, which claims the benefit of
U.S.
Provisional Application No. 60/309,841, filed August 3, 2001, and U.S.
Provisional
Application No. 60/360,061, filed February 25, 2002, and (b) a continuation-in-
part of U.S.
Application No. 09/724,553, filed November 28, 2000, which is a continuation-
in-part of
U.S. Application No. 09/547,276, filed April 11, 2000, which claims the
benefit of U.S.
Provisional Application No. 60/134,117, filed May 14, 1999. All of the
foregoing
applications are are incorporated herein by reference in their entirety for
all purposes.
1 S FIELD OF THE INVENTION
The present invention relates to the prevention and treatment of neurological
disorders, including cellular damage following stroke episodes or ischemia.
The invention
discloses methods of treating these disorders by administering inhibitors that
disrupt protein-
protein interactions involved in these disorders, screening methods to
identify such inhibitors
and specific compositions useful for treating these disorders.
BACKGROUND
Stroke is predicted to affect more than 600,000 people in the United States
this
year. In a 1999 report, over 167,000 people died from strokes, with a total
mortality of
278,000. In 1998, 3.6 billion was paid to just those Medicare beneficiaries
that were
discharged from short-stay hospitals, not including the long term care for
>1,000,000 people
that reportedly have functional limitations or difficulty with activities of
daily living resulting
from stroke (Heart and Stroke Statistical update, American Heart Association,
2002). At this
time, no therapeutics are available to reduce brain damage resulting from
stroke.
Stroke is characterized by neuronal cell death in areas of ischemia, brain
hemorrhage or trauma. Many lines of evidence have demonstrated that this cell
death is
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triggered by glutamate over-excitation of neurons, leading to increased
intracellular Caz+ and
increased nitric oxide due to an increase in nNOS (neuronal nitric oxide
synthase) activity.
Glutamate is the main excitatory neurotransmitter in the central nervous
system (CNS) and mediates neurotransmission across most excitatory synapses.
Three classes
of glutamate-gated ion channel receptors (N-methyl-D-aspartate (NMDA), alpha-
amino-3
hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and Kainate) transduce the
postsynaptic signal. Of these, NMDA receptors (NMDAR) have been shown to be
responsible for a significant portion of the excitotoxicity of glutamate. NMDA
receptors are
complex, being composed of an NR1 subunit and one or more NR2 subunits (2A,
2B, 2C or
2D) (see, e.g., McDain, C. and Mayer, M. (1994) Physiol. Rev. 74:723-760), and
less
commonly, an NR3 subunit (Chatterton et al. (2002) Nature 415:793-798). The
NR1
subunits have been shown to bind glycine, while NR2 subunits bind glutamate.
Both glycine
and glutamate binding are required to open the ion channel and allow calcium
entry into the
cell. The four NR2 receptor subunits appear to determine the pharmacology and
properties of
NMDA receptors, with further contributions from alternative splicing of the
NR1 subunit
(Kornau et al. (1995) Science 269:1737-40). Whereas NRl and NR2A subunits are
ubiquitously expressed in the brain, NR2B expression is restricted to the
forebrain, NR2C to
the cerebellum, and NR2D is rare compared to the other types.
Because of the key role these two proteins have in the excitotoxicity
response,
various approaches have been utilized to target these proteins. For example,
the NMDA
receptor contains a large number of modulatory sites and has been targeted by
many
therapeutics since the 1970's. Drugs have been developed that target the ion
channel
(ketamine, phencyclidine, PCP, MK801, amantadine), the outer channel
(magnesium), the
glycine binding site on NR1 subunits, the glutamate binding site on NR2
subunits, and
specific sites on NR2 subunits (Zinc - NR2A; Ifenprodil, Traxoprodil - NR2B).
Among
these, the non-selective antagonists of NMDA receptor have been the most
neuroprotective
agents in animal models of stroke. However, clinical trials with these drugs
in stroke and
traumatic brain injury have so far failed, generally as a result of severe
side effects such as
hallucination and even coma. Pharmaceutical companies have focused on subunit
selective
antagonists in hopes of obtaining neuroprotection without the negative side
effects that limit
the clinical utility of the compounds studied to date. These, however, have
also been
unsuccessful in the clinic thus far.
These failures have underscored the need to unravel the mechanisms of
neurotoxicity downstream from the NMDA receptors as alternative drug targets.
The goal in
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developing drugs to such targets is to identify drugs that inhibit the
glutamate excitotoxcity
response associated with glutamate activity, while not inhibiting the ability
of NMDA
receptors to function as ion channels.
SUMMARY
The present invention relates to the treatment of neuronal disorders such as
brain damage resulting from stroke, ischemia or related trauma by modulating
specific
protein:protein interactions between PDZ and PL proteins that are involved in
these diseases.
Methods for identifying specific therapeutics that modulate the specific
protein:protein
interactions involved in these disorders are also provided. Compounds and
compositions for
treating these neuronal disorders are also disclosed.
Methods of identifying the cellular PDZ proteins that are bound by the 5 main
subunits of the NMDA receptor complex (Rl, R2A, R2B, R2C, and R2D) are also
provided.
Methods are also provided to identify inhibitors that are both high affinity
for specific
subunits. Other methods are provided to determine selectivity of inhibition,
both against the
different NMDA receptor subunits and the PDZs that can bind them. Methods for
delivering
peptide inhibitors to cells such as neuron cells are also disclosed.
One class of pharmaceutical compositions that are provided include a
pharmaceutical composition comprising an isolated, recombinant or synthetic
polypeptide
inhibitor that inhibits binding between a N-methyl-D-aspartate (NMDA) receptor
and a PDZ
protein and a physiologically acceptable carrier, diluent or excipient,
wherein the polypeptide
comprises a C-terminal amino acid sequence of X-T-X-V/L/A. The C-terminal
amino acid
sequence of the polypeptide in some compositions of this type is ETEV, ETQL,
QTQV,
ETAL, QTEV, ETVA or FTDV. These compositions can be used to inhibit binding
between
an NMDA receptor and various PDZ proteins, including, for example, a PDZ
protein selected
from the group consisting of DLG1, DLG2, KIAA0973, NeDLG, Outermembrane
protein,
PSD-95, Syntrophin alpha l, TIP1, TIP2, INADL, KIAA0807, KIAA1634, Lim-
Mystique,
LIM-RIL, MAGI1, MAGI2, Syntrophin beta-1 and Syntrophin gamma-1. Certain
compositions are useful in inhibiting the interaction with PSD-95. The
polypeptides in these
compositions can be of varying lengths, such as 4-20 amino acids in length.
Other
polypeptides are fusion polypeptides, that include the C-terminal amino acid
sequence and a
segment of a transmembrane transporter sequence that is effective to
facilitate transport of the
polypeptide into the neuron cell.
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Another class of pharmaceutical compositions also include an isolated,
recombinant or synthetic polypeptide and a physiologically acceptable Garner,
diluent or
excipient, wherein the polypeptide is 3-8 amino acids in length and inhibits
binding between
a N-methyl-D-aspartate (NMDA) receptor and a PDZ protein. The polypeptides in
some of
these compositions are 3 amino acids in length. Exemplary sequences of such
polypeptides
include TEV or SDV.
Other pharmaceutical compositions include an isolated, recombinant or
synthetic polypeptide that inhibits binding between PSD-95 and N-methyl-D-
aspartate
receptor (NMDAR) 2A, NMDAR2C and/or NMDAR2D but not NMDAR2B. The
polypeptide in such compositions can be of various lengths. Some are 3-20
amino acids in
length. In some compositions, the polypeptide inhibits binding between PSD-95
and
NMDAR2A, NMDAR2C and NMDAR2D. In other compositions, the polypeptide inhibits
binding between PSD-95 and some but not all of NMDAR2A, NMDAR2C or NMDAR2D.
Still other pharmaceutical compositions include a fusion polypeptide that
inhibits binding between a N-methyl-D-aspartate (NMDA) receptor and a PDZ
protein and a
physiologically acceptable carrier, diluent or excipient. The fusion
polypeptide inhibitor in
these compositions is a fusion of (i) a 9 amino acid segment that has a C-
terminal sequence
selected from the group of amino acid sequences consisting of ETEV, ETQL,
QTQV, ETAL,
QTEV, ETVA and FTDV and (ii) an amino acid segment of a transmembrane
transporter that
is effective to transport the polypeptide into a neuron.
The polypeptide inhibitors in the foregoing pharmaceutical compositions can
be used in a variety of therapies, including treatment of a number of
neurological disorders.
Examples of such disorders include, but are not limited to, stroke, ischemia,
Parkinson's
disease, Huntington's disease, Alzheimer's disease, epilepsy and inherited
ataxias. The
inhibitors can also be used in the preparation of medicaments for use in
treating disease such
as those just listed.
Also provided are methods for determining whether a test compound inhibits
binding between a PDZ protein and a N-methyl-D-aspartate (NMDA) receptor.
Certain of
these methods initially involve contacting a PDZ -domain polypeptide
comprising a PDZ
domain from the PDZ protein and a PL peptide that comprises at least the C-
terminal 3 amino
acids of the NMDA receptor in the presence of the test compound. The PDZ
protein in these
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screening methods are typically selected from the group consisting of DLG1,
DLG2,
KIAA0973, NeDLG, Outermembrane protein, Syntrophin alpha 1, TIP1, TIP2, INADL,
KIAA0807, KIAA1634, Lim-Mystique, LIM-RIL, MAGI1, MAGI2, Syntrophin beta-1 and
Syntrophin gamma-1. The concentration of complex formed between the PDZ-domain
polypeptide and the PL peptide is then determined. The test compound is
identified as a
potential inhibitor of binding between the PDZ protein and the NMDA receptor
if a lower
concentration of the complex is detected in the presence of the test compound
relative to the
concentration of the complex in the absence of the test compound. Another
assay can be
conducted using compounds identified in the initial screen to determine
whether the
identified compound mitigates against a condition associated with a neuronal
disorder.
Examples of such assays include apoptosis assays, caspase assays, cytochrome c
assays and
cell lysis assays.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the interaction of NMDAR2A with PSD95, TIP2, DLG1, and
LIM. Light gray bars represent the background binding of NMDAR2A when 2% BSA
is
substituted for PDZ protein in the assay. Standard deviation is presented for
all data points.
Absorbance (y-axis) is measured at 450nm.
Figure 2 shows the PDZ binding profile for each NMDA receptor 2 subunit to
each of 238 PDZ proteins. Y axis indicates the A4sonm reading using the 'G'
assay described
herein; higher vertical bars are stronger interactions. The X axis indicates
individually cloned
and expressed human PDZ domains, numbered from 1 to 238.
Figure 3 demonstrates that NMDA Receptor subunits 2A, 2B and 2C can bind
PDZ domains 1 and 2 of PSD-95 (and a construct containing all three domains of
PSD-95),
but do not interact significantly with PSD-95 PDZ domain 3.
Figure 4 shows titrations of NMDA Receptor 2 subunits (A= R2A, B= R2B,
C=R2C, D=R2D) against the individual domains of PSD-95 and a construct
containing all
three domains. GST is a negative control, and PTPL/PBK is a weak positive
control for the
ELISA assay. The legend indicates the concentration in uM of the NMDA Receptor
peptide
that was added.
Figure 5 shows that binding of NMDA R2A to PSD95 domain 1 or domain 2
can be competed off by the addition of 3 amino acid peptides TEV (labeled TAT)
or SDV
(labeled 2B).
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Figure 6 demonstrates that 19 amino acid peptides corresponding to the C-
termini of TAX or HPV E6 type 16 can compete for binding of NMDA Receptor 2C
to
PSD95 domain 2 but not to domain 1 in these concentration ranges.
Figure 7 demonstrates that 3 amino acid peptides corresponding to the C-
termini of TAX or HPV E6 type 16 can compete for binding of NMDA Receptor 2C
to
PSD95 domain 2 but not to domain 1 in these concentration ranges.
Figure 8 demonstrates that 4 amino acid peptides corresponding to the C-
termini of TAX or HPV E6 type 16 can compete for binding of NMDA Receptor 2C
to
PSD95 domain 2 but not to domain 1 in these concentration ranges.
Figure 9 shows that binding of NMDA R2A to PSD95 domain 1 or domain 2
can be competed off by the addition of 19 amino acid peptides corresponding to
the C-termini
of TAX or HPV E6 type 16 in these concentration ranges.
Figure 10 demonstrates that when a TAT transporter sequence is coupled to
the C-terminal 9 amino acids of Tax binding is still mediated through the C-
terminal PDZ
Ligand motif (PL). TatTAXAA is a construct that changes the binding
specificity of TAT by
alanine substitution at the critical positions 0 and -2 of the PL. This figure
shows that the
TATTAX peptide can inhibit NMDA R2A and R2B binding to the second PDZ of PSD95
but
that the mutated PL version (TATTAXAA) cannot.
Figure 11 demonstrates that the internal PL motif of nNOS specifically binds
PDZ domain 2 of PSD95 but does not bind PDZ domain 1.
Figure 12 demonstrates that 20 amino acid and 3 amino acid peptide
inhibitors can selectively inhibit binding of one PL to PSD-95 PDZ domain 1
but not inhibit a
second PL binding to the same PDZ domain.
DETAILED DESCRIPTION
I. Definitions
"Polypeptide," "protein" and "peptide" are used interchangeably herein and
include a molecular chain of amino acids linked through peptide bonds. The
terms do not
refer to a specific length of the product. Thus, "peptides," "oligopeptides,"
and "proteins" are
included within the definition of polypeptide. The terms include post-
translational
modifications of the polypeptide, for example, glycosylations, acetylations,
phosphorylations
and the like. In addition, protein fragments, analogs, mutated or variant
proteins, fusion
proteins and the like are included within the meaning of polypeptide.
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A "fusion protein" or "fusion polypeptide" as used herein refers to a
composite protein, i.e., a single contiguous amino acid sequence, made up of
two (or more)
distinct, heterologous polypeptides which are not normally fused together in a
single amino
acid sequence. Thus, a fusion protein can include a single amino acid sequence
that contains
two entirely distinct amino acid sequences or two similar or identical
polypeptide sequences,
provided that these sequences are not normally found together in the same
configuration in a
single amino acid sequence found in nature. Fusion proteins can generally be
prepared using
either recombinant nucleic acid methods, i.e., as a result of transcription
and translation of a
recombinant gene fusion product, which fusion comprises a segment encoding a
polypeptide
of the invention and a segment encoding a heterologous protein, or by chemical
synthesis
methods well known in the art.
A "fusion protein construct" as used herein is a polynucleotide encoding a
fusion protein.
As used herein, the term "PDZ domain" refers to protein sequence (i.e.,
modular protein domain) of approximately 90 amino acids, characterized by
homology to the
brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-
Large (DLG),
and the epithelial tight junction protein ZO1 (ZOl). PDZ domains are also
known as Discs-
Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally appear
to
maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76).
PDZ domains are found in diverse membrane-associated proteins including
members of the MAGUK family of guanylate kinase homologs, several protein
phosphatases
and kinases, neuronal nitric oxide synthase, and several dystrophin-associated
proteins,
collectively known as syntrophins.
Exemplary PDZ domain-containing proteins and PDZ domain sequences are
shown in TABLE 4. The term "PDZ domain" also encompasses variants (e.g.,
naturally
occurring variants) of the sequences of TABLE 4 (e.g., polymorphic variants,
variants with
conservative substitutions, and the like). Typically, PDZ domains are
substantially identical
to those shown in TABLE 4, e.g., at least about 70%, at least about 80%, or at
least about
90% amino acid residue identity when compared and aligned for maximum
correspondence.
As used herein, the term "PDZ protein" refers to a naturally occurring protein
containing a PDZ domain. Exemplary PDZ proteins include CASK, MPPI, DLG1,
PSD95,
NeDLG, TIP33, SYNIa, TIP43, LDP, LIM, LIMK1, LIMK2, MPP2, NOSI, AF6, PTN-4,
prILl6, 41.8kD, KIAA0559, RGS12, KIAA0316, DVLl, TIP40, TIAM1, MINTI,
KIAA0303, CBP, MINT3, TIP2, KIAA0561, and those listed in TABLE 4.
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As used herein, the term "PDZ-domain polypeptide" refers to a polypeptide
containing a PDZ domain, such as a fusion protein including a PDZ domain
sequence, a
naturally occurring PDZ protein, or an isolated PDZ domain peptide.
As used herein, the term "PL protein" or "PDZ Ligand protein" refers to a
naturally occurring protein that forms a molecular complex with a PDZ-domain,
or to a
protein whose carboxy-terminus, when expressed separately from the full length
protein (e.g.,
as a peptide fragment of 4-25 residues, e.g., 8, 10, 12, 14 or 16 residues),
forms such a
molecular complex. The molecular complex can be observed in vitro using the "A
assay" or
"G assay" described infra, or in vivo. Exemplary NMDA receptor PL proteins
listed in
TABLE 2 are demonstrated to bind specific PDZ proteins. This definition is not
intended to
include anti-PDZ antibodies and the like.
As used herein, the terms "NMDA receptor," "NMDAR," or "NMDA receptor
protein" refer to a membrane associated protein that is known to interact with
NMDA. The
term thus includes the various subunit forms, including for example, those
listed in TABLE
2. The receptor can be a non-human mammalian NMDAR (e.g., mouse, rat, rabbit,
monkey)
or a human NMDAR, for example.
As used herein, the term "NMDAR-PL" or "NMDA receptor-PL" refers to a
NMDA receptor that forms a molecular complex with a PDZ domain or to a NMDAR
protein
whose carboxy-terminus, when expressed separately from the full length protein
(e.g., as a
peptide fragment of 4-25 residues, e.g., 8, 10, 12, 14 or 16 residues), forms
such a molecular
complex.
As used herein, a "PL sequence" refers to the amino acid sequence of the C-
terminus of a PL protein (e.g., the C-terminal 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,
14, 16, 20 or 25
residues) ("C-terminal PL sequence") or to an internal sequence known to bind
a PDZ
domain ("internal PL sequence")
As used herein, a "PL peptide" is a peptide of having a sequence from, or
based on, the sequence of the C-terminus of a PL protein. Exemplary PL
peptides
(biotinylated) are listed in TABLE 2.
As used herein, a "PL fusion protein" is a fusion protein that has a PL
sequence as one domain, typically as the C-terminal domain of the fusion
protein. An
exemplary PL fusion protein is a tat-PL sequence fusion.
As used herein, the term "PL inhibitor peptide sequence" refers to PL peptide
amino acid sequence that (in the form of a peptide or PL fusion protein)
inhibits the
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interaction between a PDZ domain polypeptide and a PL peptide (e.g., in an A
assay or a G
assay).
As used herein, a "PDZ-domain encoding sequence" means a segment of a
polynucleotide encoding a PDZ domain. In various embodiments, the
polynucleotide is
DNA, RNA, single stranded or double stranded.
As used herein, the terms "antagonist" and "inhibitor," when used in the
context of modulating a binding interaction (such as the binding of a PDZ
domain sequence
to a PL sequence), are used interchangeably and refer to an agent that reduces
the binding of
the, e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain sequence
(e.g., PDZ
protein, PDZ domain peptide).
As used herein, the terms "agonist" and "enhancer," when used in the context
of modulating a binding interaction (such as the binding of a PDZ domain
sequence to a PL
sequence), are used interchangeably and refer to an agent that increases the
binding of the,
e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain sequence (e.g.,
PDZ protein,
PDZ domain peptide).
The terms "isolated" or "purified" means that the object species (e.g., a
polypeptide) has been purified from contaminants that are present in a sample,
such as a
sample obtained from natural sources that contain the object species. If an
object species is
isolated or purified it is the predominant macromolecular (e.g., polypeptide)
species present
in a sample (i.e., on a molar basis it is more abundant than any other
individual species in the
composition), and preferably the object species comprises at least about 50
percent (on a
molar basis) of all macromolecular species present. Generally, an isolated,
purified or
substantially pure composition comprises more than 80 to 90 percent of all
macromolecular
species present in a composition. Most preferably, the object species is
purified to essential
homogeneity (i.e., contaminant species cannot be detected in the composition
by
conventional detection methods), wherein the composition consists essentially
of a single
macromolecular species.
The term "recombinant" when used with respect to a polypeptide refers to a
polypeptide that has been prepared be expressing a recombinant nucleic acid
molecule in
which different nucleic acid segments have been joined together using
molecular biology
techniques.
The term "synthesized" when used with respect to a polypeptide generally
means that the polypeptide has been prepared by means other than simply
purifying the
polypeptide from naturally occurnng sources. A synthesized polypeptide can
thus be
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prepared by chemical synthesis, recombinant means, or by a combination of
chemical
synthesis and recombinant means. Segments of a synthesized polypeptide,
however, may be
obtained from naturally occurring sources.
The term "biological function" or "biological activity" in the context of a
cell,
refers to a detectable biological activity normally carried out by the cell,
e.g., a phenotypic
change such as proliferation, cell activation, excitotoxicity responses,
neurotransmitter
release, cytokine release, degranulation, tyrosine phosphorylation, ion (e.g.,
calcium) flux,
metabolic activity, apoptosis, changes in gene expression, maintenance of cell
structure, cell
migration, adherence to a substrate, signal transduction, cell-cell
interactions, and others
described herein or known in the art.
As used herein, the terms "peptide mimetic, " "peptidomimetic," and "peptide
analog" are used interchangeably and refer to a synthetic chemical compound
which has
substantially the same structural and/or functional characteristics of an PL
inhibitory or PL
binding peptide of the invention. The mimetic can be either entirely composed
of synthetic,
non-natural analogues of amino acids, or, is a chimeric molecule of partly
natural peptide
amino acids and partly non-natural analogs of amino acids. The mimetic can
also incorporate
any amount of natural amino acid conservative substitutions as long as such
substitutions also
do not substantially alter the mimetic's structure and/or inhibitory or
binding activity. As
with polypeptides of the invention which are conservative variants, routine
experimentation
will determine whether a mimetic is within the scope of the invention, i.e.,
that its structure
and/or function is not substantially altered. Thus, a mimetic composition is
within the scope
of the invention if it is capable of binding to a PDZ domain and/or inhibiting
a PL-PDZ
interaction.
Polypeptide mimetic compositions can contain any combination of nonnatural
structural components, which are typically from three structural groups: a)
residue linkage
groups other than the natural amide bond ("peptide bond") linkages; b) non-
natural residues
in place of naturally occurring amino acid residues; or c) residues which
induce secondary
structural mimicry, i.e., to induce or stabilize a secondary structure, e.g.,
a beta turn, gamma
turn, beta sheet, alpha helix conformation, and the like.
A polypeptide can be characterized as a mimetic when all or some of its
residues are joined by chemical means other than natural peptide bonds.
Individual
peptidomimetic residues can be joined by peptide bonds, other chemical bonds
or coupling
means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters,
bifunctional maleimides,
N,N=-dicyclohexylcarbodiimide (DCC) or N,N=-diisopropylcarbodiimide (DIC).
Linking
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groups that can be an alternative to the traditional amide bond ("peptide
bond") linkages
include, e.g., ketomethylene (e.g., -C(=O)-CHZ- for -C(=O)-NH-),
aminomethylene (CHZ-
NH), ethylene, olefin (CH=CH), ether (CHZ-O), thioether (CHz-S), tetrazole
(CN4-), thiazole,
retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and
Biochemistry of
Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, A Peptide Backbone
Modifications,
Marcell Dekker, NY).
A polypeptide can also be characterized as a mimetic by containing all or
some non-natural residues in place of naturally occurring amino acid residues.
Nonnatural
residues are well described in the scientific and patent literature; a few
exemplary nonnatural
compositions useful as mimetics of natural amino acid residues and guidelines
are described
below.
Mimetics of aromatic amino acids can be generated by replacing by, e.g., D-
or L- naphylalanine; D- or L- phenylglycine; D- or L-2 thieneylalanine; D- or
L-1, -2, 3-, or
4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine;
D- or L-(3-
1 S pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-
phenylglycine; D-
(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-
fluorophenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-
methoxybiphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and, D- or L-
alkylainines,
where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl,
butyl, pentyl,
isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids.
Aromatic rings of a
nonnatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,
benzimidazolyl,
naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
Mimetics of acidic amino acids can be generated by substitution by, e.g., non-
carboxylate amino acids while maintaining a negative charge;
(phosphono)alanine; sulfated
threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be
selectively modified
by reaction with carbodiimides (R=-N-C-N-R=) such as, e.g., 1-cyclohexyl-3(2-
morpholinyl-
(4-ethyl) carbodiimide or 1-ethyl-3(4-azonia- 4,4- dimetholpentyl)
carbodiimide. Aspartyl or
glutamyl can also be converted to asparaginyl and glutaminyl residues by
reaction with
ammonium ions.
Mimetics of basic amino acids can be generated by substitution with, e.g., (in
addition to lysine and arginine) the amino acids ornithine, citrulline, or
(guanidino)-acetic
acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile
derivative (e.g.,
containing the CN-moiety in place of COOH) can be substituted for asparagine
or glutamine.
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Asparaginyl and glutaminyl residues can be deaminated to the corresponding
aspartyl or
glutamyl residues.
Arginine residue mimetics can be generated by reacting arginyl with, e.g., one
or more conventional reagents, including, e.g., phenylglyoxal, 2,3-
butanedione, 1,2-
cyclohexanedione, or ninhydrin, preferably under alkaline conditions.
Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g.,
aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and
tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro
derivatives,
respectively.
Cysteine residue mimetics can be generated by reacting cysteinyl residues
with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide
and
corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
Cysteine
residue mimetics can also be generated by reacting cysteinyl residues with,
e.g., bromo-
trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl
phosphate, N-
alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-
chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-
oxa-1,3-
diazole.
Lysine mimetics can be generated (and amino terminal residues can be
altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid
anhydrides. Lysine
and other alpha-amino-containing residue mimetics can also be generated by
reaction with
imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal,
chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4,
pentanedione, and
transamidase-catalyzed reactions with glyoxylate.
Mimetics of methionine can be generated by reaction with, e.g., methionine
sulfoxide. Mimetics of proline include, e.g., pipecolic acid, thiazolidine
carboxylic acid, 3
or 4- hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-
dimethylproline.
Histidine residue mimetics can be generated by reacting histidyl with, e.g.,
diethylprocarbonate or para-bromophenacyl bromide.
Other mimetics include, e.g., those generated by hydroxylation of proline and
lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues;
methylation of
the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-
terminal amine;
methylation of main chain amide residues or substitution with N-methyl amino
acids; or
amidation of C-terminal carboxyl groups.
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A component of a natural polypeptide (e.g., a PL polypeptide or PDZ
polypeptide) can also be replaced by an amino acid (or peptidomimetic residue)
of the
opposite chirality. Thus, any amino acid naturally occurring in the L-
configuration (which
can also be referred to as the R or S, depending upon the structure of the
chemical entity) can
be replaced with the amino acid of the same chemical structural type or a
peptidomimetic, but
of the opposite chirality, generally referred to as the D- amino acid, but
which can
additionally be referred to as the R- or S- form.
The mimetics of the invention can also include compositions that contain a
structural mimetic residue, particularly a residue that induces or mimics
secondary structures,
such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the
like. For
example, substitution of natural amino acid residues with D-amino acids; N-
alpha-methyl
amino acids; C-alpha-methyl amino acids; or dehydroamino acids within a
peptide can induce
or stabilize beta turns, gamma turns, beta sheets or alpha helix
conformations. Beta turn
mimetic structures have been described, e.g., by Nagai (1985) Tet. Lett.
26:647-650; Feigl
(1986) J. Amer. Chem. Soc. 108:181-182; Kahn (1988) J. Amer. Chem. Soc.
110:1638-1639;
Kemp (1988) Tet. Lett. 29:5057-5060; Kahn (1988) J. Molec. Recognition 1:75-
79. Beta
sheet mimetic structures have been described, e.g., by Smith (1992) J. Amer.
Chem. Soc.
114:10672-10674. For example, a type VI beta turn induced by a cis amide
surrogate,
1,5-disubstituted tetrazol, is described by Beusen (1995) Biopolymers 36:181-
200.
Incorporation of achiral omega-amino acid residues to generate polymethylene
units as a
substitution for amide bonds is described by Banerjee (1996) Biopolymers
39:769-777.
Secondary structures of polypeptides can be analyzed by, e.g., high-field 1H
NMR or 2D
NMR spectroscopy, see, e.g., Higgins (1997) J. Pept. Res. 50:421-435. See
also, Hruby
(1997) Biopolymers 43:219-266, Balaji, et al., U.S. Pat. No. 5,612,895.
As used herein, "peptide variants" and "conservative amino acid substitutions"
refer to peptides that differ from a reference peptide (e.g., a peptide having
the sequence of
the carboxy-terminus of a specified PL protein) by substitution of an amino
acid residue
having similar properties (based on size, polarity, hydrophobicity, and the
like). Thus,
insofar as the compounds that are encompassed within the scope of the
invention are partially
defined in terms of amino acid residues of designated classes, the amino acids
may be
generally categorized into three main classes: hydrophilic amino acids,
hydrophobic amino
acids and cysteine-like amino acids, depending primarily on the
characteristics of the amino
acid side chain. These main classes may be further divided into subclasses.
Hydrophilic
amino acids include amino acids having acidic, basic or polar side chains and
hydrophobic
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amino acids include amino acids having aromatic or apolar side chains. Apolar
amino acids
may be further subdivided to include, among others, aliphatic amino acids. The
definitions of
the classes of amino acids as used herein are as follows:
"Hydrophobic Amino Acid" refers to an amino acid having a side chain that is
uncharged at physiological pH and that is repelled by aqueous solution.
Examples of
genetically encoded hydrophobic amino acids include Ile, Leu and Val. Examples
of non-
genetically encoded hydrophobic amino acids include t-BuA.
"Aromatic Amino Acid" refers to a hydrophobic amino acid having a side
chain containing at least one ring having a conjugated electron system
(aromatic group). The
aromatic group may be further substituted with groups such as alkyl, alkenyl,
alkynyl,
hydroxyl, sulfanyl, nitro and amino groups, as well as others. Examples of
genetically
encoded aromatic amino acids include Phe, Tyr and Trp. Commonly encountered
non-
genetically encoded aromatic amino acids include phenylglycine, 2-
naphthylalanine, (3-2-
thienylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chloro-
phenylalanine, 2-
fluorophenyl-alanine, 3-fluorophenylalanine and 4-fluorophenylalanine.
"Apolar Amino Acid" refers to a hydrophobic amino acid having a side chain
that is generally uncharged at physiological pH and that is not polar.
Examples of genetically
encoded apolar amino acids include Gly, Pro and Met. Examples of non-encoded
apolar
amino acids include Cha.
"Aliphatic Amino Acid" refers to an apolar amino acid having a saturated or
unsaturated straight chain, branched or cyclic hydrocarbon side chain.
Examples of
genetically encoded aliphatic amino acids include Ala, Leu, Val and Ile.
Examples of non-
encoded aliphatic amino acids include Nle.
"Hydr~hilic Amino Acid" refers to an amino acid having a side chain that is
attracted by aqueous solution. Examples of genetically encoded hydrophilic
amino acids
include Ser and Lys. Examples of non-encoded hydrophilic amino acids include
Cit and
hCys.
"Acidic Amino Acid" refers to a hydrophilic amino acid having a side chain
pK value of less than 7. Acidic amino acids typically have negatively charged
side chains at
physiological pH due to loss of a hydrogen ion. Examples of genetically
encoded acidic
amino acids include Asp and Glu.
"Basic Amino Acid" refers to a hydrophilic amino acid having a side chain pK
value of greater than 7. Basic amino acids typically have positively charged
side chains at
physiological pH due to association with hydronium ion. Examples of
genetically encoded
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basic amino acids include Arg, Lys and His. Examples of non-genetically
encoded basic
amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic
acid, 2,4-
diaminobutyric acid and homoarginine.
"Polar Amino Acid" refers to a hydrophilic amino acid having a side chain that
is uncharged at physiological pH, but which has a bond in which the pair of
electrons shared
in common by two atoms is held more closely by one of the atoms. Examples of
genetically
encoded polar amino acids include Asx and Glx. Examples of non-genetically
encoded polar
amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.
"Cysteine-Like Amino Acid" refers to an amino acid having a side chain
capable of forming a covalent linkage with a side chain of another amino acid
residue, such
as a disulfide linkage. Typically, cysteine-like amino acids generally have a
side chain
containing at least one thiol (SH) group. Examples of genetically encoded
cysteine-like
amino acids include Cys. Examples of non-genetically encoded cysteine-like
amino acids
include homocysteine and penicillamine.
As will be appreciated by those having skill in the art, the above
classification
are not absolute -- several amino acids exhibit more than one characteristic
property, and can
therefore be included in more than one category. For example, tyrosine has
both an aromatic
ring and a polar hydroxyl group. Thus, tyrosine has dual properties and can be
included in
both the aromatic and polar categories. Similarly, in addition to being able
to form disulfide
linkages, cysteine also has apolar character. Thus, while not strictly
classified as a
hydrophobic or apolar amino acid, in many instances cysteine can be used to
confer
hydrophobicity to a peptide.
Certain commonly encountered amino acids which are not genetically encoded
of which the peptides and peptide analogues of the invention may be composed
include, but
are not limited to, (3-alanine (b-Ala) and other omega-amino acids such as 3-
aminopropionic
acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth;
a-
aminoisobutyric acid (Aib); E-aminohexanoic acid (Aha); 8-aminovaleric acid
(Ava); N-
methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-
butylalanine (t-BuA);
t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);
cyclohexylalanine
(Cha); norleucine (Nle); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine
(Phe(4-Cl));
2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-
fluorophenylalanine
(Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid (Tic); (3-2-
thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-
acetyl lysine
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WO 2004/045535 PCT/US2003/036698
(AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu);
p-aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVal); homocysteine (hCys)
and
homoserine (hSer). These amino acids also fall conveniently into the
categories defined
above.
The classifications of the above-described genetically encoded and non-
encoded amino acids are summarized in TABLE 1, below. It is to be understood
that
TABLE 1 is for illustrative purposes only and does not purport to be an
exhaustive list of
amino acid residues which may comprise the peptides and peptide analogues
described
herein. Other amino acid residues which are useful for making the peptides and
peptide
analogues described herein can be found, e.g., in Fasman, 1989, CRC Practical
Handbook of
Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited
therein.
Amino acids not specifically mentioned herein can be conveniently classified
into the above-
described categories on the basis of known behavior and/or their
characteristic chemical
and/or physical properties as compared with amino acids specifically
identified.
TABLE 1
Classification Genetically Genetically Non-Encoded
Encoded
Hydrophobic
Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4-C1), Phe(2-F), Phe(3-
F), Phe(4-F), Pyridyl Ala, Benzothienyl Ala
Apolar M, G, P
Aliphatic A, V, L, I t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, bAla,
MeGly, Aib
Hydrophilic
Acidic D, E
Basic H, K, R Dpr, Orn, hArg, Phe(p-NHZ), DBU, AZBU
Polar Q, N, S, T, Y Cit, AcLys, MSO, hSer
Cysteine-Like C Pen, hCys, p-methyl Cys
As used herein, a "detectable label" has the ordinary meaning in the art and
refers to an atom (e.g., radionuclide), molecule (e.g., fluorescein), or
complex, that is or can
be used to detect (e.g., due to a physical or chemical property), indicate the
presence of a
molecule or to enable binding of another molecule to which it is covalently
bound or
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WO 2004/045535 PCT/US2003/036698
otherwise associated. The term "label" also refers to covalently bound or
otherwise
associated molecules (e.g., a biomolecule such as an enzyme) that act on a
substrate to
produce a detectable atom, molecule or complex. Detectable labels suitable for
use in the
present invention include any composition detectable by spectroscopic,
photochemical,
biochemical, immunochemical, electrical, optical or chemical means. Labels
useful in the
present invention include biotin for staining with labeled streptavidin
conjugate, magnetic'
beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red,
rhodamine, green
fluorescent protein, enhanced green fluorescent protein, and the like),
radiolabels (e.g., 3H,
~ZSI, 355, ~4C, or 32P), enzymes ( e.g., hydrolases, particularly phosphatases
such as alkaline
phosphatase, esterases and glycosidases, or oxidoreductases, particularly
peroxidases such as
horse radish peroxidase, and others commonly used in ELISAs), substrates,
cofactors,
inhibitors, chemiluminescent groups, chromogenic agents, and colorimetric
labels such as
colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene,
latex, etc.) beads.
Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837;
3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means of detecting
such labels
are well known to those of skill in the art. Thus, for example, radiolabels
and
chemiluminescent labels may be detected using photographic film or
scintillation counters,
fluorescent markers may be detected using a photodetector to detect emitted
light (e.g., as in
fluorescence-activated cell sorting). Enzymatic labels are typically detected
by providing the
enzyme with a substrate and detecting the reaction product produced by the
action of the
enzyme on the substrate, and colorimetric labels are detected by simply
visualizing the
colored label. Thus, a label is any composition detectable by spectroscopic,
photochemical,
biochemical, immunochemical, electrical, optical or chemical means. The label
may be
coupled directly or indirectly to the desired component of the assay according
to methods
well known in the art. Non-radioactive labels are often attached by indirect
means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to the
molecule. The ligand
then binds to an anti-ligand (e.g., streptavidin) molecule which is either
inherently detectable
or covalently bound to a signal generating system, such as a detectable
enzyme, a fluorescent
compound, or a chemiluminescent compound. A number of ligands and anti-ligands
can be
used. Where a ligand has a natural anti-ligand, for example, biotin,
thyroxine, and cortisol, it
can be used in conjunction with the labeled, naturally occurring anti-ligands.
Alternatively,
any haptenic or antigenic compound can be used in combination with an
antibody. The
molecules can also be conjugated directly to signal generating compounds,
e.g., by
conjugation with an enzyme or fluorophore. Means of detecting labels are well
known to
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WO 2004/045535 PCT/US2003/036698
those of skill in the art. Thus, for example, where the label is a radioactive
label, means for
detection include a scintillation counter, photographic film as in
autoradiography, or storage
phosphor imaging. Where the label is a fluorescent label, it may be detected
by exciting the
fluorochrome with the appropriate wavelength of light and detecting the
resulting
fluorescence. The fluorescence may be detected visually, by means of
photographic film, by
the use of electronic detectors such as charge coupled devices (CCDs) or
photomultipliers
and the like. Similarly, enzymatic labels may be detected by providing the
appropriate
substrates for the enzyme and detecting the resulting reaction product. Also,
simple
colorimetric labels may be detected by observing the color associated with the
label. It will
be appreciated that when pairs of fluorophores are used in an assay, it is
often preferred that
they have distinct emission patterns (wavelengths) so that they can be easily
distinguished.
As used herein, the term "substantially identical" in the context of comparing
amino acid sequences, means that the sequences have at least about 70%, at
least about 80%,
or at least about 90% amino acid residue identity when compared and aligned
for maximum
correspondence. An algorithm that is suitable for determining percent sequence
identity and
sequence similarity is the FASTA algorithm, which is described in Pearson,
W.R. & Lipman,
D.J., 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 2444. See also W. R. Pearson,
1996, Methods
Enzymol. 266: 227-258. Preferred parameters used in a FASTA alignment of DNA
sequences to calculate percent identity are optimized, BL50 Matrix 15: -5, k-
tuple = 2;
joining penalty = 40, optimization = 28; gap penalty -12, gap length penalty =-
2; and width =
16.
As used herein, the terms "test compound" or "test agent" are used
interchangeably and refer to a candidate agent that may have enhancer/agonist,
or
inhibitor/antagonist activity, e.g., inhibiting or enhancing an interaction
such as PDZ-PL
binding. The candidate agents or test compounds may be any of a large variety
of
compounds, both naturally occurring and synthetic, organic and inorganic, and
including
polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and
polynucleotides), small
molecules, antibodies (as broadly defined herein), sugars, fatty acids,
nucleotides and
nucleotide analogs, analogs of naturally occurring structures (e.g., peptide
mimetics, nucleic
acid analogs, and the like), and numerous other compounds. In certain
embodiment, test
agents are prepared from diversity libraries, such as random or combinatorial
peptide or non-
peptide libraries. Many libraries are known in the art that can be used, e.g.,
chemically
synthesized libraries, recombinant (e.g., phage display libraries), and in
vitro translation-
based libraries. Examples of chemically synthesized libraries are described in
Fodor et al.,
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WO 2004/045535 PCT/US2003/036698
1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et
al., 1991,
Nature 354:82-84; Medynski, 1994, BiolTechnology 12:709-710; Gallop et al.,
1994, J.
Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad.
Sci. USA
90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426;
Houghten et
al., 1992, Biotechnigues 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad.
Sci. USA
91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712;
PCT
Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad.
Sci. USA
89:5381-5383. Examples of phage display libraries are described in Scott and
Smith, 1990,
Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian,
R.B., et al., 1992,
J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay
et al., 1993,
Gene 128:59-65; and PCT Publication No. WO 94/18318 dated August 18, 1994. In
vitro
translation-based libraries include but are not limited to those described in
PCT Publication
No. WO 91/05058 dated April 18, 1991; and Mattheakis et al., 1994, Proc. Natl.
Acad. Sci.
USA 91:9022-9026. By way of examples of nonpeptide libraries, a benzodiazepine
library
(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be
adapted for
use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-
9371) can also
be used. Another example of a library that can be used, in which the amide
functionalities in
peptides have been permethylated to generate a chemically transformed
combinatorial
library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA
91:11138-11142).
The term "specific binding" refers to binding between two molecules, for
example, a ligand and a receptor, characterized by the ability of a molecule
(ligand) to
associate with another specific molecule (receptor) even in the presence of
many other
diverse molecules, i.e., to show preferential binding of one molecule for
another in a
heterogeneous mixture of molecules. Specific binding of a ligand to a receptor
is also
evidenced by reduced binding of a detectably labeled ligand to the receptor in
the presence of
excess unlabeled ligand (i.e., a binding competition assay).
As used herein, a "plurality" of PDZ proteins (or corresponding PDZ domains
or PDZ fusion polypeptides) has its usual meaning. In some embodiments, the
plurality is at
least S, and often at least 25, at least 40, or at least 60 different PDZ
proteins. In some
embodiments, the plurality is selected from the list of PDZ polypeptides
listed in TABLE 4.
In some embodiments, the plurality of different PDZ proteins are from (i.e.,
expressed in) a
particular specified tissue or a particular class or type of cell. In some
embodiments, the
plurality of different PDZ proteins represents a substantial fraction (e.g.,
typically at least
50%, more often at least 80%) of all of the PDZ proteins known to be, or
suspected of being,
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WO 2004/045535 PCT/US2003/036698
expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be
present in neurons.
In some embodiments, the plurality is at least 50%, usually at least 80%, at
least 90% or all of
the PDZ proteins disclosed herein as being expressed in a particular cell.
When referring to PL peptides (or the corresponding proteins, e.g.,
corresponding to those listed in TABLE 2, or elsewhere herein) a "plurality"
may refer to at
least 5, at least 10, and often at least 25 PLs such as those specifcally
listed herein, or to the
classes and percentages set forth supra for PDZ domains.
The term "neurological disorder," "neurological injury", "neurological
disease" and other related terms generally refers to a disorder correlated
with some type
neuronal insult or neuronal cell death. Specific. examples of such disorders
include, but are
not limited to, stroke, ischemic stroke, Parkinson's disease, Huntington's
disease,
Alzheimer's disease, epilepsy, inherited ataxias and motor neuron diseases.
A "stroke" has the meaning normally accepted in the art and generally refers
to neurological injury resulting from impaired blood flow regardless of cause.
Potential
causes include, but are not limited to, embolism, hemorrhage and thrombosis.
An "ischemic
stroke" refers more specifically to a type of stroke that is of limited extent
and caused due to
blockage of blood flow. ,
A difference is in general is typically considered to be "statistically
significant" if the difference is less than experimental error. Thus a
difference is considered
statistically significant if the probability of the observed difference
occurring by chance (the
p-value) is less than some predetermined level. As used herein a
"statistically significant
difference" can refer to a p-value that is < 0.05, preferably < 0.01 and most
preferably
< 0.001.
II. General
The present inventors have identified a large number of interactions between
PDZ proteins and proteins that contain a PL motif that are involved in various
biological
functions in different types of cells. Some of these interactions involve
PDZ:PL protein
interactions between proteins that have important roles in neuronal cells. As
such,
modulation of these interactions have direct implications for the treatment of
various
neurological disorders, including stroke and ischemia.
Based upon the PDZ:PL interactions that have been detected, the inventors
have identified a number of distinct strategies for treating various
neurological disorders.
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One strategy is based upon the finding that the interaction between NMDAR
proteins (which
contain a PL sequence) and PSD-95 (a PDZ protein) is an important factor in
triggering an
excitotoxicity response in neuron cells. The inventors have determined common
structural
features of a class of polypeptides that are effective in disnipting the
interaction between
NMDAR proteins and PSD-95; polypeptides with these features are thus useful in
treating
neurological disorders associated with excitotoxicity. The second strategy is
based upon the
recognition that nNOS also has an important role in excitotoxicity responses.
nNOS has an
interesting structure in that it includes a PDZ domain, as well as an internal
PL sequence.
The current inventors determined that the internal PL sequence in nNOS binds
to PSD-95.
Thus, the second strategy involves the use of inhibitors to interfere with
this interaction as a
means to modulate biological activity in neurons. The third strategy is based
upon the
identification of specific PL proteins that bind to the PDZ domain of nNOS.
Inhibitors can
also be utilized to disrupt interactions between these protein binding
combinations to affect
biological activity in neurons.
The current inventors have thus identified compounds that inhibit the
interactions between these different proteins, as well as developed methods
for designing
additional compounds. One general class of inhibitors are those that mimic the
carboxy
terminus of a PL protein and thus interfere with the ability of the carboxy
terminus of the PL
protein to bind its cognate PDZ protein. Another general class of inhibitors
include the PDZ
domain from a PDZ protein that is involved in an interaction that is to be
disrupted. These
inhibitors bind the PL protein that is the cognate ligand for the PDZ protein
of interest and
thus prevent binding between the PL protein and PDZ protein. Because the
PDZ:PL protein
interactions that are described herein are involved in the biological activity
of neuronal cells,
the inhibitors that are provided can be used to inhibit PDZ:PL protein
interactions for the
treatment of neurological disorders such as stroke, ischemia, Parkinson's
disease,
Huntington's disease, Alzheimer's disease, epilepsy, inherited ataxias and
motor neuron
diseases. Methods for determining whether a test compound acts a modulator of
a particular
PDZ protein and PL protein binding pair are also described.
For those PDZ proteins containing multiple PDZ domains, the methods that
are provided can be utilized to determine to which specific domains) a
particular PL protein
of interest binds. The methods can thus be utilized to identify or design
inhibitors that have
increased selectivity for a particular PDZ domain. For instance, as described
in greater detail
below, the inventors have found that inhibitors with certain structural motifs
preferentially
inhibit binding between NMDR2 and the second PDZ domain of PSD-95, whereas
inhibitors
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with different structural motifs preferentially inhibit binding between NMDR2
and the first
PDZ domain of PSD-95. The methods that are disclosed can also be used to
identify
inhibitors with high binding affinity.
Because NMDAR proteins play a key regulatory role in neurons, an initial set
of studies were undertaken to determine what PDZ proteins bind to the various
NMDAR
subunits (there are eight different isoforms of the NMDARI subunits, four
different
NMDAR2 subunit forms and several different NMDAR3 subunits). These analyses
were
conducted using the "A" and "G" assays described in detail below. The PDZ
proteins
identified as binding at least one NMDAR subunit protein are listed in TABLE
3. PDZ
proteins found to bind all four NMDAR2 subunits are listed on the left-hand
side of TABLE
7. Those PDZ proteins that bound at least one, but not all, of the NMDAR2
subunits are
listed separately in TABLE 7.
The C-terminal sequences of the various NMDAR subunits that contain a PL
sequence are listed in TABLE 2. Because the C-terminal region of the PL
protein is the
If region that binds to PDZ proteins, agents that include similar amino acid
motifs can be used
to inhibit binding between NMDAR proteins and the PDZ proteins that bind to
them (see
TABLE 3). As described in greater detail below, for example, certain classes
of peptide
inhibitors typically include at least 2 contiguous amino acids from the C-
terminus of the
NMDAR proteins listed in TABLE 2, but can include 3-20 or more contiguous
amino acids
from the C-terminus.
One of the PDZ proteins identified in the initial investigation as interacting
with NMDAR proteins was PSD-95 (see TABLE 7). Additional studies were
subsequently
undertaken to identify the structural motifs that were common to the
polypeptides capable of
inhibiting the interaction between NMDAR and PSD-95 (see Example 5). One class
of
compounds are polypeptides that have the following characteristics: 1 ) a
length of about 3-20
amino acids (although somewhat longer polypeptides can be used), and 2) a C-
terminal
consensus sequence of X-T-X-V/L/A (the slash separates different amino acids
that can
appear at a given position). These polypeptides also typically had ICso values
of less than
SOuM.
As alluded to above, in addition to the studies with respect to the PDZ
proteins
that bind to NMDAR proteins, the current inventors have also identified
proteins having PL
sequences that can bind to the PDZ domain of nNOS. Identification of these
interactions also
provides insight into excitotoxicity in neurons because of the key role that
nNOS also plays
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in this process. Inhibitors having sequences that mimic the C-terminal motifs
of these
proteins can be used to inhibit the interaction of these proteins with nNOS.
In another set of experiments (see Example 9 and TABLE 9) PL sequences in
addition to NMDAR2 sequences were identified as capable of binding to the PDZ
domain of
PSD-95. Thus, inhibitors incorporating these PL sequences can also be used to
disrupt
interactions between PL proteins and PDZ.
The inventors have also found that the C-terminus of PSD-95 is itself a PL
sequence (RERL) and thus can bind PDZ proteins. Accordingly, another class of
inhibitors
are those that disrupt binding between the PL sequence of PSD-95 and its PDZ
binding
partners. Interactions of this type thus provide another therapeutic target
for treatment of
various neurological diseases.
Although the foregoing classes of inhibitors are based upon the C-terminal
sequences of PL proteins that bind a PDZ protein, as alluded to above, another
class of
inhibitors includes polypeptides that include all or a part of a PDZ domain
that binds to the
1 S PL sequence of a NMDAR protein or the internal PL sequence of nNOS.
Because inhibitors
in this class typically include most or the entire PDZ domain, polypeptide
inhibitors in this
class typically are at least 50-70 amino acids in length.
The various classes of polypeptide inhibitors just described can also be
fusion
proteins. These generally include a PL inhibitor peptide sequence such as
those just listed
that is fused to another sequence that encodes a separate protein domain. One
specific
example of an inhibitory fusion protein is one in which a PL sequence (e.g.,
those listed
above) are coupled to a transmembrane transporter peptide. As described in
greater detail
infra and in Example 6, a variety of different transmembrane transporter
peptides can be
utilized.
Although certain classes of inhibitors such as those j ust described are
polypeptides, other inhibitors are peptide mimetics or variants of these
polypeptides as
described in greater detail infra. Regardless of type, the inhibitors
typically had ICSO values
less than 50 uM, 25 uM, 10 uM, 0.1 uM or 0.01 uM. In general the inhibitors
typically have
an ICSO value of between 0.1 - 1 uM. These inhibitors can be formulated as
pharmaceutical
compositions and then used in the treatment of various neurological disorders
such as those
listed above.
The following sections provide additional details regarding the identification
of PDZ:PL interactions in neuron cells, the structural characteristics of
inhibitors that disrupt
these interactions and treatment methods utilizing such inhibitors.
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III. Identification of Candidate PL Proteins and Synthesis of Peptides
A PL protein (short for PDZ Ligand protein), such as the NMDAR proteins
described herein, is a protein (or a C-terminal fragment thereof) that can
bind PDZ proteins
via its carboxy terminus. PDZ proteins, in turn, are proteins with PDZ
domains, which are
domains common to three prototypical proteins: post synaptic density protein -
95 (PSD-95),
Drosophila large disc protein and Zonula Occludin 1 protein (see, e.g.,
Gomperts et al., 1996,
Cell 84:659-662; see also, Songyang et al., 1997, Science 275:73; and Doyle et
al., 1996, Cell
88:1067-1076). Certain classes of PDZ proteins contain three PDZ domains, one
SH3
domain and one guanylate kinase domain. As described in greater detail herein,
PL proteins
have certain carboxy terminal motifs that enable these proteins to functions
as ligands to PDZ
proteins. When these carboxy terminal regions are referred to, the positioning
of the carboxy
terminal residues are sometimes referred to herein by a numbered position,
which is
illustrated in the following scheme:
Position: -3 -2 -1 0 (C- terminal)
Certain PDZ domains are bound by the C-terminal residues of PDZ-binding
proteins. To identify NMDA receptors containing a PL motif, the C-terminal
residues of
sequences were visually inspected to identify sequences that bind to PDZ-
domain containing
proteins (see, e.g., Doyle et al., 1996, Cell 85, 1067; Songyang et al., 1997,
Science 275, 73).
TABLE 2 lists these proteins, and provides corresponding C-terminal sequences
and
GenBank accession numbers. Another investigation was conducted to identify PL
motifs
that bind to the PDZ domain of nNOS. The PL C-terminal motifs of the PL
proteins binding
to the PDZ domain are listed in TABLE 8.
A. Preparation of Pe tp ides
1) Chemical Synthesis
The peptides of the invention or analogues thereof, may be prepared using
virtually any art-known technique for the preparation of peptides and peptide
analogues. For
example, the peptides may be prepared in linear form using conventional
solution or solid
phase peptide syntheses and cleaved from the resin followed by purification
procedures
(Creighton, 1983, Protein Structures And Molecular Principles, W.H. Freeman
and Co.,
N.Y.). Suitable procedures for synthesizing the peptides described herein are
well known in
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WO 2004/045535 PCT/US2003/036698
the art. The composition of the synthetic peptides may be confirmed by amino
acid analysis
or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).
In addition, analogues and derivatives of the peptides can be chemically
synthesized. The linkage between each amino acid of the peptides of the
invention may be an
S amide, a substituted amide or an isostere of amide. Nonclassical amino acids
or chemical
amino acid analogues can be introduced as a substitution or addition into the
sequence. Non-
classical amino acids include, but are not limited to, the D-isomers of the
common amino
acids, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric
acid, y-Abu,
s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic
acid,
ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
cysteic acid, t-
butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, (3-alanine,
fluoro-amino acids,
designer amino acids such as (3-methyl amino acids, Ca-methyl amino acids, Na-
methyl
amino acids, and amino acid analogues in general. Furthermore, the amino acid
can be D
(dextrorotary) or L (levorotary).
Synthetic peptides of defined sequence (e.g., corresponding to the carboxyl-
termini of the indicated proteins) can be synthesized by any standard resin-
based method
(see, e.g., U. S. Pat. No. 4,108,846; see also, Caruthers et al., 1980,
Nucleic Acids Res. Symp.
Ser., 215-223; Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232;
Roberge, et al.,
1995, Science 269:202). The peptides used in the assays described herein were
prepared by
the FMOC (see, e.g., Guy and Fields, 199?, Meth. Enz. 289:67-83; Wellings and
Atherton,
1997, Meth. Enz.289:44-67). In some cases (e.g., for use in the A and G assays
of the
invention), peptides were labeled with biotin at the amino-terminus by
reaction with a four-
fold excess of biotin methyl ester in dimethylsulfoxide with a catalytic
amount of base. The
peptides were. cleaved from the resin using a halide containing acid (e.g.
trifluoroacetic acid)
in the presence of appropriate antioxidants (e.g. ethanedithiol) and excess
solvent lyophilized.
2) Recombinant Synthesis
If the peptide is composed entirely of gene-encoded amino acids, or a portion
of it is so composed, the peptide or the relevant portion may also be
synthesized using
conventional recombinant genetic engineering techniques. For recombinant
production, a
polynucleotide sequence encoding a linear form of the peptide is inserted into
an appropriate
expression vehicle, i.e., a vector which contains the necessary elements for
the transcription
and translation of the inserted coding sequence, or in the case of an RNA
viral vector, the
CA 02505479 2005-05-10
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necessary elements for replication and translation. The expression vehicle is
then transfected
into a suitable target cell which will express the peptide. Depending on the
expression
system used, the expressed peptide is then isolated by procedures well-
established in the art.
Methods for recombinant protein and peptide production are well known in the
art (see, e.g.,
Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring
Harbor
Laboratory, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular
Biology, Greene
Publishing Associates and Wiley Interscience, N.Y.).
A variety of host-expression vector systems may be utilized to express the
peptides described herein. These include, but are not limited to,
microorganisms such as
bacteria transformed with recombinant bacteriophage DNA or plasmid DNA
expression
vectors containing an appropriate coding sequence; yeast or Iiiamentous fungi
transformed
with recombinant yeast or fungi expression vectors containing an appropriate
coding
sequence; insect cell systems infected with recombinant virus expression
vectors (e.g.,
baculovirus) containing an appropriate coding sequence; plant cell systems
infected with
recombinant virus expression vectors (e.g., cauliflower mosaic virus or
tobacco mosaic virus)
or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)
containing an
appropriate coding sequence; or animal cell systems.
The expression elements of the expression systems vary in their strength and
specificities. Depending on the host/vector system utilized, any of a number
of suitable
transcription and translation elements, including constitutive and inducible
promoters, may be
used in the expression vector. For example, when cloning in bacterial systems,
inducible
promoters such as pL of bacteriophage ~,, plac, ptrp, ptac (ptrp-lac hybrid
promoter) and the
like may be used; when cloning in insect cell systems, promoters such as the
baculovirus
polyhedron promoter may be used; when cloning in plant cell systems, promoters
derived
ZS from the genome of plant cells (e.g., heat shock promoters; the promoter
for the small subunit
of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from
plant viruses
(e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be
used;
when cloning in mammalian cell systems, promoters derived from the genome of
mammalian
cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late
promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell
lines that
contain multiple copies of expression product, SV40-, BPV- and EBV-based
vectors may be
used with an appropriate selectable marker.
In cases where plant expression vectors are used, the expression of sequences
encoding the peptides of the invention may be driven by any of a number of
promoters. For
26
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WO 2004/045535 PCT/US2003/036698
example, viral promoters such as the 35S RNA and 195 RNA promoters of CaMV
(Brisson
et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV
(Takamatsu et al.,
1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as
the small
subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al.,
1984,
Science 224:838-843) or heat shock promoters, e.g., soybean hsp17.5-E or
hsp17.3-B (Gurley
et al., 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be
introduced
into planleukocytes using Ti plasmids, Ri plasmids, plant virus vectors,
direct DNA
transformation, microinjection, electroporation, etc. For reviews of such
techniques see, e.g.,
Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic
Press, NY,
Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular
Biology, 2d Ed.,
Blackie, London, Ch. 7-9.
In one insect expression system that may be used to produce the peptides of
the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is
used as a vector
to express the foreign genes. The virus grows in Spodoptera frugiperda cells.
A coding
sequence may be cloned into non-essential regions (for example the polyhedron
gene) of the
virus and placed under control of an AcNPV promoter (for example, the
polyhedron
promoter). Successful insertion of a coding sequence will result in
inactivation of the
polyhedron gene and production of non-occluded recombinant virus (i.e., virus
lacking the
proteinaceous coat coded for by the polyhedron gene). These recombinant
viruses are then
used to infect Spodoptera frugiperda cells in which the inserted gene is
expressed. (e.g., see
Smith et al., 1983, J. Virol. 46:584; Smith, U.S. Patent No. 4,215,051).
Further examples of
this expression system may be found in Current Protocols in Molecular Biology,
Vol. 2,
Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.
In mammalian host cells, a number of viral based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, a
coding sequence
may be ligated to an adenovirus transcription/translation control complex,
e.g., the late
promoter and tripartite leader sequence. This chimeric gene may then be
inserted in the
adenovirus genome by in vitro or in vivo recombination. Insertion in a non-
essential region
of the viral genome (e.g., region El or E3) will result in a recombinant virus
that is viable and
capable of expressing peptide in infected hosts. (e.g., See Logan & Shenk,
1984, Proc. Natl.
Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may
be used,
(see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:741 S-7419;
Mackett et al.,
1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA
79:4927-4931).
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Other expression systems for producing linear peptides of the invention will
be apparent to those having skill in the art.
B. Purification of Peptides and Peptide Analogues
The peptides and peptide analogues of the invention can be purified by art-
known techniques such as high performance liquid chromatography, ion exchange
chromatography, gel electrophoresis, affinity chromatography and the like. The
actual
conditions used to purify a particular peptide or analogue will depend, in
part, on factors such
as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to
those having skill
in the art. The purified peptides can be identified by assays based on their
physical or
functional properties, including radioactive labeling followed by gel
electrophoresis,
radioimmuno-assays, ELISA, bioassays, and the like.
For affinity chromatography purification, any antibody which specifically
binds the peptides or peptide analogues may be used. For the production of
antibodies,
various host animals, including but not limited to rabbits, mice, rats, etc.,
may be immunized
by injection with a peptide. The peptide may be attached to a suitable
carrier, such as BSA or
KLH, by means of a side chain functional group or linkers attached to a side
chain functional
group. Various adjuvants may be used to increase the immunological response,
depending on
the host species, including but not limited to Freund's (complete and
incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and
potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum.
Monoclonal antibodies to a peptide may be prepared using any technique
which provides for the production of antibody molecules by continuous cell
lines in culture.
These include but are not limited to the hybridoma technique originally
described by Koehler
and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma technique,
Kosbor et
al., 1983, Immunology Today 4:?2; Cote et al., 1983, Proc. Natl. Acad. Sci.
U.S.A. 80:2026-
2030 and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). In addition, techniques
developed for
the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl.
Acad. Sci. U.S.A.
81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985,
Nature
314:452-454) by splicing the genes from a mouse antibody molecule of
appropriate antigen
r
specificity together with genes from a human antibody molecule of appropriate
biological
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WO 2004/045535 PCT/US2003/036698
activity can be used. Alternatively, techniques described for the production
of single chain
antibodies (LT.S. Patent No. 4,946,778) can be adapted to produce peptide-
specific single
chain antibodies.
Antibody fragments which contain deletions of specific binding sites may be
S generated by known techniques. For example, such fragments include but are
not limited to
F(ab')2 fragments, which can be produced by pepsin digestion of the antibody
molecule and
Fab fragments, which can be generated by reducing the disulfide bridges of the
F(ab')Z
fragments. Alternatively, Fab expression libraries may be constructed (Huse et
al., 1989,
Science 246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments
with the desired specificity for the peptide of interest.
The antibody or antibody fragment specific for the desired peptide can be
attached, for example, to agarose, and the antibody-agarose complex is used in
immunochromatography to purify peptides of the invention. See, Scopes, 1984,
Protein
Purification: Principles and Practice, Springer-Verlag New York, Inc., NY,
Livingstone,
1974, Methods Enzymology: Immunoaffinity Chromatography of Proteins 34:723-
731.
For the peptides used in the present invention, cleavage from resin and
lyophilization was followed by peptides being redissolved and purified by
reverse phase high
performance liquid chromatography (HPLC). One appropriate HPLC solvent system
involves a Vydac C-18 semi-preparative column running at 5 mL per minute with
increasing
quantities of acetonitrile plus 0.1% trifluoroacetic acid in a base solvent of
water plus 0.1%
trifluoroacetic acid. After HPLC purification, the identities of the peptides
are confirmed by
MALDI canon-mode mass spectrometry. As noted, exemplary biotinylated peptides
are
provided in TABLE 2.
IV. PDZ Protein and PL Protein Interactions
TABLES 3, 7, 8 and 9 (Dave: Don't we also want Table 9) list PDZ
proteins and other PL proteins which the current inventors have identified as
binding to one
another. Each page of TABLE 3 includes seven columns. The columns are numbered
from
left to right such that the left-most column is column 1 and the right-most
column is column
7. Thus, the first column is labeled "internal PL 1D" and lists AA numbers
that serve as
unique internal designations for each PL peptide. These ID numbers correspond
to those
listed in column 6 of TABLE 2. The second column is labeled "PL Name" and
lists the
various PL proteins/PDZ-Ligands that were examined. This column lists gene
abbreviations,
with subtypes included, for peptides corresponding to the carboxyl-terminal 20
amino acids
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WO 2004/045535 PCT/US2003/036698
of the protein listed. The third column, labeled "PL 20mer Sequence," lists
the carboxyl-
terminal 20 amino acids of the protein. All ligands are biotinylated at the
amino-terminus.
Some have been modified to eliminate cysteine amino acids from the 20mer
sequence. In
these cases, wildtype sequences are presented in TABLE 2.
The PDZ protein (or proteins) that interacts) with a PL peptide are listed in
the fourth column that is labeled "PDZ Name". This column provides the gene
name for the
PDZ portion of the GST-PDZ fusion that interacts with the PDZ-ligand to the
left. For PDZ
domain-containing proteins with multiple domains, the domain number is listed
to the right of
the PDZ (i.e., in column 5 labeled "PDZ Domain"), and indicates the PDZ domain
number
when numbered from the amino-terminus to the carboxy-terminus.
The sixth column labeled "Binding Strength" is a measure of the level of
binding. In particular, it provides an absorbence value at 450 nm which
indicates the amount
of PL peptide bound to the PDZ protein. The following numerical values have
the following
meanings: '1' - A450nm 0-1; '2' - A450nm 1-2; '3'- A450nm 2-3; '4' - A450nm 3-
4; 'S' -
A450nm of 4. All interactions have been repeated a total of at least 4 times,
and all show
A450nm values that are at least two times that of controls. Note that the
binding strength has
not been indicated for all interactions, and should not be used as a
quantitative comparison of
avidity between interactions. The last column in TABLE 3, labeled "Assay
Used," indicates
whether the interaction was detected using the "A Assay," the "G Assay," or
both assays (see
below).
Further information regarding these PL proteins and PDZ proteins is provided
in TABLES 2 and 4. In particular, TABLE 2 provides a list of known NMDA
receptors,
along with the amino acid sequence of the carboxyl-terminal 20 amino acids.
When
numbered from left to right, the first column labeled "Name" provides the
commonly used
abbreviation of the gene name. Genbank GI numbers are listed in column 2,
labeled "GI#."
Columns 3 and 4, labeled "C-terminal 20mer sequence" and "C-terminal 4mer
sequence,"
respectively, list the last 20 amino acids, and the last 4 amino acids of each
protein. Column
5, labeled "PL?" marks with an "X" those carboxy-terminal sequences that are
predicted to
display a classic PL amino acid motif. Many of the carboxyl-terminal motifs
that are not
marked in column 5 may exhibit binding to PDZ proteins, and the designation as
a classic PL
motif is in no way intended to predict or restrict NMDAR binding patterns to
PDZ proteins.
The sixth column labeled "internal PL ID" provides the internal designation
number used to
refer to a particular PL protein and correlates with the designation used in
column 1 of
TABLE 3.
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Many of the genes listed in TABLE 2 express more than one amino acid
sequence, depending on alternative exon splicing and single amino acid
changes. When the
information was available, alternatively spliced and point mutated isoforms of
the same gene
have been represented separately in TABLE 2. It is understood in the art that
many
alternatively spliced and point mutated forms of the same gene may exist in
nature. As
indicated supra, all peptides were biotinylated at the amino terminus and the
amino acid
sequences correspond to the C-terminal sequence of the gene name listed in
column 1.
TABLE 4 lists the sequences of the PDZ domains cloned into a vector
(PGEX-3X vector) for production of GST-PDZ fusion proteins (Pharmacia). More
specifically, the first column (left to right) entitled "Gene Name" lists the
name of the gene
containing the PDZ domain. The second column labeled "GI or Acc#" is a unique
Genbank
identifier for the gene used to design primers for PCR amplification of the
listed sequence.
The next column labeled "Domain#" indicates the Pfam-predicted PDZ domain
number, as
numbered from the amino-terminus of the gene to the carboxy-teminus. The last
column
entitled "Sequence fused to GST Construct" provides the actual amino acid
sequence inserted
into the GST-PDZ expression vector as determined by DNA sequencing of the
constructs.
As discussed in detail herein, the PDZ proteins listed in TABLES 3 and 4 are
naturally occurring proteins containing a PDZ domain. Only significant
interactions are
presented in this TABLE 3. Thus, the present invention is directed to the
detection and
modulation of interactions between a PDZ protein and PL protein pair listed in
TABLE 3.
As used herein the phrase "protein pair" or 'protein binding pair" when used
in reference to a
PDZ protein and PL protein refers to a PL protein and PDZ protein listed in
TABLE 3 which
bind to one another. It should be understood that TABLE 3 is set up to show
that certain PL
proteins bind to a plurality of PDZ proteins. For example, PL protein AA34.2
binds to PDZ
proteins PSD95 and DLG1.
The interactions summarized in TABLE 3 can occur in a wide variety of cell
types. Examples of such cells include hematopoietic, stem, neuronal, muscle,
epidermal,
epithelial, endothelial, and cells from essentially any tissue such as liver,
lung, placenta,
uterus, kidney, ovaries, testes, stomach, colon and intestine. Because the
interactions
disclosed herein can occur in such a wide variety of cell types, these
interactions can also
play an important role in a variety of biological functions.
Thus, for example, in certain embodiments of the invention, the PL proteins
and/or the PDZ protein to which it binds are expressed in the nervous system
(e.g., neurons).
In an embodiment, the PL proteins of the invention bind a PDZ protein that is
expressed in
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neurons. In various embodiments, the PL protein is highly expressed in
neuronal cells. In
still other instances the PL proteins and/or the PDZ protein to which it binds
are expressed in
non-neural cells (e.g., in hematopoietic cells).
In various embodiments of the invention, the PL protein is expressed or up
s regulated upon cell activation (e.g., in stimulated neurons), upon entry
into mitosis (e.g., up
regulation in rapidly proliferating cell populations), or in association with
apoptosis.
In certain other various embodiments of the invention, the PL protein is (i) a
protein that mediates the biological function of a neuronal cell, (ii) a
protein that mediates
apoptosis in a neural cell, (iii) a protein that is a N-methyl-D-aspartate
receptor, or (iv) a
protein that is a N-methyl-D-aspartate receptor and is expressed in neural
cells.
In certain various embodiments of the invention, the methods disclosed infra
are used to block the interaction between (i) NMDAR2A and an intracellular PDZ
protein,
(ii) NMDAR2B and an intracellular PDZ protein, (iii) NMDAR2C and an
intracellular PDZ
protein, and/or (iv) NMDAR2D and an intracellular PDZ protein.
In a preferred embodiment of the invention, the methods disclosed infra are
used to block an interaction between all type 2 NMDA receptors (NMDAR2) and
any
intracellular PDZ.
In one embodiment of the invention, the methods disclosed infra are used to
block an interaction between any type 2 NMDA receptor (NMDAR2) and any
intracellular
PDZ.
A. Detection of PDZ Domain-Containing Proteins
As noted supra, the present inventors have identified a number of PDZ protein
and NMDAR PL protein interactions that can play a role in modulation of a
number of
biological functions in a variety of cell types. A comprehensive list of PDZ
domain
containing proteins was retrieved from the Sanger Centre database (Pfam)
searching for the
keyword, "PDZ". The corresponding cDNA sequences were retrieved from GenBank
using
the NCBI "entrez" database (hereinafter, "GenBank PDZ protein cDNA
sequences"). The
DNA portion encoding PDZ domains was identified by alignment of cDNA and
protein
sequence using CLUSTALW. Based on the DNA/protein alignment information,
primers
encompassing the PDZ domains were designed. The expression of certain PDZ-
containing
proteins in cells was detected by polymerase chain reaction ("PCR")
amplification of cDNAs
obtained by reverse transcription ("RT") of cell-derived RNA (i.e., "RT-PCR").
PCR, RT-
PCR and other methods for analysis and manipulation of nucleic acids are well
known and
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are described generally in Sambrook et al., (1989) MOLECULAR CLONING: A
LABORATORY
MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory hereinafter,
"Sambrook"); and
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR B10LOGY, Greene Publishing and
Wiley-Interscience, New York (1997), as supplemented through January 1999
(hereinafter
"Ausubel")
Samples of cDNA for those sequences identified through the foregoing search
were obtained and then amplified. In general a sample of the cDNA (typically,
1/5 of a 20 ~1
reaction) was used to conduct PCR. PCR was conducted using primers designed to
amplify
specifically PDZ domain-containing regions of PDZ proteins of interest.
Oligonucleotide
primers were designed to amplify one or more PDZ-encoding domains. The DNA
sequences
encoding the various PDZ domains of interest were identified by inspection
(i.e., conceptual
translation of the PDZ protein cDNA sequences obtained from GenBank, followed
by
alignment with the PDZ domain amino acid sequence). TABLE 4 shows the PDZ-
encoded
domains amplified, and the GenBank accession number of the PDZ-domain
containing
proteins. To facilitate subsequent cloning of PDZ domains, the PCR primers
included
endonuclease restriction sequences at their ends to allow ligation with pGEX-
3X cloning
vector (Pharmacia, GenBank XXI13852 ) in frame with glutathione-S transferase
(GST).
TABLE 4 lists the proteins isolated for use in the aforementioned assays.
B. Production of Fusion Proteins Containing PDZ-Domains
GST-PDZ domain fusion proteins were prepared for use in the assays of the
invention. PCR products containing PDZ encoding domains (as described supra)
were
subcloned into an expression vector to permit expression of fusion proteins
containing a PDZ
domain and a heterologous domain (i.e., a glutathione-S transferase sequence,
"GST"). PCR
products (i.e., DNA fragments) representing PDZ domain encoding DNA was
extracted from
agarose gels using the "sephaglas" gel extraction system (Pharmacia) according
to the
manufacturer's recommendations.
As noted supra, PCR primers were designed to include endonuclease
restriction sites to facilitate ligation of PCR fragments into a GST gene
fusion vector (pGEX-
3X; Pharmacia, GenBank accession no. XXU13852) in-frame with the glutathione-S
transferase coding sequence. This vector contains a IPTG inducible lacZ
promoter. The
pGEX-3X vector was linearized using Bam HI and Eco RI or, in some cases, Eco
RI or Sma
I, and dephosphorylated. For most cloning approaches, double digestion with
Bam HI and
Eco RI was performed, so that the ends of the PCR fragments to clone were Bam
HI and Eco
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RI. In some cases, restriction endonuclease combinations used were Bgl II and
Eco RI, Bam
HI and Mfe I, or Eco RI only, Sma I only, or BamHI only. When more than one
PDZ domain
was cloned, the DNA portion cloned represents the PDZ domains and the cDNA
portion
located between individual domains. Precise locations of cloned fragments used
in the assays
S are indicated in TABLE 4. Examples of the primers used to generate fragments
for cloning
are presented in TABLE 5. DNA linker sequences between the GST portion and the
PDZ
domain containing DNA portion vary slightly, dependent on which of the above
described
cloning sites and approaches were used. As a consequence, the amino acid
sequence of the
GST-PDZ fusion protein varies in the linker region between GST and PDZ domain.
Protein
linkers sequences corresponding to different cloning sites/approaches are
shown below.
Linker sequences (vector DNA encoded) are bold, PDZ domain containing gene
derived
sequences are in italics.
1 ) GST-BamHI/BamHI- PDZ domain insert
Gly--Ile---PDZ domain insert
2) GST-BamHI/BgII~ PDZ domain insert
Gly-Ile---PDZ domain insert
3) GST-EcoR.I/Ecol-PDZ domain insert
Gly-Ile-Pro-Gly--Asn PDZ domain ihsert
4) GST--SmaIlSmaI-PDZ domain insert
Gly-Ile-Pro-PDZ domain insert
The PDZ-encoding PCR fragment and linearized pGEX-3X vector were
ethanol precipitated and resuspended in 10 ul standard ligation buffer.
Ligation was
performed for 4-10 hours at 7°C using T4 DNA ligase. It will be
understood that some of
the resulting constructs include very short linker sequences and that, when
multiple PDZ
domains were cloned, the constructs included some DNA located between
individual PDZ
domains.
The ligation products were transformed in DHSa or BL-21 E.coli bacteria
strains. Colonies were screened for presence and identity of the cloned PDZ
domain
containing DNA as well as for correct fusion with the glutathione S-
transferase encoding
DNA portion by PCR and by sequence analysis. Positive clones were tested in a
small scale
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assay for expression of the GST/PDZ domain fusion protein and, if expressing,
these clones
were subsequently grown up for large scale preparations of GST/PDZ fusion
protein.
GST-PDZ domain fusion protein was overexpressed following addition of
IPTG to the culture medium and purified. Detailed procedure of small scale and
large scale
fusion protein expression and purification are described in "GST Gene Fusion
System"
(second edition, revision 2; published by Pharmacia). In brief, a small
culture (3-Smls)
containing a bacterial strain (DHSa, BL21 or JM109) with the fusion protein
construct was
grown overnight in LB-media at 37°C with the appropriate antibiotic
selection (100ug/ml
ampicillin; a.k.a. LB-amp). The overnight culture was poured into a fresh
preparation of LB-
amp (typically 250-SOOmIs) and grown until the optical density (OD) of the
culture was
between 0.5 and 0.9 (approximately 2.5 hours). IPTG (isopropyl ~3-D-
thiogalactopyranoside)
was added to a final concentration of l.OmM to induce production of GST fusion
protein, and
culture was grown an additional 1.5-2.5 hours. Bacteria were collect by
centrifugation (4500
g) and resuspended in Buffer A- (SOmM Tris, pH 8.0, SOmM dextrose, 1mM EDTA,
200uM
phenylmethylsulfonylfluoride). An equal volume of Buffer A+ (Buffer A-, 4mg/ml
lysozyme) was added and incubated on ice for 3 min to lyse bacteria. An equal
volume of
Buffer B (lOmM Tris, pH 8.0, SOmM KCI, 1mM EDTA. 0.5% Tween-20, 0.5% NP40
(a.k.a.
IGEPAL CA-630), 200uM phenylmethylsulfonylfluoride) was added and incubated
for an
additional 20 min. The bacterial cell lysate was centrifuged (x20,000g), and
supernatant was
added to glutathione Sepharose 4B (Pharmacia, cat no. 17-0765-O1) previously
swelled
(rehydrated) in 1X phosphate-buffered saline (PBS). The supernatant-Sepharose
slurry was
poured into a column and washed with at least 20 bed volumes of 1X PBS. GST
fusion
protein was eluted off the glutathione sepharose by applying 0.5-1.0 ml
aliquots of SmM
glutathione and collected as separate fractions. Concentrations of fractions
were determined
using BioRad Protein Assay (cat no. 500-0006) according to manufacturer's
specifications.
Those fractions containing the highest concentration of fusion protein were
pooled and
dialyzed against 1X PBS/35% glycerol. Fusion proteins were assayed for size
and quality by
SDS gel electrophoresis (PAGE) as described in "Sambrook." Fusion protein
aliquots were
stored at minus 80°C and at minus 20°C.
C. Classification of PDZ Domain-Containin Proteins
The PDZ proteins identified herein as interacting with particular PL proteins
can be grouped into a number of different categories. Thus, as described in
greater detail
below, the methods and reagents that are provided herein can be utilized to
modulate PDZ
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interactions, and thus biological functions, that are regulated or otherwise
involve the
following classes of proteins. It should be recognized, however, that
modulation of the
interactions that are identified herein can be utilized to affect biological
functions involving
other protein classes.
1) Protein Kinases
A number of protein kinases contain PDZ domains. Protein kinases are
widely involved in cellular metabolism and regulation of protein activity
through
phosphorylation of amino acids on proteins. An example of this is the
regulation of signal
transduction pathways such as T cell activation through the T cell Receptor,
where ZAP-70
kinase function is required for transmission of the activation signal to
downstream effector
molecules. These molecules include, but are not limited to KIAA0303, KIAA0561,
KIAA0807, KIAA0973, and CASK.
2) Guanalyte Kinases
A number of guanalyte kinases contain PDZ domains. These molecules
include, but are not limited to Atrophin 1, CARD11, CARD14, DLG1, DLG2, DLGS,
FLJ12615, MPP1, MPP2, NeDLG, p55T, PSD95, ZO-1, ZO-2, and ZO-3.
3) Guanine Exchange Factors
A number of guanine exchange factors contain PDZ domains. Guanine
exchange factors regulate signal transduction pathways and other biological
processes
through facilitating the exchange of differently phosphorylated guanine
residues. These
molecules include, but are not limited to GTPase, Guanine Exchange, KIAA0313,
KIAA0380, KIAA0382, KIAA1389, KIAA1415, TIAM1, and TAM.
4) LIM PDZ's
A number of LIM PDZ's contain PDZ domains. These molecules include, but
are not limited to a-Actinin 2, ELFIN1, ENIGMA, HEMBA 1003117, KIAA0613,
KIAA0858, KIAA0631, LIM Mystique, LIM protein, LIM-RIL, LIMK1, LIMK2, and LU-
1.
5) Proteins Containing Only PDZ Domains
A number of proteins contain PDZ domains without any other predicted
functional domains. These include, but are not limited to 26s subunit p27,
AIPC, Cytohesion
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Binding Protein, EZRIN Binding Protein, FLJ00011, FLJ20075, FLJ21687, GRIP1,
HEMBA1000505, KIAA0545, KIAA0967, KIAA1202, KIAA1284, KIAA1526, KIAA1620,
KIAA1719, MAGI1, Novel PDZ gene, Outer Membrane, PAR3, PAR6, PAR6y, PDZ-73,
PDZKI, PICK1, PIST, prILl6, Shankl, SIP1, SITAC-18, Syntenin, Syntrophin y2,
TIPI,
TIP2, and TIP43.
6) Tyrosine Phosphatases
A number of Tyrosine phosphatases contain PDZ domains. Tyrosine
phosphatases regulate biological processes such as signal transduction
pathways through
removal of phosphate groups required for function of the target protein.
Examples of such
enzymes include, but are not limited to PTN-3, PTN-4, and PTPL1.
7) Serine Proteases
A number of serine proteases contain PDZ domains. Proteases affect
biological molecules by cleaving them to either activate or repress their
functional ability.
These enzymes have a variety of functions, including roles in digestion, blood
coagulation
and lysis of blood clots. These include, but are not limited to Novel Serine
Protease and
Serine Protease.
8) Viral Onco~ene Interactin~Proteins that Contain PDZ Domains
A number of TAX interacting proteins contain PDZ domains. Many of these
also bind to multiple viral oncoproteins such as Adenovirus E4, Papillomavirus
E6, and HBV
protein X. These include, but are not limited to AIPC, Cormector Enhancer,
DLG1, DLG2,
ERBIN, FLJ00011, FLJ11215, HEMBA1003117, INADL, KIAA0147, KIAA0807,
KIAA1526, KIAA1634, LIMK1, LIM Mystique, LIM-RIL, MUPP1, NeDLG, Outer
Membrane, PSD95, PTN-4, PTPL-1, Syntrophin yl, Syntrophin y2, TAX2-like
protein, TIP2,
TIP1, TIP33, and T1P43.
A number of proteins containing RA and/or RHA and/or DIL and/or IGFBP
andJor WW and/or L27 and/or SAM and/or PH and/or DIX and/or DIP and/or
Dishevelled
and/or LRR and/or Hormone 3 and/or C2 and/or RPH3A and/or zf TRAF and/or zf
C3HC4
and/or PID and/or NO Synthase and/or Flavodoxin and/or FAD binding and/or NAD
binding
and/or Kazal and/or Trypsin and/or RBD and/or RGS and/or GoLoco and/or HR1
and/or
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BRO1 contain PDZ domains. These include, but are not limited to AF6, APXL-1,
MAGI1,
DVL1, DVL2, DVL3, KIAA0417, KIAA0316, KIAA0340, KIAA0559, KIAA0751,
KIAA0902, KIAA1095, KIAA1222, KIAA1634, MINT1, NOS1, RGS12, Rhophilin-like,
Shank 3, Syntrophin 1 a, Syntrophin (32, and X11 (3.
D. Assavs for Detection of Interactions Between PDZ-Domain Polvoeptides and
NMDA Receptor PL Proteins
Two complementary assays, termed "A' and "G,"" were developed to detect
binding between a PDZ-domain polypeptide and candidate PDZ ligand. In each of
the two
different assays, binding is detected between a peptide having a sequence
corresponding to
the C-terminus of a protein anticipated to bind to one or more PDZ domains
(i.e. a candidate
PL peptide) and a PDZ-domain polypeptide (typically a fusion protein
containing a PDZ
domain). In the "A" assay, the candidate PL peptide is immobilized and binding
of a soluble
PDZ-domain polypeptide to the immobilized peptide is detected (the "A"' assay
is named for
the fact that in one embodiment an avidin surface is used to immobilize the
peptide). In the
"G" assay, the PDZ-domain polypeptide is immobilized and binding of a soluble
PL peptide
is detected (The "G" assay is named for the fact that in one embodiment a GST-
binding
surface is used to immobilize the PDZ-domain polypeptide). Preferred
embodiments of these
assays are described in detail infra. However, it will be appreciated by
ordinarily skilled
practitioners that these assays can be modified in numerous ways while
remaining useful for
the purposes of the present invention.
1) "A Assay" Detection of PDZ-Ligand Binding Using Immobilized PL
Peptide.
In one aspect, the invention provides an assay in which biotinylated candidate
PL peptides are immobilized on an avidin coated surface. The binding of PDZ-
domain
fusion protein to this surface is then measured. In a preferred embodiment,
the PDZ-domain
fusion protein is a GST/PDZ fusion protein and the assay is carried out as
follows:
(1) Avidin is bound to a surface, e.g. a protein binding surface. In one
embodiment, avidin is bound to a polystyrene 96 well plate (e.g., Nunc
Polysorb (cat
#475094) by addition of 100 pL per well of 20 yg/mL of avidin (Pierce) in
phosphate
buffered saline without calcium and magnesium, pH 7.4 ("PBS", GibcoBRL) at
4°C for 12
hours. The plate is then treated to block nonspecific interactions by addition
of 200 ~L per
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WO 2004/045535 PCT/US2003/036698
well of PBS containing 2 g per 100 mL protease-free bovine serum albumin
("PBS/BSA") for
2 hours at 4°C. The plate is then washed 3 times with PBS by repeatedly
adding 200 pL per
well of PBS to each well of the, plate and then dumping the contents of the
plate into a waste
container and tapping the plate gently on a dry surface.
(2) Biotinylated PL peptides (or candidate PL peptides, e.g. see TABLE
2) are immobilized on the surface of wells of the plate by addition of 50 ~L
per well of 0.4
~M peptide in PBSlBSA for 30 minutes at 4°C. Usually, each different
peptide is added to at
least eight different wells so that multiple measurements (e.g. duplicates and
also
measurements using different (3ST/PDZ-domain fusion proteins and a GST alone
negative
control) can be made, and also additional negative control wells are prepared
in which no
peptide is immobilized. Following immobilization of the PL peptide on the
surface, the plate
is washed 3 times with PBS.
(3) GSTIPDZ-domain fusion protein (prepared as described supra) is
allowed to react with the surface by addition of 50 ~L per well of a solution
containing 5
pg/mL GST/PDZ-domain fusion protein in PBS/BSA for 2 hours at 4°C. As a
negative
control, GST alone (i.e, not a fusion protein) is added to specified wells,
generally at least 2
wells (i.e. duplicate measurements) for each immobilized peptide. After the 2
hour reaction,
the plate is washed 3 times with PBS to remove unbound fusion protein.
(4) The binding of the GST/PDZ-domain fusion protein to the avidin-
biotinylated peptide surface can be detected using a variety of methods, and
detectors known
in the art. In one embodiment, 50 ~L per well of an anti-GST antibody in
PBS/BSA (e.g. 2.5
~g/mL of polyclonal goat-anti-GST antibody, Pierce) is added to the plate and
allowed to
react for 20 minutes at 4°C. The plate is washed 3 times with PBS and a
second, detectably
labeled antibody is added. In one embodiment, 50 pL per well of 2.5 p,g/mL of
horseradish
peroxidase (HRP)-conjugated polyclonal rabbit anti-goat immunoglobulin
antibody is added
to the plate and allowed to react for 20 minutes at 4°C. The plate is
washed 5 times with SO
mM Tris pH 8.0 containing 0.2% Tween 20, and developed by addition of 100 ~L
per well of
HRP-substrate solution (TMB, Dako) for 20 minutes at room temperature (RT).
The reaction
of the HRP and its substrate is terminated by the addition of 100 ~L per well
of 1M sulfuric
acid and the optical density (O.D.) of each well of the plate is read at 450
nm.
(5) Specific binding of a PL peptide and a PDZ-domain polypeptide is
detected by comparing the signal from the wells) in which the PL peptide and
PDZ domain
polypeptide are combined with the background signal(s). The background signal
is the signal
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WO 2004/045535 PCT/US2003/036698
found in the negative controls. Typically a specific or selective reaction
will be at least twice
background signal, more typically more than 5 times background, and most
typically 10 or
more times the background signal. In addition, a statistically significant
reaction will involve
multiple measurements of the reaction with the signal and the background
differing by at
least two standard errors, more typically four standard errors, and most
typically six or more
standard errors. Correspondingly, a statistical test (e.g, a T-test) comparing
repeated
measurements of the signal with repeated measurements of the background will
result in a p-
value < 0.05, more typically a p-value < 0.01, and most typically a p-value <
0.001 or less.
As noted, in an embodiment of the "A" assay, the signal from binding of a
GSTIPDZ-domain fusion protein to an avidin surface not exposed to (i.e. not
covered with)
the PL peptide is one suitable negative control (sometimes referred to as
"B"). The signal
from binding of GST polypeptide alone (i.e. not a fusion protein) to an avidin-
coated surface
that has been exposed to (i.e. covered with) the PL peptide is a second
suitable negative
control (sometimes referred to as "B2"). Because all measurements are done in
multiples (i.e.
at least duplicate) the arithmetic mean (or, equivalently, average) of several
measurements is
used in determining the binding, and the standard error of the mean is used in
determining the
probable error in the measurement of the binding. The standard error of the
mean of N
measurements equals the square root of the following: the sum of the squares
of the
difference between each measurement and the mean, divided by the product of
(N) and (N-1).
Thus, in one embodiment, specific binding of the PDZ protein to the plate-
bound PL peptide
is determined by comparing the mean signal ("mean S") and standard error of
the signal
("SE") for a particular PL-PDZ combination with the mean Bl and/or mean B2.
2) "G Assav"-Detection of PDZ-Lig-and Binding Using Immobilized PDZ-
Domain Fusion Polypeptide
In one aspect, the invention provides an assay in which a GST/PDZ fusion
protein is immobilized on a surface ("G" assay). The binding of labeled PL
peptide (e.g., as
listed in TABLE 2) to this surface is then measured. In a preferred
embodiment, the assay is
carried out as follows:
(1) A PDZ-domain polypeptide is bound to a surface, e.g. a protein binding
surface. In a preferred embodiment, a GST/PDZ fusion protein containing one or
more PDZ
domains is bound to a polystyrene 96-well plate. The GST/PDZ fusion protein
can be bound
to the plate by any of a variety of standard methods known to one of skill in
the art, although
some care must be taken that the process of binding the fusion protein to the
plate does not
CA 02505479 2005-05-10
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alter the ligand-binding properties of the PDZ domain. In one embodiment, the
GST/PDZ
fusion protein is bound via an anti-GST antibody that is coated onto the 96-
well plate.
Adequate binding to the plate can be achieved when:
a. 100 pL per well of 5 ~g/mL goat anti-GST polyclonal antibody
(Pierce) in PBS is added to a polystyrene 96-well plate (e.g., Nunc Polysorb)
at 4°C for 12
hours.
b. The plate is blocked by addition of 200 pL per well of PBS/BSA for 2
hours at 4°C.
c. The plate is washed 3 times with PBS.
d. SO pL per well of 5 pg/mL GST/PDZ fusion protein) or, as a negative
control, GST polypeptide alone (i.e. not a fusion protein) in PBS/BSA is added
to the plate
for 2 hours at 4°C.
e. the plate is again washed 3 times with PBS.
(2) Biotinylated PL peptides are allowed to react with the surface by
addition of 50 ~L per well of 20 ~M solution of the biotinylated peptide in
PBS/BSA for 10
minutes at 4°C, followed by an additional 20 minute incubation at
25°C. The plate is washed
3 times with ice cold PBS.
(3) The binding of the biotinylated peptide to the GST/PDZ fusion protein
surface can be detected using a variety of methods and detectors known to one
of skill in the
art. In one embodiment, 100 ~L per well of 0.5 ~g/mL streptavidin-horse radish
peroxidase
(HRP) conjugate dissolved in BSA/PBS is added and allowed to react for 20
minutes at 4°C.
The plate is then washed 5 times with 50 mM Tris pH 8.0 containing 0.2% Tween
20, and
developed by addition of 100 ~L per well of HRP-substrate solution (TMB, Dako)
for 20
mW utes at room temperature (RT). The reaction of the HRP and its substrate is
terminated
by addition of 100 pL per well of 1 M sulfuric acid, and the optical density
(O.D.) of each
well of the plate is read at 450 um.
(4) Specific binding of a PL peptide and a PDZ domain polypeptide is
determined by comparing the signal from the wells) in which the PL peptide and
PDZ
domain polypeptide are combined, with the background signal(s). The background
signal is
the signal found in the negative control(s). Typically a specific or selective
reaction will be
at least twice background signal, more typically more than 5 times background,
and most
typically 10 or more times the background signal. In addition, a statistically
significant
reaction will involve multiple measurements of the reaction with the signal
and the
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background differing by at least two standard errors, more typically four
standard errors, and
most typically six or more standard errors. Correspondingly, a statistical
test (e.g. a T-test)
comparing repeated measurements of the signal with -repeated measurements of
the
background will result in a p-value < 0.05, more typically a p-value < 0.01,
and most
typically a p-value < 0.001 or less. As noted, in an embodiment of the "G"
assay, the signal
from binding of a given PL peptide to immobilized (surface bound) GST
polypeptide alone is
one suitable negative control (sometimes referred to as "B 1 "). Because all
measurement are
done in multiples (i.e. at least duplicate) the arithmetic mean (or,
equivalently, average.) of
several measurements is used in determining the binding, and the standard
error of the mean
is used in determining the probable error in the measurement of the binding.
The standard
error of the mean of N measurements equals the square root of the following:
the sum of the
squares of the difference between each measurement and the mean, divided by
the product of
(N) and (N-1). Thus, in one embodiment, specific binding of the PDZ protein to
the
platebound peptide is determined by comparing the mean signal ("mean S") and
standard
error of the signal ("SE") for a particular PL-PDZ combination with the mean
B1.
i) "G' assay" and "G" assay"
Two specific modifications of the specific conditions described supra for the
"G assay" are particularly useful. The modified assays use lesser quantities
of labeled PL
peptide and have slightly different biochemical requirements for detection of
PDZ-ligand
binding compared to the specific assay conditions described supra.
For convenience, the assay conditions described in this section are referred
to
as the "G' assay" and the "G" assay," with the specific conditions described
in the preceding
section on G assays being referred to as the "G° assay." The "G' assay"
is identical to the
"G° assay" except at step (2) the peptide concentration is 10 uM
instead of 20 uM. This
results in slightly lower sensitivity for detection of interactions with low
affinity and/or rapid
dissociation rate. Correspondingly, it slightly increases the certainty that
detected
interactions are of sufficient affinity and half life to be of biological
importance and useful
therapeutic targets.
The "G" assay" is identical to the "G° assay" except that at step
(2) the
peptide concentration is 1 pM instead of 20 ~M and the incubation is performed
for 60
minutes at 25°C (rather than, e.g., 10 minutes at 4°C followed
by 20 minutes at 25°C). This
results in lower sensitivity for interactions of low affinity, rapid
dissociation rate, and/or
affinity that is less at 25°C than at 4°C. Interactions will
have lower affinity at 25°C than at
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4°C if (as we have found to be generally true for PDZ-ligand binding)
the reaction entropy is
negative (i.e. the entropy of the products is less than the entropy of the
reactants). In contrast,
the PDZ-PL binding signal may be similar in the "G" assay" and the "G°
assay" for
interactions of slow association and dissociation rate, as the PDZ-PL complex
will
accumulate during the longer incubation of the "G" assay." Thus comparison of
results of
the "G" assay" and the "G° assay" can be used to estimate the relative
entropies, enthalpies,
and kinetics of different PDZ-PL interactions. (Entropies and enthalpies are
related to
binding affinity by the equations delta G = RT In (Kd) = delta H - T delta S
where delta G,
H, and S are the reaction free energy, enthalpy, and entropy respectively, T
is the temperature
in degrees Kelvin, R is the gas constant, and Kd is the equilibrium
dissociation constant). In
particular, interactions that are detected only or much more strongly in the
"G° assay"
generally have a rapid dissociation rate at 25°C (tl/2 < 10 minutes)
and a negative reaction
entropy, while interactions that are detected similarly strongly in the "G"
assay" generally
have a slower dissociation rate at 25°C (tl/2 > 10 minutes). Rough
estimation of the
thermodynamics and kinetics of PDZ-PL interactions (as can be achieved via
comparison of
results of the "G° assay" versus the "G" assay" as outlined supra) can
be used in the design
of efficient inhibitors of the interactions. For example, a small molecule
inhibitor based on
the chemical structure of a PL that dissociates slowly from a given PDZ domain
(as
evidenced by similar binding in the "G" assay" as in the "G° assay")
may itself dissociate
slowly and thus be of high affinity.
In this manner, variation of the temperature and duration of step (2) of the
"G
assay" can be used to provide insight into the kinetics and thermodynamics of
the PDZ-ligand
binding reaction and into design of inhibitors of the reaction.
3) Assay Variations
As discussed supra, it will be appreciated that many of the steps in the above-
described assays can be varied, for example, various substrates can be used
for binding the
PL and PDZ-containing proteins; different types of PDZ containing fusion
proteins can be
used; different labels for detecting PDZ/PL interactions can be employed; and
different ways
of detection can be used.
The PL protein used in the assay is not intended to be limited to a 20 amino
acid peptide. Full length or partial protein may be used, either alone or as a
fusion protein.
For example, a GST-PL protein fusion may be bound to the anti-GST antibody,
with PDZ
protein added to the bound PL protein or peptide.
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The PDZ-PL detection assays can employ a variety of surfaces to bind the PL
and PDZ-containing proteins. For example, a surface can be an "assay plate"
which is
formed from a material (e.g. polystyrene) which optimizes adherence of either
the PL protein
or PDZ-containing protein thereto. Generally, the individual wells of the
assay plate will have
a high surface area to volume ratio and therefore a suitable shape is a flat
bottom well (where
the proteins of the assays are adherent). Other surfaces include, but are not
limited to,
polystyrene or glass beads, polystyrene or glass slides, and the like.
For example, the assay plate can be a "microtiter" plate. The term
"microtiter"
plate when used herein refers to a multiwell assay plate, e.g., having between
about 30 to 200
individual wells, usually 96 wells. Alternatively, high density arrays can be
used. Often, the
individual wells of the microtiter plate will hold a maximum volume of about
250 ul.
Conveniently, the assay plate is a 96 well polystyrene plate (such as that
sold by Becton
Dickinson Labware, Lincoln Park, N.J.), which allows for automation and high
throughput
screening. Other surfaces include polystyrene microtiter ELISA plates such as
that sold by
Nunc Maxisorp, Inter Med, Denmark. Often, about SO ul to 300 ul, more
preferably 100 ul to
200 ul, of an aqueous sample comprising buffers suspended therein will be
added to each
well of the assay plate.
The detectable labels of the invention can be any detectable compound or
composition which is conjugated directly or indirectly with a molecule (such
as described
above). The label can be detectable by itself (e.g., radioisotope labels or
fluorescent labels)
or, in the case of an enzymatic label, can catalyze a chemical alteration of a
substrate
compound or composition which is detectable. The prefeared label is an
enzymatic one
which catalyzes a color change of a non-radioactive color reagent.
Sometimes, the label is indirectly conjugated with the antibody. One of skill
is
aware of various techniques for indirect conjugation. For example, the
antibody can be
conjugated with biotin and any of the categories of labels mentioned above can
be conjugated
with avidin, or vice versa (see also "A" and "G" assay above). Biotin binds
selectively to
avidin and thus, the label can be conjugated with the antibody in this
indirect manner. See,
Ausubel, supra, for a review of techniques involving biotin-avidin conjugation
and similar
assays. Alternatively, to achieve indirect conjugation of the label with the
antibody, the
antibody is conjugated with a small hapten (e.g. digoxin) and one of the
different types of
labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-
digoxin
antibody). Thus, indirect conjugation of the label with the antibody can be
achieved.
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Assay variations can include different washing steps. By "washing" is meant
exposing the solid phase to an aqueous solution (usually a buffer or cell
culture media) in
such a way that unbound material (e.g., non-adhering cells, non-adhering
capture agent,
unbound ligand, receptor, receptor construct, cell lysate, or HRP antibody) is
removed
therefrom. To reduce background noise, it is convenient to include a detergent
(e.g., Triton
X) in the washing solution. Usually, the aqueous washing solution is decanted
from the wells
of the assay plate following washing. Conveniently, washing can be achieved
using an
automated washing device. Sometimes, several washing steps (e.g., between
about 1 to 10
washing steps) can be required.
Various buffers can also be used in PDZ-PL detection assays. For example,
various blocking buffers can be used to reduce assay background. The term
"blocking
buffer" refers to an aqueous, pH buffered solution containing at least one
blocking compound
which is able to bind to exposed surfaces of the substrate which are not
coated with a PL or
PDZ-containing protein. The blocking compound is normally a protein such as
bovine serum
albumin (BSA), gelatin, casein or milk powder and does not cross-react with
any of the
reagents in the assay. The block buffer is generally provided at a pH between
about 7 to 7.5
and suitable buffering agents include phosphate and TRIS.
Various enzyme-substrate combinations can also be utilized in detecting PDZ-
PL interactions. Examples of enzyme-substrate combinations include, for
example:
(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,
wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. orthophenylene
diamine
[OPD] or 3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]) (as described
above).
(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as
chromogenic substrate.
(iii) 13-D-galactosidase (13 D-Gal) with a chromogenic substrate (e.g. p-
nitrophenyl- 13-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-
f3-D-
galactosidase.
Numerous other enzyme-substrate combinations are available to those skilled
in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and
4,318,980, both of
which are herein incorporated by reference.
Further, it will be appreciated that, although, for convenience, the present
discussion primarily refers antagonists of PDZ-PL interactions, agonists of
PDZ-PL
interactions can be identified using the methods disclosed herein or readily
apparent
variations thereof.
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E. Detecting PDZ-PL Interactions
The present inventors were able in part to identify the interactions
summarized
in TABLE 3 and TABLE 8. by developing new high throughput screening assays
which are
described supra. Various other assay formats known in the art can be used to
select ligands
that are specifically reactive with a particular protein. For example, solid-
phase ELISA
immunoassays, immunoprecipitation, Biacore, Fluorescence Polarization (FP),
Fluorescence
Resonance Energy Transfer (FRET) and Western blot assays can be used to
identify peptides
that specifically bind PDZ-domain polypeptides. As discussed supra, two
different,
complementary assays were developed to detect PDZ-PL interactions. In each,
one binding
partner of a PDZ-PL pair is immobilized, and the ability of the second binding
partner to bind
is determined. These assays, which are described supra, can be readily used to
screen for
hundreds to thousands of potential PDZ-ligand interactions in a few hours.
Thus these assays
can be used to identify yet more novel PDZ-PL interactions in neuronal cells.
In addition,
they can be used to identify antagonists of PDZ-PL interactions (see infra).
In various embodiments, fusion protein are used in the assays and devices of
the invention. Methods for constructing and expressing fusion proteins are
well known.
Fusion proteins generally are described in Ausubel et al., supra, Kroll et
al., 1993, DNA Cell.
Biol. 12:441, and Imai et al., 1997, Cell 91:521-30. Usually, the fusion
protein includes a
domain to facilitate immobilization of the protein to a solid substrate ("an
immobilization
domain"). Often, the immobilization domain includes an epitope tag (i.e., a
sequence
recognized by a antibody, typically a monoclonal antibody) such as
polyhistidine (Bush et al,
1991, J. Biol Chem 266:13811-14), SEAP (Berger et al, 1988, Gene 66:1-10), or
M1 and M2
flag (see, e.g, U.S. Pat. Nos. 5,011,912; 4,851,341; 4,703,004; 4,782,137). In
an
embodiment, the immobilization domain is a GST coding region. It will be
recognized that,
in addition to the PDZ-domain and the particular residues bound by an
immobilized antibody,
protein A, or otherwise contacted with the surface, the protein (e.g., fusion
protein), will
contain additional residues. In some embodiments these are residues naturally
associated
with the PDZ-domain (i.e., in a particular PDZ-protein) but they may include
residues of
synthetic (e.g., poly(alanine)) or heterologous origin (e.g., spacers of,
e.g., between 10 and
300 residues).
PDZ domain-containing polypeptide used in the methods of the invention
(e.g., PDZ fusion proteins) of the invention are typically made by (1)
constructing a vector
(e.g., plasmid, phage or phagemid) comprising a polynucleotide sequence
encoding the
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desired polypeptide, (2) introducing the vector into an suitable expression
system (e.g., a
prokaryotic, insect, mammalian, or cell free expression system), (3)
expressing the fusion
protein and (4) optionally purifying the fusion protein.
In one embodiment, expression of the protein comprises inserting the coding
S sequence into an appropriate expression vector (i.e., a vector that contains
the necessary
elements for the transcription and translation of the inserted coding sequence
required for the
expression system employed, e.g., control elements including enhancers,
promoters,
transcription terminators, origins of replication, a suitable initiation codon
(e.g., methionine),
open reading frame, and translational regulatory signals (e.g., a ribosome
binding site, a
termination codon and a polyadenylation sequence. Depending on the vector
system and host
utilized, any number of suitable transcription and translation elements,
including constitutive
and inducible promoters, can be used.
The coding sequence of the fusion protein includes a PDZ domain and an
immobilization domain as described elsewhere herein. Polynucleotides encoding
the amino
acid sequence for each domain can be obtained in a variety of ways known in
the art;
typically the polynucleotides are obtained by PCR amplification of cloned
plasmids, cDNA
libraries, and cDNA generated by reverse transcription of RNA, using primers
designed
based on sequences determined by the practitioner or, more often, publicly
available (e.g.,
through GenBank). The primers include linker regions (e.g., sequences
including restriction
24 sites) to facilitate cloning and manipulation in production of the fusion
construct. The
polynucleotides corresponding to the PDZ and immobilization regions are joined
in-frame to
produce the fusion protein-encoding sequence.
The fusion proteins of the invention may be expressed as secreted proteins
(e.g., by including the signal sequence encoding DNA in the fusion gene; see,
e.g., Lui et al,
1993, PNAS USA, 90:8957-61) or as nonsecreted proteins.
In some embodiments, the PDZ-containing proteins are immobilized on a
solid surface. The substrate to which the polypeptide is bound may in any of a
variety of
forms, e.g., a microtiter dish, a test tube, a dipstick, a microcentrifuge
tube, a bead, a
spinnable disk, and the like. Suitable materials include glass, plastic (e.g.,
polyethylene, PVC,
polypropylene, polystyrene, and the like), protein, paper, carbohydrate, lipip
monolayer or
supported lipid bilayer, and other solid supports. Other materials that may be
employed
include ceramics, metals, metalloids, semiconductive materials, cements and
the like.
In some embodiments, the fusion proteins are organized as an array. The term
"array," as used herein, refers to an ordered arrangement of immobilized
fusion proteins, in
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which particular different fusion proteins (i.e., having different PDZ
domains) are located at
different predetermined sites on the substrate. Because the location of
particular fusion
proteins on the array is known, binding at that location can be correlated
with binding to the
PDZ domain situated at that location. Immobilization of fusion proteins on
beads
(individually or in groups) is another particularly useful approach. In one
embodiment,
individual fusion proteins are immobilized on beads. 1n one embodiment,
mixtures of
distinguishable beads are used. Distinguishable beads are beads that can be
separated from
each other on the basis of a property such as size, magnetic property, color
(e.g., using
FACS) or affinity tag (e.g., a bead coated with protein A can be separated
.from a bead not
coated with protein A by using IgG affinity methods). Binding to particular
PDZ domain
may be determined; similarly, the effect of test compounds (i.e., agonists and
antagonists of
binding) may be determined.
Methods for immobilizing proteins are known, and include covalent and non-
covalent methods. One suitable immobilization method is antibody-mediated
immobilization. According to this method, an antibody specific for the
sequence of an
"immobilization domain" of the PDZ-domain containing protein is itself
immobilized on the
substrate (e.g., by adsorption). One advantage of this approach is that a
single antibody may
be adhered to the substrate and used for immobilization of a number of
polypeptides (sharing
the same immobilization domain). For example, an immobilization domain
consisting of
poly-histidine (Bush et al, 1991, J. Biol Chem 266:13811-14) can be bound by
an anti-
histidine monoclonal antibody (R&D Systems, Minneapolis, MN); an
immobilization domain
consisting of secreted alkaline phosphatase ("SEAP") (Berger et al, 1988, Gene
66:1-10) can
be bound by anti-SEAP (Sigma Chemical Company, St. Louis, MO); an
immobilization
domain consisting of a FLAG epitope can be bound by anti-FLAG. Other ligand-
antiligand
immobilization methods are also suitable (e.g., an immobilization domain
consisting of
protein A sequences (Harlow and Lane, 1988, Antibodies A Laboratory Manual,
Cold Spring
Harbor Laboratory; Sigma Chemical Co., St. Louis, MO) can be bound by IgG; and
an
immobilization domain consisting of streptavidin can be bound by biotin
(Harlow & Lane,
supra; Sigma Chemical Co., St. Louis, MO). In a preferred embodiment, the
immobilization
domain is a GST moiety, as described herein.
When antibody-mediated immobilization methods are used, glass and plastic
are especially useful substrates. The substrates may be printed with a
hydrophobic (e.g.,
Teflon) mask to form wells. Preprinted glass slides with 3, 10 and 21 wells
per 14.5 cmZ
slide "working area" are available from, e.g., SPI Supplies, West Chester, PA;
also see U.S.
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CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Pat. No. 4,011,350). In certain applications, a large format (12.4 em x 8.3
cm) glass slide is
printed in a 96 well format is used; this format facilitates the use of
automated liquid handling
equipment and utilization of 96 well format plate readers of various types
(fluorescent,
colorimetric, scintillation). However, higher densities may be used (e.g.,
more than 10 or 100
polypeptides per cm2). See, e.g., MacBeath et al, 2000, Science 289:1760-63.
Typically, antibodies are bound to substrates (e.g., glass substrates) by
adsorption. Suitable adsorption conditions are well known in the art and
include incubation
of 0.5-SO~g/ml (e.g., 10 pg/ml) mAb in buffer (e.g., PBS, or SO to 300 mM
Tris, MOPS,
HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4°C) to 37°C and
from lhr to more than 24
hours.
Proteins may be covalently bound or noncovalently attached through
nonspecific bonding. If covalent bonding between a the fusion protein and the
surface is
desired, the surface will usually be polyfunctional or be capable of being
polyfunctionalized.
Functional groups which may be present on the surface and used for linking can
include
1 S carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups,
hydroxyl groups,
mercapto groups and the like. The manner of linking a wide variety of
compounds to various
surfaces is well known and is amply illustrated in the literature.
F. Results of PDZ-PL Interaction Assays
TABLE 3 shows the results of assays in which specific binding was detected
between NMDAR proteins and PDZ proteins using the "G"' assay described herein.
TABLE
8 summarizes. the results of interactions between a number of PL proteins with
nNOS.
TABLE 9 lists PL sequences that bind to the PDZ domain of PSD-95.
G. Measurement of PDZ-Ligand Binding Affinity
The "A" and "G" assays of the invention can be used to determine the
"apparent affinity" of binding of a PDZ ligand peptide to a PDZ-domain
polypeptide.
Apparent affinity is determined based on the concentration of one molecule
required to
saturate the binding of a second molecule (e.g., the binding of a ligand to a
receptor). Two
particularly useful approaches for quantitation of apparent affinity of PDZ-
ligand binding are
provided infra.
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WO 2004/045535 PCT/US2003/036698
Approach 1:
(1) A GST/PDZ fusion protein, as well as GST alone as a negative control, are
bound to a surface (e.g., a 96-well plate) and the surface blocked and washed
as described
supra for the "G" assay.
(2) 50 p,L per well of a solution of biotinylated PL peptide (e.g. as shown in
TABLE 2) is added to the surface in increasing concentrations in PBS/BSA (e.g.
at 0.1 pM,
0.33 pM, 1 ~M, 3.3 ~M, 10 pM, 33 pM, and 100 pM). In one embodiment, the PL
peptide is
allowed to react with the bound GST/PDZ fusion protein (as well as the GST
alone negative
control) for 10 minutes at 4°C followed by 20 minutes at 25°C.
The plate is washed 3 times
with ice cold PBS to remove unbound labeled peptide.
(3) The binding of the PL peptide to the immobilized PDZ-domain
polypeptide is detected as described supra for the "G" assay.
(4) For each concentration of peptide, the net binding signal is determined by
subtracting the binding of the peptide to GST alone from the binding of the
peptide to the
GST/PDZ fusion protein. The net binding signal is then plotted as a function
of ligand
concentration and the plot is fit (e.g. by using the Kaleidagraph software
package curve
fitting algorithm) to the following equation, where "Slgnaly;ga"d]" is the net
binding signal at
PL peptide concentration "[ligand]," "Kd" is the apparent affinity of the
binding event, and
"Saturation Binding" is a constant determined by the curve fitting algorithm
to optimize the
fit to the experimental data:
Slgnah~;ga"~~ = Saturation Binding x ([ligand] / ([ligand] + Kd))
For reliable application of the above equation it is necessary that the
highest
peptide ligand concentration successfully tested experimentally be greater
than, or at least
similar to, the calculated Kd (equivalently, the maximum observed binding
should be similar
to the calculated saturation binding). In cases where satisfying the above
criteria proves
difficult, an alternative approach (infra) can be used.
Approach 2:
(1) A fixed concentration of a PDZ-domain polypeptide and increasing
concentrations of a labeled PL peptide (labeled with, for example, biotin or
fluorescein, see
TABLE 2for representative peptide amino acid sequences) are mixed together in
solution and
allowed to react. In one embodiment, preferred peptide concentrations are 0.1
~M, 1 ~,M, 10
~M, 100 p.M, 1 mM. In various embodiments, appropriate reaction times can
range from 10
so
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WO 2004/045535 PCT/US2003/036698
minutes to 2 days at temperatures ranging from 4°C to 37°C. In
some embodiments, the
identical reaction can also be carried out using a non-PDZ domain-containing
protein as a
control (e.g., if the PDZ-domain polypeptide is fusion protein, the fusion
partner can be
used).
(2) PDZ-ligand complexes can be separated from unbound labeled peptide
using a variety of methods known in the ark. For example, the complexes can be
separated
using high performance size-exclusion chromatography (HPSEC, gel filtration)
(Rabinowitz
et al., 1998, Immunity 9:699), affinity chromatography(e.g. using glutathione
Sepharose
beads), and affinity absorption (e.g., by binding to an anti-GST-coated plate
as described
supra).
(3) The PDZ-ligand complex is detected based on presence of the label on the
peptide ligand using a variety of methods and detectors known to one of skill
in the art. For
example, if the label is fluorescein and the separation is achieved using
HPSEC, an in-line
fluorescence detector can be used. The binding can also be detected as
described supra for
the G assay.
(4) The PDZ-ligand binding signal is plotted as a function of ligand
concentration and the plot is fit. (e.g., by using the Kaleidagraph software
package curve
fitting algorithm) to the following equation, where "Signal~~;ga"a~" is the
binding signal at PL
peptide concentration "[ligand]," "Kd" is the apparent affinity of the binding
event, and
"Saturation Binding" is a constant determined by the curve fitting algorithm
to optimize the
fit to the experimental data:
Signah~;ga~d~ = Saturation Binding x ([ligand] / ([ligand + Kd))
Measurement of the affinity of a labeled peptide ligand binding to a PDZ-
domain polypeptide is useful because knowledge of the affinity (or apparent
affinity) of this
interaction allows rational design of inhibitors of the interaction with known
potency. The
potency of inhibitors in inhibition would'be similar to (i.e. within one-order
of magnitude of)
the apparent affinity of the labeled peptide ligand binding to the PDZ-domain.
Thus, in one aspect, the invention provides a method of determining the
apparent affinity of binding between a PDZ domain and a ligand by immobilizing
a
polypeptide comprising the PDZ domain and a non-PDZ domain on a surface,
contacting the
immobilized polypeptide with a plurality of different concentrations of the
ligand,
determining the amount of binding of the ligand to the immobilized polypeptide
at each of
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the concentrations of ligand, and calculating the apparent affinity of the
binding based on that
data. Typically, the polypeptide comprising the PDZ domain and a non-PDZ
domain is a
fusion protein. In one embodiment, the e.g., fusion protein is GST-PDZ fusion
protein, but
other polypeptides can also be used (e,g., a fusion protein including a PDZ
domain and any of
a variety of epitope tags, biotinylation signals and the like) so long as the
polypeptide can be
immobilized In an orientation that does not abolish the ligand binding
properties of the PDZ
domain, e.g, by tethering the polypeptide to the surface via the non-PDZ
domain via an anti-
domain antibody and leaving the PDZ domain as the free end. It was discovered,
for
example, reacting a PDZ-GST fusion polypeptide directly to a plastic plate
provided
suboptimal results. The calculation of binding affinity itself can be
determined using any
suitable equation (e.g., as shown supra; also see Cantor and Schimmel (1980)
BtoPFtYSICaL
CHEMISTRY WH Freeman & Co., San Francisco) or software.
Thus, in a preferred embodiment, the polypeptide is immobilized by binding
the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain
(e.g., an
anti-GST antibody when a GST-PDZ fusion polypeptide is used). In a preferred
embodiment, the step of contacting the ligand and PDZ-domain polypeptide is
carried out
under the conditions provided suprca in the description of the "G" assay. It
will be
appreciated that binding assays are conveniently carried out in multiwell
plates (e.g., 24-well,
96-well plates, or 384 well plates).
The present method has considerable advantages over other methods for
measuring binding affinities PDZ-PL affinities, which typically involve
contacting varying
concentrations of a GST-PDZ fusion protein to a ligand-coated surface. For
example, some
previously described methods for determining affinity (e.g., using immobilized
ligand and
GST-PDZ protein in solution) did not account for oligomerization state of the
fusion proteins
used, resulting in potential errors of more than an order of magnitude.
Although not sufficient for quantitative measurement of PDZ-PL binding
affinity, an estimate of the relative strength of binding of different PDZ-PL
pairs can be made
based on the absolute magnitude of the signals observed in the "G assay." This
estimate will
reflect several factors, including biologically relevant aspects of the
interaction, including the
affinity and the dissociation rate. For comparisons of different ligands
binding to a given
PDZ domain-containing protein, differences in absolute binding signal likely
relate primarily
to the affinity andlor dissociation rate of the interactions of interest.
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H. Assays to Identify Novel PDZ Domain Binding Moieties and to Identify
Modulators of PDZ Protein-PL Protein Binding
Although described supra primarily in terms of identifying interactions
between PDZ-domain polypeptides and PL proteins, the assays described supra
and other
S assays can also be used to identify the binding of other molecules (e.g.,
peptide mimetics,
small molecules, and the like) to PDZ domain sequences. For example, using the
assays
disclosed herein, combinatorial and other libraries of compounds can be
screened, e.g., for
molecules that specifically bind to PDZ domains. Screening of libraries can be
accomplished
by any of a variety of commonly known methods. See, e.g., the following
references, which
disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp.
Med. Biol.
251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992;
BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acccd. Sci. USA
89:5393-
5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-
580; Bock et al.,
1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA
89:6988-6992;
Ellington et al., 1992, Nature 355:850-852; U.S. Patent No. 5,096,815, U.S.
Patent No.
5,223,409, and U.S. Patent No. 5,198,346, all to Ladner et al.; Rebar and
Pabo, 1993, Science
263:671-673; and PCT Publication No. WO 94/18318.
In a specific embodiment, screening can be carried Ollt by contacting the
library members with a PDZ-domain polypeptide immobilized on a solid support
(e.g. as
described supra in the "G" assay) and harvesting those library members that
bind to the
protein. Examples of such screening methods, termed "panning" techniques are
described by
way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al.,
1992,
BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references
cited
hereinabove.
In another embodiment, the two-hybrid system for selecting interacting
proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al.,
1991, Proc. Natl.
Acad. Sci. USA 88:9578-9582) can be used to identify molecules that
specifically bind to a
PDZ domain-containing protein. Furthermore, the identified molecules are
further tested for
their ability to inhibit transmembrane receptor interactions with a PDZ
domain.
In one aspect of the invention, antagonists of an interaction between a PDZ
protein and a PL protein are identified. In one embodiment, a modification of
the "A" assay
described supra is used to identify antagonists. In one embodiment, a
modification of the
"G" assay described supra is used to identify antagonists.
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In one embodiment, screening assays are used to detect molecules that
specifically bind to PDZ domains. Such molecules are useful as agonists or
antagonists of
PDZ-protein-mediated cell function (e.g., cell activation, e.g., T cell
activation, vesicle
transport, cytokine release, growth factors, transcriptional changes,
cytoskeleton
rearrangement, cell movement, chemotaxis, and the like). In one embodiment,
such assays
are performed to screen for leukocyte activation inhibitors for drug
development. The
invention thus provides assays to detect molecules that specifically bind to
PDZ domain-
containing proteins. For example, recombinant cells expressing PDZ domain-
encoding
nucleic acids can be used to produce PDZ domains in these assays and to screen
for
molecules that bind to the domains. Molecules are contacted with the PDZ
domain (or
fragment thereof) under conditions conducive to binding, and then molecules
that specifically
bind to such domains are identified. Methods that can be used to carry out the
foregoing are
commonly known in the art.
It will be appreciated by the ordinarily skilled practitioner that, in one
embodiment, antagonists are identified by conducting the A or G assays in the
presence and
absence of a known or candidate antagonist. When decreased binding is observed
in the
presence of a compound, that compound is identified as an antagonist.
Increased binding in
the presence of a compound signifies that the compound is an agonist.
For example, in one assay, a test compound can be identified as an inhibitor
(antagonist) of binding between a PDZ protein and a PL protein by contacting a
PDZ domain
polypeptide and a PL peptide or protein in the presence and absence of the
test compound,
under conditions in which they would (but for the presence of the test
compound) form a
complex, and detecting the formation of the complex in the presence and
absence of the test
compound. It will be appreciated that less complex formation in the presence
of the test
compound than in the absence of the compound indicates that the test compound
is an
inhibitor of a PDZ protein -PL protein binding.
In one embodiment, the "G" assay is used in the presence or absence of an
candidate inhibitor. In one embodiment, the "A" assay is used in the presence
or absence of a
candidate inhibitor.
In one embodiment (in which a G assay is used), one or more PDZ domain-
containing GST-fusion proteins are bound to the surface of wells of a 96-well
plate as
described supra (with appropriate controls including nonfusion GST protein).
All fusion
proteins are bound in multiple wells so that appropriate controls and
statistical analysis can be
done. A test compound in BSA/PBS (typically at multiple different
concentrations) is added
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to wells. Immediately thereafter, 30 uL of a detectably labeled (e.g.,
biotinylated) PL peptide
or protein known to bind to the relevant PDZ domain (sec, e.g., TABLE 2) is
added in each
of the wells at a final concentration of, e.g., between about 2 pM and about
40 pM, typically
pM, 15 pM, or 25 pM. This mixture is then allowed to react with the PDZ fusion
protein
5 bound to the surface for 10 minutes at 4°C followed by 20 minutes at
25°C. The surface is
washed free of unbound PL polypeptide three times with ice cold PBS and the
amount of
binding of the polypeptide in the presence and absence of the test compound is
determined.
Usually, the level of binding is measured for each set of replica wells (e.g.
duplicates) by
subtracting the mean GST alone background from the mean of the raw measurement
of
polypeptide binding in these wells.
In an alternative embodiment, the A assay is carried out in the presence or
absence of a test candidate to identify inhibitors of PL-PDZ interactions.
If assays are conducted in the presence of test compound and compared
against binding in the absence of test compound, then the assay can be
conducted to
determine if the difference between binding in the presence and absence of the
test compound
is a statistically significant difference.
In certain screening assays, assays are conducted to identify compounds that
can inhibit a binding interaction between a NMDA receptor protein and a PDZ
listed in
TABLE 7. In other screening assays involve screening to identify an inhibitor
that interferes
with binding between a NMDA receptor protein (e.g., NMDAR2) and a PDZ listed
in
TABLE 7 other than PSD-95.
In one embodiment, a test compound is determined to be a specific inhibitor of
the binding of the PDZ domain (P) and a PL (L) sequence when, at a test
compound
concentration of less than or equal to 1 mM (e.g., less than or equal to: 500
~M, 100 ~M, 10
wM, 1 pM, 100 nM or 1 nM) the binding of P to L in the presence of the test
compound less
than about SO% of the binding in the absence of the test compound, (in various
embodiments,
less than about 25%, less than about 10%, or less than about 1 %). Preferably,
the net signal
of binding of P to L in the presence of the test compound plus six (6) times
the standard error
of the signal in the presence of the test compound is less than the binding
signal in the
absence of the test compound.
In one embodiment, assays for an inhibitor are carried out using a single PDZ
protein-PL protein pair (e.g., a PDZ domain fusion protein and a PL peptide or
protein). In a
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related embodiment, the assays are carried out using a plurality of pairs,
such as a plurality of
different pairs listed in TABLES 3, 8 and 9.
In some embodiments, it is desirable to identify compounds that, at a given
concentration, inhibit the binding of one PL-PDZ pair, but do not inhibit (or
inhibit to a lesser
degree) the binding of a specified second PL-PDZ pair. These antagonists can
be identified
by carrying out a series of assays using a candidate inhibitor and different
PL-PDZ pairs (e.g.,
as shown in TABLES 3, 8 and 9) and comparing the results of the assays. All
such pairwise
combinations are contemplated by the invention (e.g., test compound inhibits
binding of PL,
to PDZ1 to a greater degree than it inhibits binding of PL, to PDZZ or PLZ to
PDZZ).
Importantly, it will be appreciated that, based on the data provided in TABLES
3, 8 and 9
and disclosed herein (and additional data that can be generated using the
methods described
herein) inhibitors with different specificities can readily be designed.
For example, according to the invention, the Ki ("potency") of an inhibitor of
a PDZ-PL interaction can be determined. Ki is a measure of the concentration
of an inhibitor
required to have a biological effect. For example, administration of an
inhibitor of a PDZ-PL
interaction in an amount sufficient to result in an intracellular inhibitor
concentration of at
least between about 1 and about 100 Ki is expected to inhibit the biological
response
mediated by the target PDZ-PL interaction. In one aspect of the invention, the
Kd
measurement of PDZ-PL binding as determined using the methods supra is used in
determining Ki.
Thus, in one aspect, the invention provides a method of determining the
potency (Ki) of an inhibitor or suspected inhibitor of binding between a PDZ
domain and a
ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ
domain on
a surface, contacting the immobilized polypeptide with a plurality of
different mixtures of the
ligand and inhibitor, wherein the different mixtures comprise a fixed amount
of ligand and
different concentrations of the inhibitor, determining the amount of ligand
bound at the
different concentrations of inhibitor, and calculating the Ki of the binding
based on the
amount of ligand bound in the presence of different concentrations of the
inhibitor. In an
embodiment, the polypeptide is immobilized by binding the polypeptide to an
immobilized
immunoglobulin that binds the non-PDZ domain. This method, which is based on
the "G"
assay described supra, is particularly suited for high-throughput analysis of
the Ki for
inhibitors of PDZ-ligand interactions. Further, using this method, the
inhibition of the PDZ-
ligand interaction itself is measured, without distortion of measurements by
avidity effects.
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Typically, at least a portion of the ligand is detectably labeled to permit
easy
quantitation of ligand binding.
It will be appreciated that the concentration of ligand and concentrations of
inhibitor are selected to allow meaningful detection of inhibition. Thus, the
concentration of
the ligand whose binding is to be blocked is close to or less than its binding
affinity (e.g.,
preferably less than the Sx Kd of the interaction, more preferably less than
2x Kd, most
preferably less than lx Kd). Thus, the ligand is typically present at a
concentration of less
than 2 Kd (e.g., between about 0.01 Kd and about 2 Kd) and the concentrations
of the test
inhibitor typically range from 1 nM to 100 pM (e.g. a 4-fold dilution series
with highest
concentration 10 ~M or 1 mM). In a preferred embodiment, the Kd is determined
using the
assay disclosed supra.
The Ki of the binding can be calculated by any of a variety of methods
routinely used in the art, based on the amount of ligand bound in the presence
of different
concentrations of the inhibitor. in an illustrative embodiment, for example, a
plot of labeled
ligand binding versus inhibitor concentration is fit to the equation:
sinhibitor = So*Ki/([I]+Ki)
where Sinhibitor is the signal of labeled ligand binding to immobilized PDZ
domain in the
presence of inhibitor at concentration [I] and So is the signal in the absence
of inhibitor (i.e.,
[I] = 0). Typically [I] is expressed as a molar concentration.
In another aspect of the invention, an enhancer (sometimes referred to as,
augmentor or agonist) of binding between a PDZ domain and a ligand is
identified by
immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a
surface,
contacting the immobilized polypeptide with the ligand in the presence of a
test agent and
determining the amount of ligand bound, and comparing the amount of ligand
bound in the
presence of the test agent with the amount of ligand bound by the polypeptide
in the absence
of the test agent. At least two-fold (often at least 5-fold) greater binding
in the presence of
the test agent compared to the absence of the test agent indicates that the
test agent is an agent
that enhances the binding of the PDZ domain to the ligand. As noted supra,
agents that
enhance PDZ-ligand interactions are useful for disruption (dysregulation) of
biological events
requiring normal PDZ-ligand function (e.g., cancer cell division and
metastasis, and
activation and migration of immune cells).
The invention also provides methods for determining the "potency" or
"~nhancer~~ of an enhancer of a PDZ- ligand interaction. For example,
according to the
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invention, the Ke"ha"cer of an enhancer of a PDZ-PL interaction can be
determined, e.g., using
the Kd of PDZ-PL binding as determined using the methods described supra.
ICe"nancer is a
measure of the concentration of an enhancer expected to have a biological
effect. For
example, administration of an enhancer of a PDZ-PL interaction in an amount
sufficient to
result in an intracellular inhibitor concentration of at least between about
0.1 and about 100
Kenhancer (e.g., between about 0.5 and about SO Kenhancer) is expected to
disrupt the biological
response mediated by the target PDZ-PL interaction.
Thus, in one aspect the invention provides a method of determining the
potency (Kenhan~er) of an enhancer or suspected enhancer of binding between a
PDZ domain
and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-
PDZ
domain on a surface, contacting the immobilized polypeptide with a plurality
of different
mixtures of the ligand and enhancer, wherein the different mixtures comprise a
fixed amount
of ligand, at least a portion of which is detectably labeled, and different
concentrations of the
enhancer, determining the amount of ligand bound at the different
concentrations of
1 S enhancer, and calculating the potency (Kenhancer) of the enhancer from the
binding based on
the amount of ligand bound in the presence of different concentrations of the
enhancer.
Typically, at least a portion of the ligand is detectably labeled to permit
easy quantitation of
ligand binding. This method, which is based on the "G" assay described supra,
is particularly
suited for high-throughput analysis of the Ke~h~ncer for enhancers of PDZ-
ligand interactions.
It will be appreciated that the concentration of ligand and concentrations of
enhancer are selected to allow meaningful detection of enhanced binding. Thus,
the ligand is
typically present at a concentration of between about 0.01 Kd and about 0.5 Kd
and the
concentrations of the test agent/enhancer typically range from 1 nM to 1 mM
(e.g. a 4-fold
dilution series with highest concentration 10 pM or 1 mM). In a preferred
embodiment, the
Kd is determined using the assay disclosed supra.
The potency of the binding can be determined by a variety of standard
methods based on the amount of ligand bound in the presence of different
concentrations of
the enhancer or augmentor. For example, a plot of labeled ligand binding
versus enhancer
concentration can be fit to the equation:
s([E]) - s(0) + (S(~)*(Denhancerl)*[E]i(CE]+ Ke~,n~"~~~~)
where "Kenna"ccr" is the potency of the augmenting compound, and "Denhancer"
is the fold-
increase in binding of the labeled ligand obtained with addition of saturating
amounts of the
enhancing compound, [E] is the concentration of the enhancer. It will be
understood that
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saturating amounts are the amount of enhancer such that further addition does
not
significantly increase the binding signal. Knowledge of "Ke"~,dn~~,." is
useful because it
describes a concentration of the augmenting compound in a target cell that
will result in a
biological effect due to dysregulation of the PDZ-PL interaction. Typical
therapeutic
S concentrations are between about 0.1 and about 100 K~nhance~-.
V. Validation of Binding Assay
Compounds identified in the foregoing binding assays can be further analyzed
using a variety of biological assays to confirm that the ability of the
compound to inhibit a
PDZ:PL protein interaction actually inhibits a cellular activity correlated
with the PDZ:PL
binding interaction. Alternatively, these assays can be used directly to assay
the activity of a
potential inhibitory compound without conducting a binding assay beforehand.
These assays
can be conducted using various in vitro assays, or in vivo assays using
various appropriate
animal model systems.
The PDZ:PL binding interactions described herein include those involved in
various biological activities in neurons. As already noted, one set of
cellular activities of
interest are those associated with various types of neurological disorders or
injury, such as
cellular responses associated with stroke and ischemia. Because neurological
injury is often
associated with cell death, apoptosis and excitotoxicity responses, assays for
each of these
responses can be conducted to validate the inhibitory activity of a compound
identified
through a binding assay.
For example, a variety of different parameters can be monitored to assess
toxicity. Examples of such parameters include, but are not limited to, cell
proliferation,
monitoring activation of cellular pathways for toxicological responses by gene
or protein
expression analysis, DNA fragmentation, changes in the composition of cellular
membranes,
membrane permeability, activation of components of death-receptors or
downstream
signaling pathways (e.g., caspases), generic stress responses, NF-kappaB
activation and
responses to mitogens. Related assays are used to assay for apoptosis (a
programmed process
of cell death) and necrosis, including cGMP formation and NO formation. The
following are
illustrative of the type of biological assays that can be conducted to assess
whether a
inhibitory agent has a protective effect against neuronal injury or disease.
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A. Morphological Changes
Apoptosis in many cell types is correlated with altered morphological
appearances. Examples of such alterations include, but are not limited to,
plasma membrane
blebbing, cell shape change, loss of substrate adhesion properties. Such
changes are readily
detectable with a light microscope. Cells undergoing apoptosis can also be
detected by
fragmentation and disintegration of chromosomes. These changes can be detected
using light
microscopy and/or DNA or chromatin specific dyes.
B. Altered Membrane Permeability
Often the membranes of cells undergoing apoptosis become increasingly
permeable. This change in membrane properties can be readily detected using
vital dyes
(e.g., propidium iodide and trypan blue). Dyes can be used to detect the
presence of necrotic
cells. For example, certain methods utilize a green-fluorescent LIVEJDEAD
Cytotoxicity Kit
#2, available from Molecular Probes. The dye specifically reacts with cellular
amine groups.
In necrotic cells, the entire free amine content is available to react with
the dye, thus resulting
in intense fluorescent staining. In contrast, only the cell-surface amines of
viable cells are
available to react with the dye. Hence, the fluorescence intensity for viable
cells is reduced
significantly relative to necrotic cells (see, e.g., Haugland, 1996 Handbook
of Fluorescent
Probes and Research Chemicals, 6th ed., Molecular Probes, OR).
C. Dysfunction of Mitochondria) Membrane Potential
Mitochondria provide direct and indirect biochemical regulation of diverse
cellular processes as the main energy source in cells of higher organisms.
These process
include the electron transport chain activity, which drives oxidative
phosphorylation to
produce metabolic energy in the form of adenosine triphosphate (i.e., ATP).
Altered or
defective mitochondria) activity can result in mitochondria) collapse called
the "permeability
transition" or mitochondria) permeability transition. Proper mitochondria)
functioning
requires maintenance of the membrane potential established across the
membrane.
Dissipation of the membrane potential prevents ATP synthesis and thus halts or
restricts the
production of a vital biochemical energy source.
Consequently, a variety of assays designed to assess toxicity and cell death
involve monitoring the effect of a test agent on mitochondria) membrane
potentials or on the
mitochondria) permeability transition. One approach is to utilize fluorescent
indicators(see,
e.g., Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals,
6th ed.,
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Molecular Probes, OR, pp. 266-274 and 589-594). Various non-fluorescent probes
can also
be utilized (see, e.g., Kamo et al. (1979) J. Membrane Biol. 49:105).
Mitochondria)
membrane potentials can also be determined indirectly from mitochondria)
membrane
permeability (see, e.g., Quinn (1976) The Molecular Biology of Cell Membranes,
University
Park Press, Baltimore, Maryland, pp. 200-217). Further guidance on methods for
conducting
such assays is provided in PCT publication WO 00/19200 to Dykens et al.
D. Caspase Activation
Apoptosis is the process of programmed cell death and involves the activation
of a genetic program when cells are no longer needed or have become seriously
damaged.
Apoptosis involves a cascade of biochemical events and is under the regulation
of a number
of different genes. One group of genes act as effectors of apoptosis and are
referred to as the
interleukin-l.beta.converting enzyme (ICE) family of genes. These genes encode
a family of
cysteine proteases whose activity is increased in apoptosis. The ICE family of
proteases is
1 S generically referred to as caspase enzymes. The "c" in the name reflects
the fact that the
enzymes are cysteine proteases, while "aspase" refers to the ability of these
enzymes to
cleave after aspartic acid residues.
Consequently, some assays for apoptosis are based upon the observation that
caspases are induced during apoptosis. Induction of these enzymes can be
detected by
monitoring the cleavage of specifically-recognized substrates for these
enzymes. A number
of naturally occurring and synthetic protein substrates are known (see, e.g.,
Ellerby et al.
(1997) J. Neurosci. 17:6165; Kluck, et al. (1997) Science 275:1132; Nicholson
et al. (1995)
Nature 376:37; and Rosen and Casciola-Rosen (1997) J. Cell Biochem. 64:50).
Methods for
preparing a number of different substrates that can be utilized in these
assays are described in
U.S. Patent No. 5,976,822. This patent also describes assays that can be
conducted using
whole cells that are amendable to certain of the microfluidic devices
described herein. Other
methods using FRET techniques are discussed in Mahajan, et al. (1999) Chem.
Biol. 6:401-9;
and Xu, et al. (1998) Nucl. Acids. Res. 26:2034-5.
E. Cytochrome c Release
In healthy cells, the inner mitochondria) membrane is impermeable to
macromolecules. Thus, one indicator of cell apoptosis is the release or
leakage of
cytochrome c from the mitochondria. Detection of cytochrome c can be performed
using
spectroscopic methods because of the inherent absorption properties of the
protein. Thus,
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one detection option with the present devices is to place the cells within a
holding space and
monitor absorbance at a characteristic absorption wavelength for cytochrome c.
Alternatively, the protein can be detected using standard immunological
methods (e.g.,
ELISA assays) with an antibody that specifically binds to cytochrome c (see,
e.g., Liu et al.
(1996) Cell 86:147).
F. Assays for Cell Lysis
The final stage of cell death is typically lysis of the cell. When cells die
they
typically release a mixture of chemicals, including nucleotides, and a variety
of other
substances (e.g., proteins and carbohydrates) into their surroundings. Some of
the substances
released include ADP and ATP, as well as the enzyme adenylate cyclase, which
catalyzes the
conversion of ADP to ATP in the presence of excess ADP. Thus, certain assays
involve
providing sufficient ADP in the assay medium to drive the equilibrium towards
the
generation of ATP which can subsequently be detected via a number of different
means. One
such approach is to utilize a luciferin/luciferase system that is well known
to those of
ordinary skill in the art in which the enzyme luciferase utilizes ATP and the
substrate
luciferin to generate a photometrically detectable signal. Further details
regarding certain cell
lysis assays that can be performed are set forth in PCT publication WO
00/70082.
G. Ischemic Model S stY ems
Methods for assaying whether a compound can confer protective neurological
effects against ischemia and stroke are discussed by Aarts, et al. (Science
298:846-850,
2002). In general, this assay involves subjecting rats to a middle cerebral
artery occlusion
(MCAO) for a relatively short period of time (e.g., about 90 minutes). MCAO
can be
induced using various methods, including an intraluminal suture method (see,
e.g., Longa,
E.Z. et al. (1989) Stroke 20:84; and Belayev, L., et al. (1996) Stroke
27:1616). A
composition containing the putative inhibitor is introduced into the rat using
conventional
methods (e.g., via intravenous injection). To evaluate the compositions
prophylactic effect,
the composition is administered before performing MCAO. If the compound is to
be
evaluated for its ability to mitigate against an ischemic event that has
already occurred, the
composition with the compound is introduced after MCAO has been initiated. The
extent of
cerebral infarction is then evaluated using various measures of neurological
function.
Examples of such measures include the postural reflex test (Bcderson, J.B. et
al. (1986)
Stroke 17:472) and the forelimb placing test (De Ryck, M. et al. (1989) Stroke
20:1383).
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Methods are also described in Aarts et al assessing the effects of NMDA-
induced
excitotoxicity using in vitro assays.
VI. Global Analysis of PDZ-PL Interactions
S As described supra, the present invention provides powerful methods for
analysis of PDZ-ligand interactions, including high-throughput methods such as
the "G"
assay and affinity assays described supra. In one embodiment of the invention,
the affinity is
determined for a particular ligand and a plurality of PDZ proteins. Typically
the plurality is
at least S, and often at least 25, or at least 40 different PDZ proteins. In a
preferred
embodiment, the plurality of different PDZ proteins are from a particular
tissue (e.g., central
nervous system) or a particular class or type of cell, (e.g., a neuron) and
the like. In a most
preferred embodiment, the plurality of different PDZ proteins represents a
substantial fraction
(e.g., typically a majority, more often at least 80%) of all of the PDZ
proteins known to be, or
suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ
proteins known to be
present in neuronal cells. In an embodiment, the plurality is at least 50%,
usually at least
80%, at least 90% or all of the PDZ proteins disclosed herein as being
expressed in neuronal
cells.
In one embodiment of the invention, the binding of a ligand to the plurality
of
PDZ proteins is determined. Using this method, it is possible to identify a
particular PDZ
domain bound with particular specificity by the ligand. The binding may be
designated as
"specific" if the affinity of the ligand to the particular PDZ domain is at
least 2-fold that of
the binding to other PDZ domains in the plurality (e.g., present in that cell
type). The binding
is deemed "very specific" if the affinity is at least 10-fold higher than to
any other PDZ in the
plurality or, alternatively, at least 10-fold higher than to at least 90%,
more often 95% of the
other PDZs in a defined plurality. Similarly, the binding is deemed
"exceedingly specific" if
it is at least 100-fold higher. For example, a ligand could bind to 2
different PDZs with an
affinity of 1 uM and to no other PDZs out of a set 40 with an affinity of less
than 100 uM.
This would constitute specific binding to those 2 PDZs. Similar measures of
specificity are
used to describe binding of a PDZ to a plurality of PLs.
It will be recognized that high specificity PDZ-PL interactions represent
potentially more valuable targets for achieving a desired biological effect.
The ability of an
inhibitor or enhancer to act with high specificity is often desirable. In
particular, the most
specific PDZ-ligand interactions are also the best therapeutic targets,
allowing specific
inhibition of the interaction.
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Thus, in one embodiment, the invention provides a method of identifying a
high specificity interaction between a particular PDZ domain and a ligand
known or
suspected of binding at least one PDZ domain, by providing a plurality of
different
immobilized polypeptides, each of said polypeptides comprising a PDZ domain
and a non-
PDZ domain; determining the affinity of the ligand for each of said
polypeptides, and
comparing the affinity of binding of the ligand to each of said polypeptides,
wherein an
interaction between the ligand and a particular PDZ domain is deemed to have
high
specificity when the ligand binds an immobilized polypeptide comprising the
particular PDZ
domain with at least 2-fold higher affinity than to immobilized polypeptides
not comprising
the particular PDZ domain.
In a related aspect, the affinity of binding of a specific PDZ domain to a
plurality of ligands (or suspected ligands) is determined. For example, in one
embodiment,
the invention provides a method of identifying a high specificity interaction
between a PDZ
domain and a particular ligand known or suspected of binding at least one PDZ
domain, by
providing an immobilized polypeptide comprising the PDZ domain and a non-PDZ
domain;
determining the affinity of each of a plurality of ligands for the
polypeptide, and comparing
the affinity of binding of each of the ligands to the polypeptide, wherein an
interaction
between a particular ligand and the PDZ domain is deemed to have high
specificity when the
ligand binds an immobilized polypeptide comprising the PDZ domain with at
least 2-fold
higher affinity than other ligands tested. Thus, the binding may be designated
as "specific" if
the affinity of the PDZ to the particular PL is at least 2-fold that of the
binding to other PLs in
the plurality (e.g., present in that cell type). The binding is deemed "very
specific" if the
affinity is at least 10-fold higher than to any other PL in the plurality or,
alternatively, at least
10-fold higher than to at least 90%, more often 95% of the other PLs in a
defined plurality.
Similarly, the binding is deemed "exceedingly specific" i f it is at least 100-
fold higher.
Typically the plurality is at least S different ligands, more often at least
10.
1. Use of Array for Global Predictions
One discovery of the present inventors relates to the important and extensive
roles played by interactions between PDZ proteins and PL proteins,
particularly in the
biological function of neuronal cells. Further, it has been discovered that
valnahle
information can be ascertained by analysis (e.g., simultaneous analysis) of a
large number of
PDZ-PL interactions. In a preferred embodiment, the analysis encompasses all
of the PDZ
proteins expressed in a particular tissue (e.g., brain) or type or class of
cell (e.g., neuron).
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Alternatively, the analysis encompasses at least about 5, or at least about
10, or at least about
12, or at least about 15 and often at least 50 different polypeptides, up to
about 60, about 80,
about 100, about 150, about 200, or even more different polypeptides; or a
substantial
fraction (e.g., typically a majority, more often at least 80%) of all of the
PDZ proteins known
S to be, or suspected of being, expressed in the tissue or cell(s), (e.g., all
of the PDZ proteins
known to be present in neurons).
It will be recognized that the arrays and methods of the invention are
directed
to analyze of PDZ and PL interactions, and involve selection of such proteins
for analysis.
While the devices and methods of the invention may include or involve a small
number of
control polypeptides, they typically do not include significant numbers of
proteins or fusion
proteins that do not include either PDZ or PL domains (e.g., typically, at
least about 90% of
the arrayed or immobilized polypeptides in a method or device of the invention
is a PDZ or
PL sequence protein, more often at least about 95%, or at least about 99%).
It will be apparent from this disclosure that analysis of the relatively large
number of different interactions preferably takes place simultaneously. In
this context,
"simultaneously" means that the analysis of several different PDZ-PL
interactions (or the
effect of a test agent on such interactions) is assessed at the same time.
Typically the analysis
is carried out in a high throughput (e.g., robotic) fashion. One advantage of
this method of
simultaneous analysis is that it permits rigorous comparison of multiple
different PDZ-PL
interactions. For example, as explained in detail elsewhere herein,
simultaneous analysis
(and use of the arrays described infra) facilitates, for example, the direct
comparison of the
effect of an agent (e.g., an potential interaction inhibitor) on the
interactions between a
substantial portion of PDZs and/or PLs in a tissue or cell.
Accordingly, in one aspect, the invention provides an array of immobilized
polypeptide comprising the PDZ domain and a non-PDZ domain on a surface.
Typically, the
array comprises at least about S, or at least about 10, or at least about 12,
or at least about 15
and often at least 50 different polypeptides. In one preferred embodiment, the
different PDZ
proteins are from a particular tissue (e.g., central nervous system) or a
particular class or type
of cell, (e.g., a neuron) and the like. In a most preferred embodiment, the
plurality of
different PDZ proteins represents a substantial fraction (e.g., typically a
majority, more often
at least 60%, 70% or 80%) of all of the PDZ proteins known to be, or suspected
of being,
expressed in the tissue or cell(s), (e.g., all of the PDZ proteins known to be
present in
neurons).
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Certain embodiments are arrays which include a plurality, usually at least 5,
10, 25, SO PDZ proteins present in a particular cell of interest. In this
context, "array" refers
to an ordered series of immobilized polypeptides in which the identity of each
polypeptide is
associated with its location. In some embodiments the plurality of
polypeptides are arrayed
in a "common" area such that they can be simultaneously exposed to a solution
(e.g.,
containing a ligand or test agent). For example, the plurality of polypeptides
can be on a
slide, plate or similar surface, which may be plastic, glass, metal, silica,
beads or other
surface to which proteins can be immobilized. In a different embodiment, the
different
immobilized polypeptides are situated in separate areas, such as different
wells of multi-well
plate (e.g., a 24-well plate, a 96-well plate, a 384 well plate, and the
like). It will be
recognized that a similar advantage can be obtained by using multiple arrays
in tmdem.
2. Analysis of PDZ-PL Inhibition Profile
In one aspect, the invention provides a method for determining if a test
compound inhibits any PDZ-ligand interaction in large set of PDZ-ligand
interaction (e.g., a
plurality of the PDZ-ligands interactions described in TABLE 3, 8 or 9; a
majority of the
PDZ-ligands identified in a particular cell or tissue as described supra
(e.g., neurons) and the
like. In one embodiment, the PDZ domains of interest are expressed as GST-PDZ
fusion
proteins and immobilized as described herein. For each PDZ domain, a labeled
ligand that
binds to the domain with a known affinity is identified as described herein.
For any known or suspected modulator (e.g., inhibitor) of a PDL-PL
interaction(s), it is useful to know which interactions are inhibited (or
augmented). For
example, an agent that inhibits all PDZ-PL interactions in a cell (e.g., a
neuron) will have
different uses than an agent that inhibits only one, or a small number, of
specific PDZ-PL
interactions. The profile of PDZ interactions inhibited by a particular agent
is referred to as
the "inhibition profile" for the agent, and is described in detail below. The
profile of PDZ
interactions enhanced by a particular agent is referred to as the "enhancement
profile" for the
agent. It will be readily apparent to one of skill guided by the description
of the inhibition
profile how to determine the enhancement profile for an agent. The present
invention
provides methods for determining the PDZ interaction (inhibitionenhancement)
profile of an
agent in a single assay.
In one aspect, the invention provides a method for determining the PDZ-PL
inhibition profile of a compound by providing (i) a plurality of different
immobilized
polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ
domain
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and (ii) a plurality of corresponding ligands, wherein each ligand binds at
least one PDZ
domain in (i), then contacting each of said immobilized polypeptides in (i)
with a
corresponding ligand in (ii) in the presence and absence of a test compound,
and determining
for each polypeptide-ligand pair whether the test compound inhibits binding
between the
immobilized polypeptide and the corresponding ligand.
Typically the plurality is at least 5, and often at least 25, or at least 40
different
PDZ proteins. In a preferred embodiment, the plurality of different ligands
and the plurality
of different PDZ proteins are from the same tissue or a particular class or
type of cell, (e.g., a
neuron). In a most preferred embodiment, the plurality of different PDZs
represents a
substantial fraction (e.g., at least 80%) of all of the PDZs known to be, or
suspected of being,
expressed in the tissue or cell(s), e.g., all of the PDZs known to be present
in neurons (for
example, at least 80%, at least 90% or all of the PDZs disclosed herein as
being expressed in
neuronal cells).
In one embodiment, the inhibition profile is determined as follows: A
plurality
(e.g., all known) PDZ domains expressed in a cell (e.g., neurons) are
expressed as GST-
fusion proteins and immobilized without altering their ligand binding
properties as described
s~cpra. For each PDZ domain, a labeled ligand that binds to this domain with a
known
affinity is identified. If the set of PDZ domains expressed in neurons is
denoted by
~P1...Pn}, any given PDZ domain Pi binds a (labeled) ligand Li with affinity
Kdi. To
determine the inhibition profile for a test agent "compound X" the "G" assay
(supra) can be
performed as follows in 96-well plates with rows A-H and columns 1-12. Column
1 is coated
with P1 and washed. The corresponding ligand L1 is added to each washed coated
well of
column 1 at a concentration 0.5 Kdl with (rows B, D, F, H) or without (rows A,
C, E, F)
between about 1 and about 1000 uM) of test compound X. Column 2 is coated with
P2, and
L2 (at a concentration 0.5 Kd2) is added with or without inhibitor X.
Additional PDZ
domains and ligands are similarly tested.
Compound X is considered to inhibit the binding of Li to Pi if the average
signal in the wells of column i containing X is less than half the signal in
the equivalent wells
of the column lacking X. Thus, in this single assay one determines the full
set of neural
PDZs that are inhibited by compound X.
In some embodiments, the test compound X is a mixture of compounds, such
as the product of a combinatorial chemistry synthesis as described supra. In
some
embodiments, the test compound is known to have a desired biological effect,
and the assay
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is used to determine the mechanism of action (i.e., if the biological effect
is due to
modulating a PDZ-PL interaction).
It will be apparent that an agent that modulates only one, or a few PDZ-PL
interactions, in a panel (e.g., a panel of all known PDZs in neurons, a panel
of at least 10, at
least 20 or at least 50 PDZ domains) is a more specific modulator than an
agent that modulate
many or most interactions. Typically, an agent that modulates less than 20% of
PDZ
domains in a panel (e.g., TABLE 4) is deemed a "specific" inhibitor, less than
G% a "very
specific" inhibitor, and a single PDZ domain a "maximally specific" inhibitor.
It will be recognized that high specificity modulators of PDZ-PL interactions
represent potentially more valuable drug targets for achieving a desired
biological effect. The
ability of an inhibitor or enhancer to act with "maximal specificity" is most
desirable.
In one embodiment, the assays of the invention can be used to determine a
maximally specific modulator of the interaction between a NMDA receptor and a
PDZ
domain.
In a preferred embodiment, the assays of the invention are used to identify a
maximally specific modulator of the interaction between NMDA receptor 2B
(NMDAR2B)
and PSD95.
It will also be appreciated that "compound X" may be a composition
containing mixture of compounds (e.g., generated using combinatorial chemistry
methods)
rather than a single compound.
Several variations of this assay are contemplated:
In some alternative embodiments, the assay above is performed using varying
concentrations of the test compound X, rather than fixed concentration. This
allows
determination of the Ki of the X for each PDZ as described above.
In an alternative embodiment, instead of pairing each PDZ Pi with a specific
labeled ligand Li, a mixture of different labeled ligands is created that such
that for every
PDZ at least one of the ligands in the mixture binds to this PIDZ sufficiently
to detect the
binding in the "G" assay. This mixture is then used for every PDZ domain.
In one embodiment, compound X is known to have a desired biological effect,
but the chemical mechanism by which it has that effect is unknown. The assays
of the
invention can then be used to determine if compound X has its effect by
binding to a PDZ
domain.
In one embodiment, PDZ-domain containing proteins are classified in to
groups based on their biological function, e.g. into those that regulate
apoptosis versus those
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that regulate transcription. An optimal inhibitor of a particular function
(e.g., including but
not limited to an anti-apoptotic agent, an anti-T cell activation agent, cell-
cycle control,
vesicle transport, ete.) will inhibit multiple PDZ-ligand interactions
involved in the function
(e.g., apoptosis, activation) but few other interactions. Thus, the assay is
used in one
embodiment in screening and design of a drug that specifically blocks a
particular function.
For example, an agent designed to block apoptosis might be identified because,
at a given
concentration, the agent inhibits 2 or more PDZs involved in apoptosis but
fewer than 3 other
PDZs, or that inhibits PDZs involved in apoptosis with a Ki > 10-fold better
than for other
PDZs. Thus, the invention provides a method for identifying an agent that
inhibits a first
selected PDZ-PL interaction or plurality of interactions but does not inhibit
a second selected
PDZ-PL interaction or plurality of interactions. The two (or more) sets of
interactions can be
selected on the basis of the known biological function of the PDZ proteins,
the tissue
specificity of the PDZ proteins, or any other criteria. Moreover, the assay
can be used to
determine effective doses (i.e., drug concentrations) that result in desired
biological effects
while avoiding undesirable effects.
3. Side Effects of PDZ-PL Modulator Interactions
In a related embodiment, the invention provides a method for determining
likely side effects of a therapeutic that inhibits PDZ-ligand interactions.
The method entails
identifying those target tissues, organs or cell types that express PDZ
proteins and ligands
that are disrupted by a specified inhibitor. If, at a therapeutic dosage, a
drug intended to have
an effect in one organ system (e.g., central nervous system) disrupts PDZ-PL
interactions in a
different system (e.g., hematopoietic system) it can be predicted that the
drug will have
effects ("side effects") on the second system. It will be apparent that the
information
obtained from this assay will be useful in the rational design and selection
of drugs that do
not have the side-effect.
In one embodiment, for example, a comprehensive PDZ protein set is
obtained. A "perfectly comprehensive" PDZ protein set is defined as the set of
all PDZ
proteins expressed in the subject animal (e.g., humans). A comprehensive set
may be
obtained by analysis of, for example, the human genome sequence. However, a
"perfectly
comprehensive" set is not required and any reasonably large set of PDZ domain
proteins
(e.g., the set of all known PDZ proteins; or the set listed in TABLE 4) will
provide valuable
information.
In one embodiment, the method involves some of all of the following steps:
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a) For each PDZ protein, determine the tissues in which it is highly
expressed. This can be done experimentally although the information generally
will be
available in the scientific literature;
b) For each PDZ protein (or as many as possible), identify the cognate PL(s)
bound by the PDZ protein;
c) Determine the Ki at which the test agent inhibits each PDZ-PL interaction,
using the methods described supra;
d) From this information it is possible to calculate the pattern of PDZ-PL
interactions disrupted at various concentrations of the test agent.
By correlating the set of PDZ-PL interactions disrupted with the expression
pattern of the members of that set, it will be possible to identify the
tissues likely affected by
the agent.
Additional steps can also be carried out, including determining whether a
specified tissue or cell type is exposed to an agent following a particular
route of
administration. This can be determined using basis pharmacokinetic methods and
principles.
4. Modulation of Activities
The PDZ binding moieties and inhibitors described herein that disrupt PDZ:PL
protein interactions can be used to modulate biological activities or
functions of cells (e.g.,
neurons). These agents can also be utilized to treat diseases and conditions
in human and
nonhuman animals (e.g., experimental models). Exemplary biological activities
are listed
supra.
When administered to patients, the compounds of the invention (e.g., PL-PDZ
interaction inhibitors) are useful for treating (ameliorating symptoms of) a
variety of
neurological disorders, including those associated with some type of injury to
neuronal cells
or the death of neurons. Such disorders include, but are not limited to,
stroke, ischemia, brain
traumas and chronic pain. Certain inhibitors can also be used to treat other
types of
neuorological disorders like Alzheimer's disease, epilepsy, Parkinson's
disease, Huntington's
disease, motor neuron diseases and inherited ataxias.
Some other inhibitors can be utilized to treat other disease types, including,
for
instance, inflammatory and humoral immune responses, e.g., inflammation,
allergy (e.g.,
systemic anaphylaxis, hypersensitivity responses, drug allergies, insect sting
allergies);
infectious diseases (e.g., viral infection, such as HN, measles,
parainfluenza, virus-mediated
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cell fusion,), and ischemia (e.g., post-myocardial infarction complications,
joint injury,
kidney, scleroderma).
VII. Antagonists of PDZ-PL Interactions
As described herein, interactions between PDZ proteins and PL proteins in
cells (e.g., neurons) may be disrupted or inhibited by the administration of
inhibitors or
antagonists. Inhibitors can be identified using screening assays described
herein. In
embodiment, the motifs disclosed herein are used to design inhibitors. In some
embodiments,
the antagonists of the invention have a structure (e.g., peptide sequence)
based on the C-
terminal residues of PL-domain proteins listed in TABLE 2. In some
embodiments, the
antagonists of the invention have a structure (e.g., peptide sequence) based
on a PL motif
disclosed herein.
The PDZ/PL antagonists and antagonists of the invention can be any of a large
variety of compounds, both naturally occurring and synthetic, organic and
inorganic, and
including polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and
polynucleotides),
small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide
analogs, analogs
of naturally occurring structures (e.g., peptide mimetics, nucleic acid
analogs, and the like),
and numerous other compounds. Although, for convenience, the present
discussion primarily
refers antagonists of PDZ-PL interactions, it will be recognized that PDZ-PL
interaction
agonists can also be use in the methods disclosed herein.
In one aspect, the peptides and peptide mimetics or analogues of the invention
contain an amino acid sequence that binds a PDZ domain in a cell of interest.
In one
embodiment, the antagonists comprise a peptide that has a sequence
corresponding to the
carboxy-terminal sequence of a PL protein listed in TABLE 2, e.g., a peptide
listed TABLE
2. Typically, the peptide comprises at least the C-terminal two (3), three (3)
or four (4)
residues of the PL protein, and often the inhibitory peptide comprises more
than four residues
(e.g., at least five, six, seven, eight, nine, ten, twelve or fifteen
residues) from the PL protein
C-terminus.
In some embodiments, the inhibitor is a peptide, e.g., having a sequence of a
PL C-terminal protein sequence.
In some embodiments, the antagonist is a fusion protein comprising such a
sequence. Fusion proteins containing a transmembrane transporter amino acid
sequence can
be used to facilitate transport of the inhibitor into a cell.
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In some embodiments, the inhibitor is conserved variant of the PL C-terminal
protein sequence having inhibitory activity.
In some embodiments, the antagonist is a peptide mimetic of a PL C-terminal
sequence.
In some embodiments, the inhibitor is a small molecule (i.e., having a
molecular weight less than 1 kD).
A. Polypentide Antag-onists
1. Inhibitors with a PL Sequence
One class of inhibitors or antagonists that are provided comprise a peptide
that
has a sequence of a PL protein carboxy-terminus listed in TABLE 2. The PL
protein
carboxy-terminus sequences can be considered as the "core PDZ motif sequence"
because of
the ability of the short sequence from the carboxy terminus to interact with
the PDZ domain.
For example, in some inhibitors the "core PDZ motif sequence" or simply the
"PL sequence"
contains the last 2, 3 or 4 C-terminus amino acids. In other instances,
however, the core PDZ
motif comprises more than 2-4 residues (e.g., at least 5, 6, 7, 8, 9, 10, 1 l,
12, 13, 14, 15, 16,
17, 18, 19, or 20 residues) from the PL protein C-terminus. For some
inhibitors, the PDZ
motif sequence peptide is from 4-15 amino acids in length. Other inhibitors
have a PDZ
motif sequence that is 6-10 amino acids in length, or 3-8 amino acids in
length, or 3-7 amino
acids in length. Certain inhibitors have a PDZ motif sequence that is 8 amino
acids in length.
Although the residues shared by the inhibitory peptide and the PL protein are
often found at
the C-terminus of the peptide, some inhibitors incorporate a PL sequence that
is located in an
internal region of a PL protein. Similarly, in some cases, the inhibitory
peptide comprises
residues from a PL sequence that is near, but not at the C-terminus of a PL
protein (see, Gee
et al., 1998, .l Biological Chem. 273:21980-87).
Another set of inhibitors are based upon the identi .fication of amino acid
sequences that specifically disrupt binding between NMDAR proteins and PSD-95.
This
particular class of inhibitors are polypeptides that share the following
characteristics: 1) a
size ranging from 3-20 amino acids in length (although somewhat longer
polypeptides can be
used), and 2) a C-terminal consensus sequence of X-T-X-V/L/A (the slash
separates different
amino acids that can appear at a given position).
Specific examples of polypeptides that were found to be able to inhibit
NMDAR and PSD-95 interactions include:
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1) peptides 3 amino acids in length: TEV and SDV;
2) peptides 4 amino acids in length: ETEV, ETQL, QTQV, ETAL, QTEV,
ESEV, ETVA and FTDV;
3) 19 C-terminal amino acids from TAX (ISPGGLEPPSEKHFRETEV);
4) 19 C-terminal amino acids from modified HPV 16 E6
(TGRGMSGGRSSRTRRETQL); and
5) 20 C-terminal amino acids from TAX (QISPGGLEPPSEKHFRETEV).
These specific examples should not be considered as limiting but simply
illustrative of inhibitors having the general characteristics listed above.
Yet another set of inhibitors are based upon the PL sequences that were
identified as binding to the PDZ domain of nNOS (see Example 8 and TABLE 8).
Inhibitors
in this class can be polypeptides whose carboxy terminus comprises at least
two contiguous
amino acids from the C-terminus of one of the PL sequences. As with the other
classes of
inhibitors described above, the PL sequence/PDZ core sequence motif can be
longer, such as
3-20 amino acids from the C-terminus of the PL sequences listed in TABLE 8.
A third group of inhibitors include a PL sequence from the list shown in
TABLE 9. This table lists PL sequences that were identified as binding to the
PDZ domain
of PSD-95 (see Example 9). Like the other classes of inhibitors based upon PL
sequences,
these inhibitors generally include at least 2-3 continguous amino acids from
the C-terminus
of the sequences listed in this table. Typically, the PL sequence portion of
these inhibitors is
3-20 amino acids in length. Inhibitors within this class can be utilized to
disrupt binding
between PL proteins containing these sequences can PDZ proteins such as PSD-
95.
As described in greater detail below, short PL peptides, such as just
described
can be used in the rational design of other small molecules with similar
properties according
to established techniques.
Core PDZ motif sequences/PL sequences such as those just listed can
optionally be joined to additional amino acids at their amino terminus to
further increase
binding affinity and/or stability and/or transportability into cells. These
additional sequences
located at the amino terminus can be from the natural sequence of a neuronal
cell surface
receptor or from other sources. The PDZ motif sequence and additional N-
terminal
sequences can optionally be joined by a linker. The additional amino acids can
also be
conservatively substituted. The total peptide length (i.e., core PDZ motif
sequence plus
optional N-terminal segment) can be of a variety of lengths (e.g., at least 2,
3, 4, 5, 6, 8, 10,
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15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids). Typically,
the overall length
is in the range of 30-40 amino acids. For those inhibitors in which additional
sequences are
attached at the N-terminus of the core PDZ motif sequence (PL sequence), the
overall
structure is thus: N-terminal segment - core PDZ motif sequence (PL sequence),
or N-
terminal segment - linker - core PDZ motif sequence (PL sequence). As
discussed further
below, one useful class of proteins that can be fused to the core PDZ motifs
or PL sequences
are transmembrane transporter peptides. These peptides can be fused to the
inhibitory
sequences to facilitate transport into a target cell (e.g., neuron). Further
details are provided
below. Purification tags that are known in the art can also optionally be
fused to the N
terminus of the PL sequence.
2. Inhibitors with a PDZ-Domain Polypeptide
Some of the inhibitors that are provided rather than containing a PL sequence,
instead contain all or a portion of a PDZ binding domain. The PDZ-domain
sequence
included in these inhibitors is selected to mimic (i.e., have similar binding
characteristics) of
the PDZ domain in the PDZ protein of interest (i.e., the PDZ protein whose
binding
interaction with a PL protein one seeks to disrupt). The PDZ-domain sequence
is long
enough to include at least enough of the PDZ domain such that the resulting
polypeptide
inhibitor can effectively bind to the cognate PL protein. This typically means
that the PDZ-
domain sequence is at least 50, 55, 60, 65, 70, 75, 80, 85, 90 or more amino
acids long. But
certain inhibitors can include the entire PDZ-domain, or even additional amino
acids from the
PDZ protein that extend beyond the PDZ-domain.
3. Optional Features of Inhibitors
Polypeptide inhibitors such as those just described can optionally be
derivatized (e.g., acetylated, phosphorylated and/or glycoslylated) to improve
the binding
affinity of the inhibitor, to improve the ability of the inhibitor to be
transported across a cell
membrane or to improve stability. As a specific example, for inhibitors in
which the third
residue from the C-terminus is S, T or Y, this residue can be phosphorylated
prior to the use
of the peptide.
The polypeptide inhibitors can also optionally be linked directly or via a
linker
to a transmembrane transporter peptide. Specific examples of these sequences
are described
in the section on formulation and administration of the polypeptides of the
invention. But
certain polypeptide inhibitors do not include a transporter peptide.
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B. Peptide Variants
Having identified PDZ binding peptides and PDZ-PL interaction inhibitory
sequences, variations of these sequences can be made and the resulting peptide
variants can
be tested for PDZ domain binding or PDZ-PL inhibitory activity. In
embodiments, the
variants have the same or a different ability to bind a PDZ domain as the
parent peptide.
Typically, such amino acid substitutions are conservative, i.e., the amino
acid residues are
replaced with other amino acid residues having physical and/or chemical
properties similar to
the residues they are replacing. Preferably, conservative amino acid
substitutions are those
wherein an amino acid is replaced with another amino acid encompassed within
the same
designated class.
C. Peptide Mimetics
Having identified PDZ binding peptides and PDZ-PL interaction inhibitory
sequences, peptide mimetics can be prepared using routine methods, and the
inhibitory
activity of the mimetics can be confirmed using the assays of the invention.
Thus, in some
embodiments, the antagonist is a peptide mimetic of a PL C-terminal sequence.
The skilled
artisan will recognize that individual synthetic residues and polypeptides
incorporating
mimetics can be synthesized using a variety of procedures and methodologies,
which are well
described in the scientific and patent literature, e.g., Organic Syntheses
Collective Volumes,
Gilman et al. (Eds) John Wiley & Sons, Inc., NY. Polypeptides incorporating
mimetics can
also be made using solid phase synthetic procedures, as described, e.g., by Di
Marchi, et al.,
U.S. Pat. No. 5,422,426. Mimetics of the invention can also be synthesized
using
combinatorial methodologies. Various techniques for generation of peptide and
peptidomimetic libraries are well known, and include, e.g., multipin, tea bag,
and
split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol. Biotechnol.
9:205-223; Hruby
(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-
27; Ostresh
(1996) Methods Enzymol. 267:220-234.
D. Small Molecules
In some embodiments, the inhibitor is a small molecule (i.e., having a
molecular weight less than 1 kD). Methods for screening small molecules are
well known in
the art and include those described supra.
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E. Binding AffinitX
Regardless of type, the inhibitors generally have an ECso of less than 50 um.
Some inhibitors have an ECso of less than 10 uM, others have an ECSO of 1 uM,
and still
others an ECSO of less than 100 nM. The inhibitors typically have an ECSO
value of 20-100
nM.
VIII. Uses of PDZ Domain Binding and Antagonist Compounds
Because the inhibitors that are described herein are useful in interfering
with
binding between certain PDZ and PL proteins in neurons (e.g., the NMDARlPSD-95
interaction, and the interaction between nNOS and various PL proteins), the
inhibitors can be
utilized in the treatment of a variety of biological processes in neuron
cells. For instance, the
inhibitors can be utilized to treat problems associated with excitotoxicity
and apoptosis
occasioned by neuronal damage. The inhibitors can also be utilized to treat
various
neurological diseases, including those associated with stroke and ischemia.
Specific
examples of neurological diseases that can be treated with certain inhibitors
include,
Alzheimer's disease, epilepsy, Parkinson's disease, Huntington's disease,
motor neuron
diseases and inherited ataxias.
Because PDZ proteins are involved in a number of biological functions
besides involvement in excitotoxicity responses, some of the inhibitors that
are provided can
be used in the treatment of other conditions and activities correlated with
the PDZ:PL protein
interactions described herein. Examples of such activities include, but are
not limited to,
organization and regulation of multiprotein complexes, vesicular trafficking,
tumor
suppression, protein sorting, establishment of membrane polarity, apoptosis,
regulation of
immune response and organization of synapse formation. In general, PDZ
proteins have a
common function of facilitating the assembly of multi-protein complexes, often
serving as a
bridge between several proteins, or regulating the function of other proteins.
Additionally, as
also noted supra, these proteins are found in essentially all cell types.
Consequently, modulation of these interactions can be utilized to control a
wide variety of biological conditions and physiological conditions. In
particular, modulation
of interactions such as those disclosed herein can be utilized to control
movement of vesicles
within a cell, inhibition of tumor formation, as well as in the treatment of
immune disorders,
neurological disorders, muscular disorders, and intestinal disorders.
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Certain compounds which modulate binding of the PDZ proteins and PL
proteins can be used to inhibit leukocyte activation, which is mani Tested in
measurable events
including but not limited to, cytokine production, cell adhesion, expansion of
cell numbers,
apoptosis and cytotoxicity. Thus, some compounds of the invention can be used
to treat
diverse conditions associated with undesirable leukocyte activation, including
but not limited
to, acute and chronic inflammation, graft-versus-host disease, transplantation
rejection,
hypersensitivities and autoimmunity such as multiple sclerosis, rheumatoid
arthritis,
peridontal disease, systemic lupus erythematosis, juvenile diabetes mellitis,
non-insulin-
dependent diabetes, and allergies, and other conditions listed herein.
Thus, the invention also relates to methods of using such compositions in
modulating leukocyte activation as measured by, for example, cytotoxicity,
cytokine
production, cell proliferation, and apoptosis.
IX. Formulation and Route of Administration
1 S A. Introduction of Antagonists (e.~. Peptides and Fusion Proteins) into
Cells
The inhibitors disclosed herein or identified using the screening methods that
are provided can be used in the manufacture of a medicament or pharmaceutical
composition.
These can then be administered according to a number of different methods.
In one aspect, the PDZ-PL antagonists of the invention are introduced into a
cell to modulate (i.e., increase or decrease) a biological function or
activity of the cell. Many
small organic molecules readily cross the cell membranes (or can be modified
by one of skill
using routine methods to increase the ability of compounds to enter cells,
e.g., by reducing or
eliminating charge, increasing lipophilicity, conjugating the molecule to a
moiety targeting a
cell surface receptor such that after interacting with the receptor). Methods
for introducing
larger molecules, e.g., peptides and fusion proteins are also well known,
including, e.g.,
injection, liposome-mediated fusion, application of a hydrogel, conjugation to
a targeting
moiety conjugate endocytozed by the cell, electroporation, and the like).
In one embodiment, the antagonist or agent is a fusion polypeptide or
derivatized polypeptide. A fusion or derivatized protein may include a
targeting moiety that
increases the ability of the polypeptide to traverse a cell membrane or causes
the polypeptide
to be delivered to a specified cell type (e.g., a neuron) preferentially or
cell compartment
(e.g., nuclear compartment) preferentially. Examples of targeting moieties
include lipid tails,
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amino acid sequences such as antennapoedia peptide or a nuclear localization
signal (NLS;
e.g., Xenopus nucleoplasmin Robbins et al., 1991, Cell 64:615).
In one embodiment of the invention, a peptide sequence or peptide analog
determined to inhibit a PDZ domain-PL protein binding interaction as described
herein is
introduced into a cell by linking the sequence to an amino acid sequence that
facilitates its
transport through the plasma membrane (a "transmembrane transporter
sequence"). The
peptides of the invention may be used directly or fused to a transmembrane
transporter
sequence to facilitate their entry into cells. In the case of such a fusion
peptide, each peptide
may be fused with a heterologous peptide at its amino terminus directly or by
using a flexible
polylinker such as the pentamer G-G-G-G-S repeated 1 to 3 times. Such linker
has been
used in constructing single chain antibodies (scFv) by being inserted between
V,., and V~
(Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl.
Acad. Sci. U.S.A.
85:5979-5883). The linker is designed to enable the correct interaction
between two beta-
sheets forming the variable region of the single chain antibody. Other linkers
which may be
used include Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp
(Chaudhary et al.,
1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) and Lys-Glu-Ser-Gly-Ser-Val-
Ser-Ser-
Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (Bird et al., 1988, Science 242:423-
426).
A number of peptide sequences have been described in the art as capable of
facilitating the entry of a peptide linked to these sequences into a cell
through the plasma
membrane (Derossi et al., 1998, Trends in Cell Biol. 8:84). For the purpose of
this invention,
such peptides are collectively referred to as transmembrane transporter
peptides. Examples
of these peptide include, but are not limited to, tat derived from HIV (Vives
et al., 1997, J.
Biol. Chem. 272:16010; Nagahara et al., 1998, Nat. Med. 4:1449), antennapedia
from
Drosophila (Derossi et al., 1994, J. Biol. Chem. 261:10444), VP22 from herpes
simplex virus
(Elliot and D'Hare, 1997, Cell 88:223-233), complementarity-determining
regions (CDR) 2
and 3 of anti-DNA antibodies (Avrameas et al., 1998, Proc. Natl Acad. Sci.
U.S.A., 95:5601-
5606), 70 KDa heat shock protein (Fujihara, 1999, EMBO .l. 18:411-419) and
transportan
(Pooga et al., 1998, FASEB J. 12:67-77). In a preferred embodiment of the
invention, a
truncated HIV tat peptide having the sequence of GYGRKKRRQRRRG is used.
In some instances, a transmembrane transporter sequence is fused to a
neuronal cell surface receptor carboxyl terminal sequence at its amino-
terminus with or
without a linker. Generally, the C-terminus of a PDZ motif sequence (PL
sequence) is free to
interact with a PDZ domain. The transmembrane transporter sequence can be used
in whole
or in part as long as it is capable of facilitating entry of the peptide into
a cell.
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In an alternate embodiment of the invention, a neuronal cell surface receptor
C-terminal sequence can be used alone when it is delivered in a manner that
allows its entry
into cells in the absence of a transmembrane transporter sequence. For
example, the peptide
may be delivered in a liposome formulation or using a gene therapy approach by
delivering a
coding sequence for the PDZ motif alone or as a fusion molecule into a target
cell.
The compounds of the of the invention can also be administered via
liposomes, which serve to target the conjugates to a particular tissue, such
as neural tissue, or
targeted selectively to infected cells, as well as increase the half life of
the peptide
composition. Liposomes include emulsions, foams, micelles, insoluble
monolayers, liquid
crystals, phospholipid dispersions, lamellar layers and the like. In these
preparations, the
peptide to be delivered is incorporated as part of a liposome, alone or in
conjunction with a
molecule which binds to, e.g., a receptor prevalent among neural cells, such
as monoclonal
antibodies which bind to the NMDA Receptor. Thus, liposomes f Iled with a
desired peptide
or conjugate of the invention can be directed to the site of neural cells,
where the liposomes
then deliver the selected inhibitor compositions. Liposomes for use in the
invention are
formed from standard vesicle-forming lipids, which generally include neutral
and negatively
charged phospholipids and a sterol, such as cholesterol. The selection of
lipids is generally
guided by consideration of, e.g., liposome size, acid lability and stability
of the liposomes in
the blood stream. A variety of methods are available for preparing liposomes,
as described
in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat.
Nos. 4,235,871,
4,501,728 and 4,837,028.
The targeting of liposomes using a variety of targeting agents is well known
in
the art (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044). For targeting
to the neural cells,
a ligand to be incorporated into the liposome can include, e.g., antibodies or
fragments
thereof specific for cell surface determinants of the desired nervous system
cells. A liposome
suspension containing a peptide or conjugate may be administered
intravenously, locally,
topically, etc. in a dose which varies according to, inter alia, the manner of
administration, the
conjugate being delivered, and the stage of the disease being treated.
In order to specifically deliver a PDZ motif sequence (PL sequence) peptide
into a specific cell type, the peptide can be linked to a cell-specific
targeting moiety, which
include but are not limited to, ligands for diverse neuron surface molecules
such as growth
factors, hormones and cytokines, neuronal receptors, ion transporters, as well
as antibodies or
antigen-binding fragments thereof. Since a large number of cell surface
receptors have been
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identified in neurons, ligands or antibodies specific for these receptors may
be used as cell-
specific targeting moieties.
Antibodies are the most versatile cell-specific targeting moieties because
they
can be generated against any cell surface antigen. Monoclonal antibodies have
been
generated against neuron-specific markers. Antibody variable region genes can
be readily
isolated from hybridoma cells by methods well known in the art. However, since
antibodies
are assembled between two heavy chains and two light chains, it is preferred
that a scFv be
used as a cell-specific targeting moiety in the present invention. Such scFv
are comprised of
V,., and V~ domains linked into a single polypeptide chain by a flexible
linker peptide.
The PDZ motif sequence (PL sequence) may be linked to a transmembrane
transporter sequence and a cell-specific targeting moiety to produce a tri-
fusion molecule.
This molecule can bind to a neuron surface molecule, passes through the
membrane and
targets PDZ domains. Alternatively, a PDZ motif sequence (PL sequence) may be
linked to a
cell-specific targeting moiety that binds to a surface molecule that
internalizes the fusion
peptide.
In an other approach, microspheres of artificial polymers of mixed amino
acids (proteinoids) have been used to deliver pharmaceuticals. For example,
U.S. Pat. No.
4,925,673 describes drug-containing proteinoid microsphere carriers as well as
methods for
their preparation and use. These proteinoid microspheres are useful for the
delivery of a
number of active agents. Also see, U.S. Patent Nos. 5,907,030 and 6,033,884,
which are
incorporated herein by reference.
B. Introduction of Polynucleotides into Cells
By introducing gene sequences into cells, gene therapy can be used to treat
diseased cells (e.g., neuron cells that are associated with apoptosis or an
excitotoxic response
due to a neuronal insult). In one embodiment, a polynucleotide that encodes a
PL sequence
peptide of the invention is introduced into a cell where it is expressed. The
expressed peptide
then inhibits the interaction of PDZ proteins and PL proteins in the cell.
Thus, in one embodiment, the polypeptides of the invention are expressed in a
cell by introducing a nucleic acid (e.g., a DNA expression vector or mRNA)
encoding the
desired protein or peptide into the cell. Expression can be either
constitutive or inducible
depending on the vector and choice of promoter. Methods for introduction and
expression of
nucleic acids into a cell are well known in the art and described herein.
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In a specific embodiment, nucleic acids comprising a sequence encoding a
peptide disclosed herein, are administered to a human subject. In this
embodiment of the
invention, the nucleic acid produces its encoded product that mediates a
therapeutic effect.
Any of the methods for gene therapy available in the art can be used according
to the present
S invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
1993,
Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev,
1993,
Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932;
and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH
11(5):155-215. Methods commonly known in the art of recombinant DNA technology
which
can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and
Expression, A
Laboratory Manual, Stockton Press, NY.
In a preferred embodiment of the invention, the therapeutic composition
comprises a coding sequence that is part of an expression vector. In
particular, such a nucleic
acid has a promoter operably linked to the coding sequence, said promoter
being inducible or
constitutive, and, optionally, tissue-specific. In another specific
embodiment, a nucleic acid
molecule is used in which the coding sequence and any other desired sequences
are flanked
by regions that promote homologous recombination at a desired site in the
genome, thus
providing for intrachromosomal expression of the nucleic acid (Koller and
Smithies, 1989,
Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-
438).
Delivery of the nucleic acid into a patient may be either direct, in which
case
the patient is directly exposed to the nucleic acid or nucleic acid-carrying
vector, or indirect,
in which case, cells are first transformed with the nucleic acid in vitro,
then transplanted into
the patient. These two approaches are known, respectively, as in vivo or ex
vivo gene
therapy.
In a specific embodiment, the nucleic acid is directly administered in vivo,
where it is expressed to produce the encoded product. This can be accomplished
by any
methods known in the art, e.g., by constructing it as part of an appropriate
nucleic acid
expression vector and administering it so that it becomes intracellular, e.g.,
by infection using
a defective or attenuated retroviral or other viral vector (see U.S. Patent
No. 4,980,286), by
direct injection of naked DNA, by use of microparticle bombardment (e.g., a
gene gun;
Biolistic, Dupont), by coating with lipids or cell-surface receptors or
transfecting agents, by
encapsulation in liposomes, microparticles, or microcapsules, by administering
it in linkage
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to a peptide which is known to enter the nucleus, or by administering it in
linkage to a ligand
subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol.
Chem.
262:4429-4432) which can be used to target cell types specifically expressing
the receptors.
In another embodiment, a nucleic acid-ligand complex can be formed in which
the ligand
comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic
acid to avoid
lysosomal degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo
for cell specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT
Publications WO 92/06180 dated April 16, 1992; WO 92/22635 dated December 23,
1992;
W092/20316 dated November 26, 1992; W093/14188 dated July 22, 1993; WO
93120221
dated October 14, 1993). Alternatively, the nucleic acid can be introduced
intracellularly and
incorporated within host cell DNA for expression, by homologous recombination
(Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al.,
1989, Nature
342:435-438).
In a preferred embodiment of the invention, adenoviruses as viral vectors can
be used in gene therapy. Adenoviruses have the advantage of being capable of
infecting non-
dividing cells (Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development
3:499-503). Other instances of the use of adenoviruses in gene therapy can be
found in
Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell
68:143-155; and
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234. Furthermore, adenoviral
vectors with
modified tropism may be used for cell specific targeting (W098/40508). Adeno-
associated
virus (AAV) has also been proposed for use in gene therapy (Walsh et al.,
1993, Proc. Soc.
Exp. Biol. Med. 204:289-300).
In addition, retroviral vectors (see Miller et al., 1993, Meth. Enzymol.
217:581-599) have been modified to delete retroviral sequences that are not
necessary for
packaging of the viral genome and integration into host cell DNA. The coding
sequence to
be used in gene therapy is cloned into the vector, which facilitates delivery
of the gene into a
patient. More detail about retroviral vectors can be found in Boesen et al.,
1994, Biotherapy
6:291-302, which describes the use of a retroviral vector to deliver the mdrJ
gene to
hematopoietic stem cells in order to make the stem cells more resistant to
chemotherapy.
Other references illustrating the use of retroviral vectors in gene therapy
are: Clowes et al.,
1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;
Salmons and
Gunzberg, 1993, Human Gene Therapy 4: I 29-141; and Grossman and Wilson, 1993,
Curr.
Opin. in Genetics and Devel. 3:110-114.
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Another approach to gene therapy involves transferring a gene to cells in
tissue culture. Usually, the method of transfer includes the transfer of a
selectable marker to
the cells. The cells are then placed under selection to isolate those cells
that have taken up
and are expressing the transferred gene. Those cells are then delivered to a
patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be carried out
by any method known in the art, including but not limited to transfection,
electroporation,
lipofection, microinjection, infection with a viral or bacteriophage vector
containing the
nucleic acid sequences, cell fusion, chromosome-mediated gene transfer,
microcell-mediated
gene transfer, spheroplast fusion, etc. Numerous techniques are known in the
art for the
introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993,
Meth. Enzymol.
217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985,
Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention, provided
that the
necessary developmental and physiological functions of the recipient cells are
not disrupted.
The technique should provide for the stable transfer of the nucleic acid to
the cell, so that the
nucleic acid is expressible by the cell and preferably heritable and
expressible by its cell
progeny. In a preferred embodiment, the cell used for gene therapy is
autologous to the
patient.
In a specific embodiment, the nucleic acid to be introduced for purposes of
gene therapy comprises an inducible promoter operably linked to the coding
sequence, such
that expression of the nucleic acid is controllable by controlling the
presence or absence of
the appropriate inducer of transcription.
Oligonucleotides such as anti-sense RNA and DNA molecules, and ribozymes
that function to inhibit the translation of a targeted mRNA, especially its C-
terminus are also
within the scope of the invention. Anti-sense RNA and DNA molecules act to
directly block
the translation of mRNA by binding to targeted mRNA and preventing protein
translation. In
regard to antisense DNA, oligodeoxyribonucleotides derived from the
translation initiation
site, e.g., between -10 and +10 regions of a nucleotide sequence, are
preferred.
The antisense oligonucleotide may comprise at least one modified base moiety
which is selected from the group including, but not limited to, 5-
fluorouracil, 5-bromouracil,
S-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
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2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-
D-mannosylqueosine, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-
methylthio-N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-
5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil, 3-(3-amino-
3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA. The mechanism of ribozyme action involves sequence specific
hybridization of the ribozyme molecule to complementary target RNA, followed
by
endonucleolytic cleavage. Within the scope of the invention are engineered
hammerhead
motif ribozyme molecules that specifically and efficiently catalyze
endonucleolytic cleavage
of target RNA sequences.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by scanning the target molecule for ribozyme cleavage sites which
include the
following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of
between 15 and 20 ribonucleotides corresponding to the region of the target
gene containing
the cleavage site may be evaluated for predicted structural features such as
secondary
structure that may render the oligonucleotide sequence unsuitable. The
suitability of
candidate targets may also be evaluated by testing their accessibility to
hybridization with
complementary oligonucleotides, using ribonuclease protection assays.
The anti-sense RNA and DNA molecules and ribozynes of the invention may
be prepared by any method known in the art for the synthesis of nucleic acid
molecules.
These include techniques for chemically synthesizing oligodeoxyribonucleotides
well known
in the art such as for example solid phase phosphoramidite chemical synthesis.
Alternatively,
RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences
encoding the RNA molecule. Such DNA sequences may be incorporated into a wide
variety
of vectors which contain suitable RNA polymerase promoters such as the T7 or
SP6
polymerase promoters. Alternatively, antisense cDNA constructs that synthesize
antisense
RNA constitutively or inducibly, depending on the promoter used, can be
introduced stably
into cell lines.
Various modifications to the DNA molecules may be introduced as a means of
increasing intracellular stability and half life. Possible modifications
include, but are not
limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides
to the 5' and/or 3'
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ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
C. Other Pharmaceutical Compositions
The compounds of the invention, may be administered to a subject per se or in
the form of a sterile composition or a pharmaceutical composition.
Pharmaceutical
compositions comprising the compounds of the invention may be manufactured by
means of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying,
encapsulating, entrapping or lyophilizing processes. Pharmaceutical
compositions may be
formulated in conventional manner using one or more physiologically acceptable
carriers,
diluents, excipients or auxiliaries that facilitate processing of the active
peptides or peptide
analogues into preparations which can be used pharmaceutically. Proper
formulation is
dependent upon the route of administration chosen.
For topical administration the compounds of the invention can be formulated
as solutions, gels, ointments, creams, suspensions, etc. as are well-lalown in
the art.
Systemic formulations include those designed for administration by injection,
e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal
injection, as well
as those designed for transdermal, transmucosal, oral or pulmonary
administration.
For injection, the compounds of the invention can be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hanks's
solution, Ringer's
solution, or physiological saline buffer. The solution can contain formulatory
agents such as
suspending, stabilizing andlor dispersing agents.
Alternatively, the compounds can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
For transmucosal administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art. This
route of administration may be used to deliver the compounds to the nasal
cavity.
For oral administration, the compounds can be readily formulated by
combining the active peptides or peptide analogues with pharmaceutically
acceptable carriers
well known in the art. Such carriers enable the compounds of the invention to
be formulated
as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for
oral ingestion by a patient to be treated. For oral solid formulations such
as, for example,
powders, capsules and tablets, suitable excipients include fillers such as
sugars, such as
lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize
starch, wheat
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starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If
desired,
disintegrating agents may be added, such as the cross-linked
polyvinylpyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate.
If desired, solid dosage forms may be sugar-coated or enteric-coated using
standard techniques.
For oral liquid preparations such as, for example, suspensions, elixirs and
solutions, suitable earners, excipients or diluents include water, glycols,
oils, alcohols, etc.
Additionally, flavoring agents, preservatives, coloring agents and the like
may be added.
For buccal administration, the compounds may take the form of tablets,
lozenges, etc. formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present invention are conveniently delivered in the form of an aerosol spray
from pressurized
packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other
suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined by
providing a valve to
deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in
an inhaler or
insufflator may be formulated containing a powder mix of the compound and a
suitable
powder base such as lactose or starch.
The compounds may also be formulated in rectal or vaginal compositions such
as suppositories or retention enemas, e.g., containing conventional
suppository bases such as
cocoa butter or other glycerides.
In addition to the formulations descrihed previously, the compounds may also
be formulated as a depot preparation. Such long acting formulations may be
administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection.
Thus, for example, the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable oil) or ion
exchange
resins, or as sparingly soluble derivatives, for example, as a sparingly
soluble salt.
Alternatively, other pharmaceutical delivery systems may be employed.
Liposomes and emulsions are well known examples of delivery vehicles that may
be used to
deliver peptides and peptide analogues of the invention. Certain organic
solvents such as
dimethylsulfoxide also may be employed, although usually at the cost of
greater toxicity.
Additionally, the compounds may be delivered using a sustained-release system,
such as
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semipermeable matrices of solid polymers containing the therapeutic agent.
Various of
sustained-release materials have been established and are well known by those
skilled in the
art. Sustained-release capsules may, depending on their chemical nature,
release the
compounds for a few weeks up to over 100 days. Depending on the chemical
nature and the
S biological stability of the therapeutic reagent, additional strategies for
protein stabilization
may be employed.
As the compounds of the invention may contain charged side chains or
termini, they may be included in any of the above-described formulations as
the free acids or
bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable
salts are those
salts which substantially retain the biologic activity of the free bases and
which are prepared
by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble
in aqueous
and other protic solvents than are the corresponding free base forms.
D. Effective Dosages
The compounds of the invention will generally be used in an amount effective
to achieve the intended purpose (e.g., treatment of a neuronal injury). The
compounds of the
invention or pharmaceutical compositions thereof, are administered or applied
in a
therapeutically effective amount. By therapeutically effective amount is meant
an amount
effective ameliorate or prevent the symptoms, or prolong the survival of, the
patient being
treated. Determination of a therapeutically effective amount is well within
the capabilities of
those skilled in the art, especially in light of the detailed disclosure
provided herein. An
"inhibitory amount" or "inhibitory concentration" of a PL-PDZ binding
inhibitor is an
amount that reduces binding by at least about 40%, preferably at least about
50%, often at
least about 70°l0, and even as much as at least about 90%. Binding can
as measured in vitro
(e.g., in an A assay or G assay) or in situ.
For systemic administration, a therapeutically effective dose can be estimated
initially from in vitro assays. For example, a dose can be formulated in
animal models to
achieve a circulating concentration range that includes the ICSO as determined
in cell culture.
Such information can be used to more accurately determine useful doses in
humans.
Initial dosages can also be estimated from irr vivo data, e.g., animal models,
using techniques that are well known in the art. One having ordinary skill in
the art could
readily optimize administration to humans based on animal data.
Dosage amount and interval may be adjusted individually to provide plasma
levels of the compounds that are sufficient to maintain therapeutic effect.
Usual patient
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dosages for administration by injection range from about 0.1 to 5 mglkg/day,
preferably from
about 0.5 to 1 mglkg/day. Therapeutically effective serum levels may be
achieved by
administering multiple doses each day. For usual peptide therpaeutic treatment
of stroke,
acute administration of 0.03 nmol/g to 30 mnol/g within G hours of stroke or
brain ischemia is
typical. In other instances, 0.1 nmol/g to 20 nmol/g within G hours are
administered. And in
still other instances lnmollg to 10 nmol/g is administered with in G hours.
In cases of local administration or selective uptake, the effective local
concentration of the compounds may not be related to plasma concentration. One
having
skill in the art will be able to optimize therapeutically effective local
dosages without undue
experimentation.
The amount of compound administered will, of course, be dependent on the
subject being treated, on the subject's weight, the severity of the
affliction, the manner of
administration and the judgment of the prescribing physician.
The therapy may be repeated intermittently while symptoms detectable or
even when they are not detectable. The therapy may be provided alone or in
combination
with other drugs.
E. Toxicity
Preferably, a therapeutically effective dose of the compounds described herein
will provide therapeutic benefit without causing substantial toxicity.
Toxicity of the compounds described herein can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., by
determining the
LDSO (the dose lethal to 50% of the population) or the LD~oo (the dose lethal
to 100% of the
population). The dose ratio between toxic and therapeutic effect is the
therapeutic index.
Compounds which exhibit high therapeutic indices are preferred. The data
obtained from
these cell culture assays and animal studies can be used in formulating a
dosage range that is
not toxic for use in human. The dosage of the compounds described herein lies
preferably
within a range of circulating concentrations that include the effective dose
with little or no
toxicity. The dosage may vary within this range depending upon the dosage form
employed
and the route of administration utilized. The exact formulation, route of
administration and
dosage can be chosen by the individual physician in view of the patient's
condition. (See,
e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.l,
p.l).
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EXAMPLE 1
GENERATION OF EUKARYOTIC EXPRESSION CONSTRUCTS
BEARING DNA FRAGMENTS THAT ENCODE PDZ DOMAIN CONTAINING GENES
OR PORTIONS OF PDZ DOMAIN GENES
This example describes the cloning of PDZ domain containing genes or
portions of PDZ domain containing genes into prokaryotic expression vectors in
fusion with
Glutathione S-Transferase (GST). Some PDZ proteins were also cloned into
eukaryotic
expression vectors in fusion with a number of protein tags, including but not
limited to
Enhanced Green Fluorescent Protein (EGFP) or Hemagglutinin (HA).
A. Strategy
DNA fragments corresponding to PDZ domain containing genes were
generated by RT-PCR from RNA from a library of individual cell lines (CLONTECH
Cat#
K4000-1) derived RNA, using random (oligo-nucleotide) primers (Invitrogen
Cat.#
48190011). DNA fragments corresponding to PDZ domain containing genes or
portions of
PDZ domain containing genes were generated by standard PCR, using above
purified cDNA
fragments and specific primers (see TABLE 5). Primers used were designed to
create
restriction nuclease recognition sites at the PCR fragment's ends, to allow
cloning of those
fragments into appropriate expression vectors. Subsequent to PCR, DNA samples
were
submitted to agarose gel electrophoresis. Bands corresponding to the expected
size were
excised. DNA was extracted by Sephaglas Band Prep Kit (Amersham Pharmacia Cat#
27-
9285-01) and digested with appropriate restriction endonuclease. Digested DNA
samples
were purified once more by gel electrophoresis, according to the same protocol
used above.
Purified DNA fragments were coprecipitated and ligated with the appropriate
linearized
vector. After transformation into E.coli, bacterial colonies were screened by
colony PCR and
restriction digest for the presence and correct orientation of insert.
Positive clones were
innoculated in liquid culture for large scale DNA purification. Plasmid
purification was done
by mini, midi, or maxiprep (Quiagen or Mo Bio), according to the
manufacturer's protocol.
The insert and flanking vector sites from the purified plasmid DNA were
sequenced to ensure
correct sequence of fragments and junctions between the vectors and fusion
proteins.
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B. Vectors
All PDZ domain-containing genes were cloned into the vector pGEX-3X
(Amersham Pharmacia #27-4803-Ol, Genemed Acc#U13852, GI#595717), containing a
tac
promoter, GST, Factor Xa, ~3-lactamase, and lac repressor.
The amino acid sequence of the pGEX-3X coding region including GST,
Factor Xa, and the multiple cloning site is listed below. Note that linker
sequences between
the cloned inserts and GST-Factor Xa vary depending on the restriction
endonuclease used
for cloning. Amino acids in the translated region below that may change
depending on the
insertion used are indicated in small caps, and are included as changed in the
construct
sequence listed in (C).
as 1 - as 232:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEF
PNLPYYIDGDVKLTQSMAIIRYIADKIINMLGGCPKERAEISMLEGAVLDIRYG
V SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD
ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQAT
FGGGDHPPKSDLIEGRgipgnss
C. Constructs
Primers used to generate DNA fragments by PCR are listed in TABLE 5.
PCR primer combinations and restriction sites for insert and vector are listed
below, along
with amino acid translation for insert and restriction sites. Non-native amino
acid sequences
are shown in lower case.
TABLE 5. Primers used in cloning of PSD95 (all 3 domains), DLG 1 (domains 1
and 2),
TIP2 (domain 1 of 1), and LIM (domain 1 of 1) into representative expression
vectors.
Primer Primer Sequence Description
Name
1DF TCGGATCCAGGTT Forward (5' to 3') primer corresponding
to DLG 1,
AATGGCTCAGAT nucleotide numbers 815-841. Generates
a Bam H1
G site upstream (5') of the PDZ 1 boundary.
Used for
cloning into pGEX-3X.
2DR CGGAATTCGGTG Reverse (3' to 5') primer corresponding
to DLG l,
CATAGCCATC nucleotide numbers 1442-1421. Generates
an
EcoRl site downstream (3') of the
PDZ 2 boundary.
Used for cloning into pGEX-3X.
BPSF TCGGATCCTTGAG Forward (5' to 3') primer corresponding
to PSD95,
GGGGAGATGGA nucleotide numbers 1150-1173. Generates
a
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BamHl site upstream (5') of the PDZ
1 boundary.
Used for cloning into pGEX-3X.
11PSR TCGGAATTCGCTA Reverse (3' to 5') primer corresponding
to PSD95,
TACTCTTCTGG nucleotide numbers 2191-21 G8. Generates
an
EcoRl site downstrealo (3') of the
PDZ 3 boundary.
Used for cloning into pGEX-3X.
182LF TTAGGATCCTGAG Forward (5' to 3') primer corresponding
to LIM,
CAAGTACAGTGT nucleutide numbers 86-115. Generates
a Bam Hl
GTCAC site upstream (5') of the PDZ boundary.
Used for
cloning into pGEX-3X.
183LR CTTGAATTCAGCA Reverse (3' to 5') primer corresponding
to LIM,
GATGCTCTTTGCA nucleotide numbers 350-320. Generates
an EcoRl
GAGTC site downstream (3') of the PDZ boundary.
Used for
cloning into pGEX-3X.
197TF AGGGGATCCGCA Forward (5' to 3') primer corresponding
to TIP2.
AGGAGGTGGAGG Generates a Bam Hl site upstream (5')
of the PDZ
TGTTC boundary. Used for cloning into pGEX-3X.
198TR TGTGGAATTCCTT Reverse (3' to 5') primer corresponding
to TIP2,
GCGAGGCTCCGT nucleotide numbers 429-401. Generates
an EcoRl
GAGC site downstream (3') of the PDZ boundary.
Used for
cloning into pGEX-3X.
1. DLG 1, PDZ domains 1 and 2:
Acc#:U13897
~Construct: DLG l, PDZ domains I and 2-pGEX-3X
Primers: 1DF & 2DR
Vector Cloning Sites(5'/3'): Bam Hl/EcoRl
Insert Cloning Sites(5'/3'): BamHl/EcoRl
as 275- as 477
qVNGTDADYEYEEITLERGNSGLGFSIAGGTDNPHIGDDSSIFITKIITGG
AAAQDGRLRVNDCILRVNEVDVRDVTHSKAVEALKEAGSIVRLYVK
RRKPVSEKIMEIKLIKGPKGLGFSIAGGVGNQI-IIPGDNSIYVTKIIEGGA
AHKDGKLQIGDKLLAVNNVCLEEVTHEEAVTALKNTSDFVYLKVAK
PTSMYMNDGYApns
2. PSD95, PDZ domains 3 of 3:
Acc#:U83192
~Construct: PSD95, PDZ domains 3 of 3-pGEX-3X
Primers: BPSF & 11PSR
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Vector Cloning Sites(5'/3'): Bam H1/EcoRl
Insert Cloning Sites(5'/3'): BamHl/EcoRl
as 387- as 724
legEGEMEYEEITLERGNSGLGFSIAGGTDNPHIGDDPSIFITKIIP
GGAAAQDGRLRVNDSILFVNEVDVREVTHSAAVEALKEAGSI
VRLYVMRRKPPAEKVMEIKLIKGPKGLGFSIAGGVGNQHIPGD
NSIYVTKIIEGGAAHKDGRLQIGDKILAVNSVGLEDVMHEDAV
AALKNTYDVVYLKVAKPSNAYLSDSYAPPDITTSYSQHLDNEI
SHSSYLGTDYPTAMTPTSPRRYSPVAKDLLGEEDIPREPRRIVI
HRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDQILS
VNGVDLRNASHEQAAIALKNAGQTVTIIAQYKPEf v
3. TAX Interacting Protein 2 (TIP2):
Acc#:AF028824
.Construct: TIl'2, PDZ domain 1 of 1-pGEX-3X
Primers: 197TF & 198TR
Vector Cloning Sites(5'/3'): Bam H1/EcoRl
Insert Cloning Sites(5'/3'): BamHl/EcoRl
as 54- as 140
RKEVEVFKSEDALGLTITDNGAGYAFIKRIKEG S V IDHIHLIS V G
DMIEAINGQSLLGCRHYEVARLLKELPRGRTFTLKLTEPRKefiv
td
3. LIM Protein:
Acc#:AF061258
.Construct: LIM-pGEX-3X
Primers: 182LF & 183LR
Vector Cloning Sites(S'/3'): Bam H1/ Bam H1
Insert Cloning Sites(5'/3'): BamHl/ Bam H1
as 29- as 112
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1SNYSVSLVGPAPWGFRLQGGKDFNMPLTISSLKDGGKAAQA
NVRIGDVVLSIDGINAQGMTHLEAQNKIKGCTGSLNMTLQRA
Sc
D. GST Fusion Protein Production and Purification
The constructs using pGEX-3X expression vector were used to make fusion
proteins according to the protocol outlined in the "GST Gene Fusion System",
Second
Edition, Revision 2, Pharmacia Biotech. Method II was used, optimized for a 1L
LgPP.
In brief, a small culture (3-Smls) containing a bacterial strain (DH50, BL21
or
JM109) with the fusion protein construct was grown overnight in 2XYT-media at
37°C with
the appropriate antibiotic selection (100uglml ampicillin; a.k.a. LB-amp). The
overnight
culture was poured into a fresh preparation of 2XYT-amp (typically 250-500m1s)
and grown
until the optical density (OD) of the culture was between 0.5 and 0.9
(approximately 2.5
hours). IPTG was added to a final concentration of l.OmM to induce production
of GST
fusion protein, and culture was grown an additional 1-2 hours. Bacteria were
collect by
centrifugation (4500 g) and resuspended in Buffer A- (50mM Tris, pH 8.0, 50mM
dextrose,
1mM EDTA, 200uM PMSF). An equal volume of Buffer A+ (Buffer A-, 4mg/ml
lysozyme)
was added and incubated on ice for 3 min to lyse bacteria. An equal volume of
Buffer B
(lOmM Tris, pH 8.0, 50mM KCI, 1mM EDTA. 0.5% Tween-20, 0.5% NP40 (a.k.a.
IGEPAL
CA-630), 200uM PMSF) was added and incubated for an additional 20 min. The
bacterial
cell lysate was centrifuged (x20,000g), and supernatant was added to
Glutathione Sepharose
4B (Pharmacia, cat no. 17-0765-O1) previously swelled (rehydrated) in 1X
phosphate-
buffered saline (PBS). The supernatant-Sepharose slurry was poured into a
column and
washed with at least 20 bed volumes of 1X PBS. GST fusion protein was eluted
off the
glutathione sepharose by applying 0.5-1.0 ml aliquots of 5mM glutathione and
collected as
separate fractions. Concentrations of fractions were determined using BioRad
Protein Assay
(cat no. 500-0006) according to manufacturer's specifications. Those fractions
containing the
highest concentration of fusion protein were pooled and glycerol was added to
a final
concentration of 35% glycerol. Fusion proteins were assayed for size and
quality by SDS gel
electrophoresis (PAGE). Fusion protein aliquots were stored at minus
80°C.
Purified proteins were used for ELISA-based assays and antibody production.
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EXAMPLE 2
>DENTIFICATION OF N-METHYL-D-ASPARTATE RECEPTOR 2A (NMDAR2A)
INTERACTIONS WITH PSD95 TIP2, DLGI, AND LIM IN VITRO
This example describes the binding of NMDAR2A to PSD95, TIP2, DLG1,
and LIM, assessed using a modified ELISA. Briefly, a GST-PDZ fusion was
produced that
contained the entire PDZ domain of human LIM or TIP2, domains 1 and 2 of 3 in
DLG1, or
all 3 PDZ domains for PSD95 (see Example 1). In addition, biotinylated peptide
corresponding to the C-terminal 20 amino acids of NMDAR2A was synthesized and
purified
by HPLC. Binding between these entities was detected through the "G" Assay, a
colorimetric assay using avidin-HRP to bind the biotin and a peroXidase
substrate.
A. Peptide Purification
Peptide representing the C-terminal 20 amino acids of NMDAR2A, as shown in
TABLES 2 and 3 was synthesized by standard FMOC chemistry and biotinylated if
not used
as an unlabeled competitor. Peptide was purified by reverse phase high
performance liquid
chromatography (HPLC) using a Vydac 218TP C18 Reversed Phase column having the
dimensions of 10*25 mm, 5 um. Approximately 40 mg of peptide was dissolved in
2.0 ml of
aqueous solution of 49.9% acetonitrile and 0.1% Tri-Fluoro acetic acid (TFA).
This solution
was then injected into the HPLC machine through a 25 micron syringe filter
(Millipore).
Buffers used to get a good separation are (A) distilled water with 0. I % TFA
and (B) 0.1
TFA with Acetonitrile. Gradient Segment setup is listed in TABLE 6.
TABLE 6.
Time A B C Flow rate (ml/min)
0 96% 4% 0 5.00
100% 100% 0 5.00
100% 100% 0 5.00
96% 4% 0 5.00
The separation occurs based on the nature of the peptides. A peptide of
overall hydrophobic
nature will elute off later than a peptide of a hydrophilic nature. Fractions
containing the
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"pure" peptide were collected and checked by Mass Spectrometer (MS). Purified
peptides
are lyophilized for stability and later use.
B. "G" Assay for Identification of Interactions Between Peatides and Fusion
Proteins
Reagents and Materials:
~ Nunc Polysorp 96 well Immuno-plate (Nunc cat#62409-005)
(Maxisorp plates have been shown to have higher background signal)
~ PBS pH 7.4 (Gibco BRL cat#16777-148) or (AVC phosphate buffered saline, 8gm
NaCI,
0.29 gm KC1, 1.44 gm Na2HP04, 0.24gm KHZP04, add H20 to 1 L and pH 7.4; 0.2Eun
filter
~ 2% BSA/PBS (1 Ogm of bovine serum albumin, fraction V (ICN Biomedicals
cat#IC15142983) into 500 ml PBS
~ Goat anti-GST mAb stock @ S mg/ml, store at 4°C, (Amersham Pharmacia
cat#27-4577-
O1), dilute 1:1000 in PBS, final concentration 5 ~glml
~ HRP-Streptavidin, 2.Smg/2ml stock stored at 4°C (Zymed cat#43-4323),
dilute 1:2000
into 2% BSA, final concentration at 0.5 pg/ml
~ Wash Buffer, 0.2% Tween 20 in SOmM Tris pH 8.0
~ TMB ready to use (Dako cat#S 1600)
~ 1M HZS04
~ 12w multichannel pipettor,
~ 50 ml reagent reservoirs,
~ 1 S ml polypropylene conical tubes
Protocol
1 ) Coat plate with 100 pl of 5 ~g/ml goat anti GST, O/N @ 4°C
2) Dump coating antibodies out and tap dry
3) Blocking - Add 200 p.l per well 2% BSA, 2 hrs at 4°C
4) Prepare proteins in 2% BSA
(2ml per row or per two columns)
5) 3 washes with cold PBS (must be cold through entire experiment)
(at last wash leave PBS in wells until immediately adding next step)
6) Add proteins at SOpI per well on ice (1 to 2 hrs at 4°C)
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7) Prepare Peptides in 2% BSA (2 ml/row or /columns)
8) 3 X wash with cold PBS
9) Add peptides at 50 pl per well on ice (time on / time off)
a. keep on ice after last peptide has been added for 10 minutes exactly
b. place at room temp for 20 minutes exactly
10) Prepare 12 ml/plate of HRP-Streptavidin (1:2000 dilution in 2%BSA)
11 ) 3 X wash with cold PBS
12) Add HRP-Streptavidin at 100 ul per well on ice, 20 minutes at 4°C
13) Turn on plate reader and prepare files
14) 5 X washes, avoid bubbles
15) Using gloves, add TMB substrate at 100 pl per well
a. incubate in dark at room temp
b. check plate periodically (5, 10, & 20 minutes)
c. take early readings, if necessary, at 650 nm (blue)
d. at 20 minutes, stop reaction with 100 ul of 1M H2S04
e. take last reading at 450nm (yellow)
C. Results of Binding Experiments
Results of peptides representing the carboxy-terminal 20 amino acids of
NMDAR2A binding to PSD95, TIP2, DLG1, and LIM are shown in Figure 1. NMDAR2A
binds GST-PSD95 and GST-DLGI with much higher affinity than it does to GST-LIM
or
GST-Tll'2 at equivalent peptide concentrations and with an equivalent amount
of GST-PDZ
fusion protein. Because the interaction between NMDAR2A and LIM is not
significantly
higher than background, this particular experiment indicates that LIM PDZ's
may not interact
with NMDAR2A PL peptide.
D. Conclusions and Summary
PSD-95 and DLG1 bind to NMDAR2A better than TlP2 and LIM bind to the
same peptide. Thus, they are more likely to be in vivo interactions and
binding of PDZ
domains to the C-terminus of NMDA R2A will strongly favor PSD-95 and DLG1 over
T1P2
or Lim.
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EXAMPLE 3
TREATMENT OF ISCHEMIC BRAIN DAMAGE BY MODULATING NMDA-
RECEPTOR PSD-95 INTERACTIONS
Recent experiments performed by Aarts et al. (Science 298:846-850, 2002,
which is incorporated herein by reference in its entirety) are consistent with
the interactions
identified herein, specifically the interactions between NMDAR and PSD-95.
Aarts et al. conducted studies with a fusion polypeptide in which the C-
terminal 9 amino acids of NMDA Receptor 2B were fused to a Tat transmembrane
transporter peptide and found that this fusion polypeptide could inhibit
binding between
NMDAR2 with domain 2 of PSD-95. The sequence of the inhibitory peptide was
YGRKKRRQRRRKLSSIESDV. These researchers also demonstrated that this peptide,
when labeled with dansyl chloride, could penetrate cells in the coronal
section of the brain in
a short amount of time. It was also found that administration of the
polypeptide to rats either
before or up to one hour after induction of transient middle cerebral artery
occlusion
(MCAO) significantly protected the rat brain from ischemic damage due to the
occlusion.
For example, in the presence of the inhibitory polypeptide, the infarct area
in the cortical area
of the brain following inducement of MCAO was reduced to below 20% of the
infarct area of
untreated rats.
EXAMPLE 4
NMDA RECEPTOR 2 SUBUNITS BIND A NUMBER OF PDZ DOMAINS
The selectivity of NMDA Receptor 2 (NR2) subunit binding to PDZ domains was
assayed using the G assay described supra. Biotinylated peptides corresponding
to the C-
terminal 19 or 20 amino acids of NR2A, NR2B, NR2C, and NR2D were synthesized
and
tested for their ability to specifically interact with 238 independent PDZ
domain constructs.
Figure 2 shows the results of these interactions. Each binds a similar subset
of
approximately 16 to 20 PDZ domains. PDZ interactions that are common to all
NMDA R2
subunits or to only a subset are listed in TABLE 7.
TABLE 7: PDZ domains that interact strongly with NMDA R2 subunits
PDZ domains that interact with all I PDZ domains that interact with a subset
of
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NR2 s NR2 s
DLG1 d2 DLGl dl
DLG2 d2 INADL d8
KIAA0973 KIAA0807
NeDLG KIAA 1634 d 1
Outermembrane Protein Lim-Mystique
PSD-95 d2 L1M-RIL
Syntrophin alpha 1 MAGII d2,d4,d5
TIP1 MAGI2 d5
TIP2 PSD-95 dl
Syntrophin beta-1
Syntrophin gamma 1
Table 7 legend: Domain numbers of PDZ proteins that contain multiple Y1~G
domains are
indicated as dl or d2 etc.
Concurrent binding tests were performed with the main R2 subunits indicated in
neuroprotection (R2A, R2B, R2C) and the individual and complexed PDZ domains
of PSD95
(Figure 3). All three NR2 subunits bind PSD95 domains 1 and 2 but fail to bind
PSD95
domain 3. Peptides corresponding to the C-termini of all 4 NR2 subunits were
titrated
against a constant amount of PSD95 PDZ domain proteins to determine relative
binding
affinilties for the PL-mimicking peptides and each domain of PSD95. Results
are shown in
Figure 4.
These experiments show that NR2 subunits can bind a number of different PDZ
domains, and that the highest relative affinity interaction occurs between
NR2C and PSD95
domain 2. Thus, peptides as described in Example 3 may inhibit a number of
interactions.
In addition to previous demonstrations that NMDA Receptor antagonists are
neuroprotective,
previous research has demonstrated that reduction of PSD95 protein in neuronal
cells is
neuroprotective. The methods for identifying inhibitors disclosed herein can
be used to
identify inhibitors that are specific for PSD-95 as well as inhibitors
specific to other NMDA
R2 PDZ interactions. Using such specific inhibitors, one can ascertain whether
the
neuroprotective effect of inhibitors is due wholly or partially to the NMDA R2
PSD95
interactions. Specific inhibitors that block only the necessary interactions)
are extremely
valuable in the reduction of side effects which often occur during clinical
testing.
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EXAMPLE 5
1NDENTIFICATION OF 3,4 AND 19 AMINO ACID INHIBITORS OF NR2
SUBLINIT/PSD95 PDZ INTERACTIONS
A number of peptides of different length were synthesized and tested for their
ability
to inhibit NMDA R2 interactions with PSD95 domain 1 or domain 2. These
peptides were
tested using the G assay as described supra and results are shown in Figures 5-
9.
Figure 5 shows the ability of N-terminal acetylated peptides corresponding to
the C-
terminal 3 amino acids of the TAX oncoprotein (Ac-TEV) and NMDA R2B (Ac-SDV)
to
inhibit the interaction between NMDA R2A and PSD95 domain 1 or domain 2. Both
peptides are able to inhibit the interactions of NR2A and PSD95 domain 2, and
only at the
highest concentration (1mM) is any inhibition seen with PSD95 domain 1 and
NR2A.
Figure 6 shows the ability of N-terminal peptides corresponding to the C-
terminal 19
or 20 amino acids of the TAX oncoprotein and HPV 16 E6 protein to inhibit the
interaction
between NMDA R2C and PSD95 domain 1 or domain 2. Both peptides are able to
inhibit the
interactions of NR2C and PSD95 domain 2, and no inhibition between PSD95
domain 1 and
NR2C is seen in this concentration range (up to 100uM). Peptides corresponding
to the C-
terminus of TAX (ending ETEV) show better inhibition that those of E6 (ending
ETQL).
Figure 7 shows the ability of N-terminal acetylated peptides corresponding to
the C-
terminal 3 amino acids of the TAX oncoprotein (Ac-TEV) and NMIDA R2B (Ac-SDV)
to
inhibit the interaction between NMDA R2C and PSD95 domain 1 or domain 2. Both
peptides are able to inhibit the interaction between NR2C and PSD95 domain 2,
and no
inhibition between PSD95 domain 1 and NR2C is seen in this concentration range
(up to
1 mM).
Figure 8 shows the ability of N-terminal acetylated peptides corresponding to
the C-
terminal 4 amino acids of the TAX oncoprotein (Ac-ETEV) and NMDA R2B (Ac-ESDV)
to
inhibit the interaction between NMDA R2C and PSD95 domain 1 or domain 2. Both
peptides are able to inhibit the interaction between NR2C and PSD95 domain 2,
and no
inhibition between PSD95 domain 1 and NR2C is seen in this concentration range
(up to
1mM). These 4 amino acid inhibitors both demonstrate a slightly better Ki than
the 3 amino
acid variants.
Figure 9 shows the ability of N-terminal peptides corresponding to the C-
terminal 19
or 20 amino acids of the TAX oncoprotein and HPV 16 E6 protein to inhibit the
interaction
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between NMDA R2A and PSD95 domain 1 or domain 2. Both peptides are able to
inhibit
the interactions of NR2A and PSD95 domain 2, and for TAX sequences inhibition
of the
interaction between PSD95 domain 1 and NR2A is seen at 1 to 10 peptide
concentration
equivalents ([NR2A] for PSD95 domain 1 = lOuM; inhibition seen at Tax
concentrations of
lOuM and 100uM). Peptides corresponding to the C-terminus of TAX (ending ETEV)
show
better inhibition that those of E6 (ending ETQL) for both domain 1 and 2 of
PSD95.
Figure 12 shows that although both NMDA R2A and NMDA R2C can bind the 1st
PDZ of PSD-95, either a 20 amino acid or a three amino acid inhibitor
corresponding to the
C-terminus of the TAX oncoprotein can selectively block the ability of NMDA
R2A (PL1) to
bind PSD-95 dl without blocking the ability of NMDA R2C (PL2) to bind PSD-95
dl.
Summary
Peptide inhibitors of NMDA Receptor 2 subunit interactions with PSD95 PDZ
domains 1 and 2 have been identified. These inhibitors can function with as
little as 3 amino
acids to 20 amino acids with increasing affinity. Many more sequences were
tested in this
manner, and peptide inhibitors terminating in ETEV, ETQL, QTQV, ETAL, QTEV and
ESEV showed the best ability to block interactions between NMDA R2's and PSD95
domain
2 (with varying concurrent ability to inhibit PSD95 domain 1 interactions).
Peptides
sequences terminating in ETVA and FTDV had greater ability to inhibit PSD95
domain 1
interactions. Grouping these findings, peptides with consensus X-T-X-(V,L, or
A) can inhibit
PSD95 domain 1 and 2 interactions with NMDA Receptor 2 subunits. Figure 12
shows the
ability to achieve selective inhibition, where either the 20 amino acid or the
three amino acid
inhibitors corresponding to the C-terminus of Tax (TEV) are able to
selectively inhibit the
interaction of NR2A with PSD95 domain 1 without inhibiting the ability of NR2C
to interact
with domain 1. Thus, using approaches such as described herein, one can design
inhibitors to
selectively modulate interactions to treat specific phenotypes without
disrupting all potential
activities of the PDZ domain.
EXAMPLE 6
ADDITION OF TRANSPORTER PEPTIDES TO 9 AMINO ACID INHIBITORS DOES
NOT NEGATIVELY AFFECT INHIBITORY ABILITY
Since peptides corresponding to the TAX oncoprotein were effective inhibitors
of
NMDA R2 interactions with PSD95 PDZ domains, transporter peptide-coupled
versions with
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native or disrupted PL sequences were synthesized. These peptides were
assessed for their
ability to inhibit interactions between PSD95 domain 2 and either NMDAR2A or
R2B. The
TatTAX construct with the native PL was able to show inhibition at
concentrations as low as
nM (Figure 10), with half maximal inhibition between .l and 1.0 uM. When the
PL was
5 mutated by alanine substitution at residues 0 and -2, no inhibition was seen
for NMDA R2A
or R2B with PSD95 domain 2, indicating that the inhibition is dependent on the
PL sequence
and not the additional transporter sequences. In a similar manner, other
transporters
described supra were also effective.
10 EXAMPLE 7
AN INTERNAL PDZ LIGAND 1N NNOS BINDS TO THE 2N° PDZ DOMAIN OF
PSD95
WITHOUT BINDING THE 1sT OR 3R° PDZS
Excitotoxicity stemming from ischemia or neurotrauma is highly correlated with
Nitric Oxide (NO) production. Inhibition of nNOS, the major neuronal enzyme
producing
NO, is neuroprotective, and nNOS itself has been shown to interact with PSD95.
An
expression construct was made comprising the Maltose Binding Protein (MBP) and
amino
acids 1-120 of nNOS (gi:10835173). This protein was used in the G assay in
place of a
labeled peptide to assess the interaction between PSD95 domains 1 and 2 and
nNOS, and
detection was performed using an HRP-conjugated antibody against MBP. Figure
11 shows
that the internal PL of MBP-nNOS can specfically recognize PDZ domain 2 of
PSD95 but
fails to interact with PSD95 domain 1. MBP/PSD95 indicates a negative control
containing
MBP without the nNOS sequences.
Since PSD95 and nNOS have each been implicated in neuroprotection, modulating
of
the interaction between these proteins provides an alternative therapeutic
target for
neuroprotection.
EXAMPLE 8
THE PDZ DOMAIN OF NNOS BINDS A NUMBER OF DIFFFERNT PL SEQUENCES
AND CAN RECRUIT ADDITIONAL PROTEINS TO NMDAR/PSD95/NNOS
COMPLEXES
The G assay was used to identify PL sequences able to bind the PDZ domain of
nNOS, and these sequences and binding values are shown in TABLE 8. These
sequences
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can be used to identify similar sequences in the genome (methods described
supra) that may
be important nNOS interactions involved in neurotoxicity or neuroprotection.
Designing
inhibitors that block the PDZ domain of nNOS in neurons or neuronal tissues
could provide
and attractive alternative therapeutic target for neuroprotection.
S
TABLE 8: PL sequences that bind the PDZ domain of nNOS by G-assay.
PL Name Average Relative Sequence
OD STDV
AdenoE4 typ9 4.00 0.0000 VGTLLLERVIFPSVKIATLV
AdenoE4 typ9 4.00 0.0000 VGTLLLERVIFPSVKIATLV
ephrin A2 4.00 0.0000 RIAYSLLGLKDQVNTVGIPI
HPV-E6 #63 4.00 0.0000 VHKVRNKFKAKCSLCRLYII
HPV-E6 #63 4.00 0.0000 VHKVRNKFKAKCSLCRLYII
M I NT-1 4.OO O.OOOO KTMPAAMYRLLTAQEQPVYI
MINT-1 4.OO O.OOOO KTMPAAMYRLLTAQEQPVYI
MINT-1 4.00 0.0163 KTMPAAMYRLLTAQEQPVYI
MINT-1 ~ 3.76 0.0047 KTMPAAMYRLLTAQEQPVYI
AdenoE4 typ9 3.64 0.0259 VGTLLLERVIFPSVKIATLV
a-2B Adrenergic receptor3.54 0.1835 QDFRRAFRR1LARPWTQTAW
a-2C Adrenergic receptor3.02 0.4586 DFRPSFKHILFRRARRGFRQ
AdenoE4 typ9 2.73 0.0005 VGTLLLERVIFPSVKIATLV
HPV-E6 #63 2.55 0.0385 VHKVRNKFKAKCSLCRLYII
a-2B Adrenergic receptor2.52 0.0208 QDFRRAFRRILARPWTQTAW
CSPG4 (chondroitin 2.43 0.0134 ELLQFCRTPNPALKNGQYWV
sulfae
proteoglycan 4, melanoma-
associated
DNAM-1 2.40 0.0871 TREDIYVNYPTFSRRPKTRV
HPV-E6 #18 2.14 0.1098 HSCCNRARQERLQRRRETQV
catenin - delta 2 2.10 0.0967 PYSELNYETSHYPASPDSWV
alpha-2C Adrenergic 2.09 0.0186 DFRPSFKHILFRRARRGFRQ
receptor
Fas Ligand 1.99 0.0515 SSKSKSSEESQTFFGLYKL
Fas Ligand 1.93 0.1362 SSKSKSSEESQTFFGLYKL
ephrin A2 1.83 0.0186 RIAYSLLGLKDQVNTVGIPI
presenilin-1 1.80 0.0173 ATDYLVQPFMDQLAFHQFYI
ephrin B2 1.68 0.0644 ILNSIQVMRAQMNQIQSVEV
DNAM-1 1.67 0.1086 TREDIYVNYPTFSRRPKTRV
Dopamine transporter 1.38 0.0471 RELVDRGEVRQFTLRHWLKV
CSPG4 (chondroitin 1.27 0.0627 ELLQFCRTPNPALKNGQYWV
sulfae
proteoglycan 4, melanoma-
associated
Dopamine transporter 1.27 0.0545 RELVDRGEVRQFTLRHWLKV
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PL Name Average Relative Sequence
OD STDV
Dopamine transporter 1.10 0.0155 RELVDRGEVRQFTLRHWLKV
noradrenaline transporter1.08 0.0864 HHLVAQRDIRQFQLQHWLAI
presenilin-1 1.06 0.1494 ATDYLVQPFMDQLAFHQFYI
Serotonin receptor 1.02 0.1510 LAVLAYSITLVMLWSIWQYA
3a
claudin 7 1.01 0.1490 KAGYRAPRSYPKSNSSKEYV
alpha-2A Adrenergic 0.96 0.0471 HDFRRAFKKILARGDRKRIV
receptor
DNAM-1 0.96 0.1079 TREDIYVNYPTFSRRPKTRV
claudin 7 0.93 0.0053 KAGYRAPRSYPKSNSSKEYV
claudin 1 0.90 0.1158 SYPTPRPYPKPAPSSGKDYV
DNAM-1 0.86 0.0148 TREDIYVNYPTFSRRPKTRV
beta-2 Adrenergic Receptor0.85 0.9946 VPSDNIDSQGRNASINDSLL
LPAP 0.77 0.1007 AWDDSARAAGGQGLHVTAL
GLUR7 (metabotropic 0.75 0.1655 VDPNSPAAKKKYVSYNNLVI
lutamate rece for
HPV E6 #35 (cysteine-free)0.74 0.0411 GRWTGRAMSAWKPTRRETE
V
alpha-2A Adrenergic 0.73 0.1252 HDFRRAFKKILARGDRKRIV
receptor
KIAA 1481 0.71 0.0961 PIPAGGCTFSGIFPTLTSPL
claudin 2 0.69 0.1976 PGQPPKVKSEFNSYSLTGYV
CD68 0.67 0.0242 ALVLIAFC11RRRPSAYQAL
KV1.3 0.67 0.0011 TTNNNPNSAVNIKKIFTDV
GIuR5-2 (rat) 0.65 0.0186 SFTSILTCHQRRTQRKETVA
GLUR7 (metabotropic 0.64 0.0759 VDPNSPAAKKKYVSYNNLVI
lutamate rece for
HPV E6 #35 (cysteine-free)0.62 0.0366 GRWTGRAMSAWKPTRRETE
V
CD6 0.59 0.0528 SPQPDSTDNDDYDDISAA
HPV E6 58 (modified) 0.59 0.0624 AVGGRPARGGRLQGRRQTQ
V
GLUR7 (metabotropic 0.59 0.4410 VDPNSPAAKKKYVSYNNLVI
lutamate rece for
Nectin 2 0.54 0.1487 SSPDSSYQGKGFVMSRAMYV
ephrin B2 0.54 0.0183 ILNSIQVMRAQMNQIQSVEV
claudin 9 0.51 0.0498 LGYSIPSRSGASGLDKRDYV
CD62E 0.50 0.0225 SSSQSLESDGSYQKPSYIL
NMDA Glutamate Receptor0.49 0.1629 TQGFPGPATWRRISSLESEV
2C c steine-free
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EXAMPLE 9
THE PDZ DOMAINS OF PSD-95 BIND A NUMBER OF DIFFFERNT PL SEQUENCES
AND CAN RECRUIT ADDITIONAL PROTEINS TO NMDAR/PSD95/NNOS
COMPLEXES
S
The G assay was used to identify PL sequences able to bind the three PDZ
domains of
PSD-95, and these sequences and binding values are shown in TABLE 9. These
sequences
can be used to identify similar sequences in the genome (methods described
supra) that may
be important PSD-95 interactions involved in neurotoxicity or neuroprotection.
Inhibitors
based on these sequences that block the PDZ domains of PSD-95 in neurons or
neuronal
tissues can provide an attractive alternative therapeutic target for
neuroprotection. Sequences
with higher OD binding to the 3 domains of PSD-95 are potentially stronger
interactions.
TABLE 9: Peptide sequences that bind the PDZ domains of PSD-95
Gene DomainAverage Sequence
Stri OD
n
PSD95 1 4.17 QISPGGLEPPSEKHFRETEV
PSD95 1 4.07 LNSCSNRRVYKKMPSIESDV
PSD95 1 4.06 VGTLLLERVIFPSVKIATLV
PSD95 1 4.04 VGTLLLERVIFPSVKIATLV
PSD95 1 4.00 LNSCSNRRVYKKMPSIESDV
PSD95 1 4.00 QISPGGLEPPSEKHFRETEV
PSD95 1 4.00 VHKVRNKFKAKCSLCRLYII
PSD95 1 4.00 VHKVRNKFKAKCSLCRLYII
PSD95 1 4.00 GRWTGRAMSAWKPTRRETEV
PSD95 1 4.00 GRWTGRAMSAWKPTRRETEV
PSD95 1 4.00 AVGGRPARGGRLQGRRQTQV
PSD95 1 4.00 GRWTGRAMSAWKPTRRETEV
PSD95 1 4.00 TQGFPGPATWRRISSLESEV
PSD95 1 4.00 AVGGRPARGGRLQGRRQTQV
PSD95 1 4.00 VGTLLLERVIFPSVKIATLV
PSD95 1 3.87 YGRKKRRQRRRKLSSIESDV
PSD95 1 3.86 TGSALQAWRHTSRQATESTV
PSD95 1 3.15 VGTLLLERVIFPSVKIATLV
PSD95 1 3.12 SFTSILTCHQRRTQRKETVA
PSD95 1 3.08 GRWTGRAMSAWKPTRRETEV
PSD95 1 2.81 YGRKKRRQRRREKHFRETEV
PSD95 1 2.76 AAGGRSARGGRLQGRRETAL
PSD95 1 2.67 VHKVRNKFKAKCSLCRLYII
PSD95 1 2.66 TQGFPGPATWRRISSLESEV
PSD95 1 2.66 AAGGRSARGGRLQGRRETAL
PSD95 1 2.66 TQGFPGPATWRRISSLESEV
PSD95 1 2.51 AAGGRSARGGRLQGRRETAL
PSD95 1 2.47 TQGFPGPATWRRISSLESEV
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Gene DomainAverage Sequence
' StrinOD
PSD951 2.28 TTNNNPNSAVNIKKIFTDV
PSD951 2.26 SFTSILTCHQRRTQRKETVA
PSD951 2.13 YGRKKRRQRRRKLSSIESDV
PSD951 2.07 LNSSSNRRVYKKMPSIESAV
PSD951 2.05 DFRPSFKHILFRRARRGFRQ
PSD951 2.02 LNSSSNRRVYKKMPSIESAV
PSD951 1.83 AAGGRSARGGRLQGRRETAL
PSD951 1.67 LNSSSNRRVYKKMPSIESAV
PSD951 1.59 QDFRRAFRRILARPWTQTAW
PSD951 1.49 AAGGRSARGGRLQGRRETAL
PSD951 1.40 YGRKKRRQRRRKLSSIESDV
PSD951 1.28 KAGYRAPRSYPKSNSSKEYV
PSD951 1.26 DFRPSFKHILFRRARRGFRQ
PSD951 1.25 DGGARTEDEVQSYPSKHDYV
PSD951 1.23 SSKSKSSEESQTFFGLYKL
PSD951 1.18 PYSELNYETSHYPASPDSWV
PSD951 1.17 LNSSSNRRVYKKMPSIESAV
PSD951 1.12 TTNNNPNSAVNIKKIFTDV
PSD951 1.08 TQGFPGPATWRRISSLESEV
PSD951 1.08 KAGYRAPRSYPKSNSSKEYV
PSD951 1.03 QISPGGLEPPSEKHFRETEV
PSD951 1.03 AVGGRPARGGRLQGRRQTQV
PSD951 1.03 PGQPPKVKSEFNSYSLTGYV
PSD951 1.01 SSPDSSYQGKGFVMSRAMYV
PSD951 0.98 RNIEEVYVGGKQVVPFSSSV
PSD951 0.96 AAGGRSARGGRLQGRRETAL
PSD951 0.95 SYPTPRPYPKPAPSSGKDYV
PSD951 0.94
PSD951 0.87 QDFRRAFRRILARPWTQTAW
PSD951 0.86 GGDLGTRRGSAHFSSLESEV
PSD951 0.85 VDPNSPAAKKKYVSYNNLVI
PSD951 0.85 PGQPPKVKSEFNSYSLTGYV
PSD951 0.82 LGYSIPSRSGASGLDKRDYV
PSD951 0.82 LNSSSNRRVYKKMPSIESAV
PSD951 0.80 RNIEEVYVGGKQVVPFSSSV
PSD951 0.77 GGDLGTRRGSAHFSSLESEV
PSD952 4.18 AAGGRSARGGRLQGRRETAL
PSD952 4.06 YGRKKRRQRRRKLSSIESDV
PSD952 4,00 VHKVRNKFKAKCSLCRLYII
PSD952 4.00 VHKVRNKFKAKCSLCRLYII
PSD952 3.56 TQGFPGPATWRRISSLESEV
PSD952 3.53 YGRKKRRQRRRKLSSIESDV
PSD952 3.37 LNSSSNRRVYKKMPSIESAV
PSD952 3.11 GGDLGTRRGSAHFSSLESEV
PSD952 3.10 YGRKKRRQRRRKLSSIESDV
PSD952 2.89 TQGFPGPATWRRISSLESEV
PSD952 2.82 GRWTGRAMSAWKPTRRETEV
PSD952 2.79 YGRKKRRQRRREKHFRETEV
PSD952 2.67 LNSSSNRRVYKKMPSIESAV
PSD952 2.62 VHKVRNKFKAKCSLCRLYII
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Gene DomainAverage Sequence
StrinOD
PSD952 2.39 QISPGGLEPPSEKHFRETEV
PSD952 2.32 FNGSSNGHVYEKLSSIESDV
PSD952 2.29 LNSSSNRRVYKKMPSIESAV
PSD952 2.28 FNGSSNGHVYEKLSSIESDV
PSD952 2.22 GGDLGTRRGSAHFSSLESEV
PSD952 2.17 AAGGRSARGGRLQGRRETAL
PSD952 2.07 TQGFPGPATWRRISSLESEV
PSD952 2.04 FNGSSNGHVYEKLSSIESDV
PSD952 1.86 FNGSSNGHVYEKLSSIESDV
PSD952 1.85 AVGGRPARGGRLQGRRQTQV
PSD952 1.59 FNGSSNGHVYEKLSSIESDV
PSD952 1.35 KDITSDSENSNFRNEIQSLV
PSD952 1.31 GGDLGTRRGSAHFSSLESEV
PSD952 1.23 RSGATIPLVGQDIIDLQTEV
PSD952 1.12 FNGSSNGHVYEKLSSIESDV
PSD952 1.06 TTNNNPNSAVNIKKIFTDV
PSD952 0.80 YGRKKRRQRRREKHFREAEA
PSD953 4.00 SFTSILTCHQRRTQRKETVA
PSD953 4.00 SFTSILTCHQRRTQRKETVA
PSD953 4.00 VHKVRNKFKAKCSLCRLYII
PSD953 4.00 VHKVRNKFKAKCSLCRLYII
PSD953 4.00 GRWTGRAMSAWKPTRRETEV
PSD953 4.00 AAGGRSARGGRLQGRRETAL
PSD953 4.00 AVGGRPARGGRLQGRRQTQV
PSD953 4.00 SFTSILTCHQRRTQRKETVA
PSD953 4.00 GRWTGRAMSAWKPTRRETEV
PSD953 4.00 GRWTGRAMSAWKPTRRETEV
PSD953 4.00 AAGGRSARGGRLQGRRETAL
PSD953 4.00 AVGGRPARGGRLQGRRQTQV
PSD953 3.74 QDFRRAFRRILARPWTQTAW
PSD953 2.98 QDFRRAFRRILARPWTQTAW
PSD953 2.93 YGRKKRRQRRREKHFRETEV
PSD953 1.72 DFRPSFKHILFRRARRGFRQ
PSD953 1.46 TQGFPGPATWRRISSLESEV
PSD953 1.19 DFRPSFKHILFRRARRGFRQ
PSD953 1.17 AGAVRTPLSQVNKVWDQSSV
PSD953 1.17 TQGFPGPATWRRISSLESEV
PSD953 1.07 SSKSKSSEESQTFFGLYKL
PSD953 1.05 DGGARTEDEVQSYPSKHDYV
PSD953 1.00 TTNNNPNSAVNIKKIFTDV
PSD953 0.92 KAGYRAPRSYPKSNSSKEYV
PSD953 0.91 SSPDSSYQGKGFVMSRAMYV
PSD953 0.89 AGAVRTPLSQVNKVWDQSSV
PSD953 0.86 KAGYRAPRSYPKSNSSKEYV
PSD953 0.85 SYPTPRPYPKPAPSSGKDYV
PSD953 0.82 VDPNSPAAKKKYVSYNNLVI
PSD953 0.82 HHLVAQRDIRQFQLQHWLAI
PSD953 0.79 PGQPPKVKSEFNSYSLTGYV
PSD953 0.79 PGQPPKVKSEFNSYSLTGYV
PSD953 0.78 TGSALQAWRHTSRQATESTV
c~~:%b
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Gene DomainAverage Sequence
OD
Strin
PSD953 0.70 HHLVAQRDIRQFQLQHWLAI
PSD953 0.70 ESSGTQSPKRHSGSYLVTSV
***
The present invention is not to be limited in scope by the exemplified
embodiments which are intended as illustrations of single aspects of the
invention and any
sequences which are functionally equivalent are within the scope of the
invention. Indeed,
various modifications of the invention in addition to those shown and
described herein will
become apparent to those skilled in the art from the foregoing description and
accompanying
drawings. Such modifications are intended to fall within the scope of the
appended claims.
All publications, patents and patent applications cited herein are hereby
incorporated by reference in their entirety for all purposes to the same
extent as if each
individual publication, patent or patent application were specifically and
individually
indicated to be so incorporated by reference.
10'7
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TABLE 2: NMDA Receptors with PL Sequences
Name GI# C-terminal 20mer C-terminalPL? internal
sequence PL
4mer sequence ID
NMDARl 307302 HPTDITGPLNLSDPSVST STVV X AA216
VV
NMDAR1-1 292282 HPTDITGPLNLSDPSVST STVV X AA216
VV
NMDARI-4 472845 HPTDITGPLNLSDPSVST STVV X AA216
VV
NMDAR1- 2343286 HPTDITGPLNLSDPSVST STVV X AA216
3b VV
NMDAR1- 2343288 HPTDITGPLNLSDPSVST STVV X AA216
4b V V
NMDAR1-2 11038634RRAIEREEGQLQLCSRH HRES
RES
NMDAR1-3 11038636RRAIEREEGQLQLCSRH HRES
RES
NMDAR2C 6006004 TQGFPGPCTWRRISSLES ESEV X AA180
EV
NMDAR3 560546 FNGSSNGHVYEKLSSIES ESDV X AA34.1
DV
NMDAR3A 17530176AVSRKTELEEYQRTSRT TOES
CES
NMDAR2B 4099612 FNGSSNGHVYEKLSSIES ESDV X
DV
NMDAR2A 558748 LNSCSNRRVYKKMPSIE ESDV X AA34.2
SDV
NMDAR2D 4504130 GGDLGTRRGSAHFSSLE ESEV X
SEV
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TABLE 3: PDZ Proteins that Interact with NMDA Receptor Proteins (a PL protein)
internalPL Name PL 20Mer Sequence PDZ Name PDZ Bind-Assa
pL Dom ing y
~D ain StrengUse
th d
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV MPPI I A
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV DLGI 1,2 A/G
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV PSD95 1,2,3 AlG
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV NeDLG 1,2 A/G
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV Syntrophin1 G
I
al ha
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV TIP43 1 A
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV LIMK1 1 A
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV MPP2 1 G
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV PTN4 1 A/G
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV 41.8 1 A/G
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV RGS 12 1 A/G
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV DVLI 1 A
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV MINTI 1,2 A/G
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV TIP2 1 A
AA34.2NMDAR2A LNSCSNRRVYKKMPSIESDV KIAA561 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV FLJ00011 1 2 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Magi2 3 2 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV DLGI 1,2 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MAGI 1 2 4 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MAGI 1 5 4 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA0807 1 4 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV DLG1 1 S G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV DLG1 2 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV DLG2 1 4 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Erbin 1 4 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV FLJ 112151 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV INADL 3 2 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV 1NADL 8 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA0147 2 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV K1AA0147 3 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA0380 1 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA0382 1 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA0973 1 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA1634 2 3 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAAIG34 5 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV M1NT1 1,2 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA1634 1 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV LIMK1 1 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV NeDLG 1,2 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV PSD95 1,2,35 G
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internalPL Name PL 20Mer Sequence PDZ Name PDZ Bind-Assa
PL Dom ing y
ID ain StrengUse
th d
AA180 NMDAR2C TQGFPGPATWRRISSLESEV NeDLG 3 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV NOSl 1 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV PSD95 3 2 G
.
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Syntrophin1 4 G
1
alpha
AA180 NMDAR2C TQGFPGPATWRRISSLESEV TIP1 1 5 G
AA180 NMDARZC TQGFPGPATWRRISSLESEV Syntrophin1 2 G
gamma-2
AA180 NMDAR2C TQGFPGPATWRRISSLESEV TAX IP 1 4 G
2
AA180 NMDAR2C TQGFPGPATWRRISSLESEV LIM RIL 1 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Syntrophin1 4 G
~
gamma-1
AA180 NMDAR2C TQGFPGPATWRRISSLESEV PTPLl 2 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV AIPC 1 3 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV NeDLG 2 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA1526 1 3 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MUPPI 13 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV LIM- 1 4 G
Mysti
q a a
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Outer 1 5 G
Membrane
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA0807 1 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Magi2 1 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA0147 1 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV PSD95 1 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Magi2 5 S G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV DLG2 2 5 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MAGI 1 4 3 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MAGI I 3 1 G'
AA180 NMDAR2C TQGFPGPATWRRISSLESEV Mint 1 2 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MUPPI 10 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA1634 4 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MAGI 1 6 2 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV MUPP1 5 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV NeDLG 1 2 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV APXLI 1 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV KIAA1095 1 1 G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV INADL 5 I G
AA180 NMDAR2C TQGFPGPATWRRISSLESEV PTN-4 1 2 G
AA216 NMDA HPTDITGLPNLSDPSVSTVV NeDLG 1,2 2 G
R2C
AA216 NMDARI HPTDITGLPNLSDPSVSTVV PTPLI 2 1 G
AA216 NMDAR1 HPTDITGLPNLSDPSVSTVV DLG1 1,2 ~ G
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internalPL Name PL 20Mer Sequence PDZ Name PDZ Bind-Assa
PL Dom ing y
ID
ain StrengUse
th d
AA2I6 NMDAR1 HPTDITGLPNLSDPSVSTVV KIAAlG34 2 1 G
AA2I6 NMDARl HPTDITGLPNLSDPSVSTVV PSD95 1,2,31 G
I ~
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TABLE 4: Sequences of PDZ Domains Cloned to Produce GST-PDZ Fusions
Gene Name GI or DomSequence fused to GST Construct
Acc# ain#
26s subunit9184389 1 RDMAEAHKEAMSRKLGQSESQGPPRAFAKVNSIS
p27 PGSPSIAGLQVDDEIVEFGSVNTQNFQSLHNIGSV
VQHSEGALAPTILLSVSM
AF6 430993 1 LRKEPEIITVTLKKQNGMGLSIVAAKGAGQDKLGI
YVKSVVKGGAADVDGRLAAGDQLLS VDGRSLV
GLSQERAAELMTRTS S V V TLE V AKQG
AIPC 127514511 LIRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMG
IFVKTIFPNGSAAEDGRLKEGDEILDVNGIPIKGLT
FQEAIHTFKQIRSGLFVLTVRTK.LVSPSLTNS S
AIPC 127514512 GISSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGAC
CLALENSPPGIYIHSLAPGS VAKMESNLSRGDQIL
EVNSVNVRHAALSKVHAILSKCPPGPVRLVIGRH
PNPKV SEQEMDEVIARSTYQESKEANSS
AIPC 127514513 QSENEEDVCFIVLNRKEGSGLGFSVAGGTDVEPK
SITVHRVFSQGAASQEGTMNRGDFLLSVNGASLA
GLAHGNVLKVLHQAQLHKDALV V IKKGMDQPR
PSNSS
AIPC 127514514 LGRSVAVHDALCVEVLKTSAGLGLSLDGGKSSVT
GDGPLVIKRVYKGGAAEQAGIIEAGDEILAINGKP
LV GLMHFDAWNIMKS VPEGP V QLLIRKHRNS
S
alpha actinin-22773059 1 QTVILPGPAAWGFRLSGGIDFNQPLV1TRITPGSKA
associated AAANLCPGDVILAIDGFGTESMTHADGQDRIKAA
LIM
protein EFIV
APXL-1 136512631 ILVEV QLSGGAPW GFTLKGGREHGEPLVITKIEEG
SKAAAVDKLLAGDEIVGTNDIGLSGFRQEAICLVK
GSHKTLKLV VKRNS S
Atrophin-1 2947231 1 REKPLFTRDASQLKGTFLSTTLItKSNMGFGFT1IG
Interacting GDEPDEFLQVKS V1PDGPAAQDGKMETGDVIVYI:
Protein NEVCVLGHTHADVVKLFQSVPIGQSVNLVLCRG
YP
Atrophin-1 2947231 2 LSGATQAELMTLTIVKGAQGFGFTIADSPTGQRV
Interacting KQILDIQGCPGLCEGDLIVEINQQNVQNLSHTEVV
Protein DILKDCPIGSETSLIIHRGGFF
Atrophin-I 2947231 3 HYKELDVHLRRMESGFGFRILGGDEPGQPILIGAV
Interacting IAMGSADRDGRLHPGDELVYVDGIP VAGKTHRY
Protein VIDLMHHAARNGQVNLTVRRKV LCG
Atrophin-1 2947231 4 EGRGISSHSLQTSDAVIHRKENEGFGFVIISSLNRP
Interacting ESGSTITVPHKIGRIIDGSPADRCAKLKVGDRILAV
Protein NGQSIINMPHADIVKLIKDAGLS VTLRIIPQEEL
Atrophin-1 2947231 5 LSDYRQPQDFDYFTVDMEKGAKGFGFSIRGGREY
Interacting KMDLYVLRLAEDGPAIRNGRMRVGDQIIEINGES
Protein TRDMTHARAIELIKSGGRRVRLLLKRGTGQ
Atrophin-1 2947231 6 HESVIGRNPEGQLGFELKGGAENGQFPYLGEVKP
Interacting GKVAYESGSKLV SEELLLE VNETP V AGLTIRD
V L
Protein AVIKHCKDPLRLKCVKQGGIHR
112
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or DomSequence fused to GST Construct
Acc# ain#
CARD11 123827721 NLMFRKFSLERPFRPSVTSVGHVRGPGPSVQHTTL
NGDSLTSQLTLLGGNARGSFVHSVKPGSLAEKAG
LREGHQLLLLEGCIRGERQS VPLDTCTKEEAH W
TI
QRCSGPVTLHYKVNHEGYRKLV
CARD14 131291231 ILSQVTMLAFQGDALLEQISVIGGNLTGIFIHRVTP
GSAADQMALRPGTQ1VMVDYEASEPLFKAVLED
TTLEEAVGLLRRVDGFCCLS VKVNTDGYKRL
CASK 3087815 1 TRVRLVQFQKNTDEPMGITLKMNELNHCIVARIM
HGGMIHRQGTLHVGDEIRE1NGISVANQTVEQLQ
KMLREMRGSITFKIVPSYRTQS
Connector 3930780 1 LEQKAVLEQVQLDSPLGLEIHTTSNCQHFVSQVD
Enhancer TQVPTDSRLQIQPGDEVVQiNEQV VVGWPRKNM
VRELLREPAGLSLVLKKIPIP
Cytohesin 3192908 1 QRKLVTVEKQDNETFGFEIQSYRPQNQNACSSEM
Binding FTLICKIQEDSPAHCAGLQAGDVLANINGV STEGF
Protein TYKQVVDLIRSSGNLLTIETLNG
Densin 180 167558921 RCLIQTKGQRSMDGYPEQFCVRIEKNPGLGFSISG
GISGQGNPFKPSDKGIFVTRVQPDGPASNLLQPGD
KILQANGHSFVHMEHEKAVLLLKSFQNTVDLVIQ
RELTV
DLG 1 475816 1 IQVNGTDADYEYEEITLERGNSGLGFSIAGGTDNP
HIGDDSSIFITKIITGGAAAQDGRLRVNDCILQVNE
VDVRDVTHSKAVEALKEAGSIVRLYVKRRN
DLG1 475816 2 IQLIKGPKGLGFSIAGGVGNQH1PGDNSIYVTKIIE
GGAAHKDGKLQIGDKLLAVNNVCLEEVTHEEAV
TALKNTSDFVYLKVAKPTSMYMNDGN
DLGI 475816 3 ILHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLS
GELRKGDRIISVNSVDLRAASHEQAAAALKNAGQ
AVTIVAQYRPEEYSR
DLG2 127365521 ISYVNGTEIEYEFEEITLERGNSGLGFSIAGGTDNP
HIGDDPGIFITKIIPGGAAAEDGRLRVNDCILRVNE
VDVSEVSHSKAVEALKEAGSIVRLYVRRR
DLG2 127365522 ISVVEIKLFKGPKGLGFSIAGGVGNQHIPGDNSIYV
TKIIDGGAAQKDGRLQVGDRLLMVNNYSLEEVT
HEEAVAILKNTSEVVYLKVGNPTTI
DLG2 127365523 IWAVSLEGEPRKVVL;HKGSTGLGFNIVGGEDGEG
IFVSFILAGGPADLSGELQRGDQILSVNGIDLRGAS
HEQAAAALKGAGQTVTIIAQYQPED
DLGS 3650451 1 GIPYVEEPRHVKVQKGSEPLGIS1VSGEKGGIYVS
KVTVGSIAHQAGLEYGDQLLEFNG1NLRSATEQQ
ARLIIGQQCDTITILAQYNPHVHQLRNSSZLTD
DLGS 3650451 2 GILAGDANKKTLEPRVVFIKKSQLELGVHLCGGN
LHGVFVAEVEDDSPAKGPDGLVPGDLILEYGSLD
VRNKTVEEVYVEMLKPRDGVRLKVQYRPEEFIVT
D
113
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or DomSequence fused to GST Construct
Acc# ain#
DLG6, splice146471401 PTSPEIQELRQMLQAPHFKALLSAHDTIAQKDFEP
variant LLPPLPDNIPESEEAMRIVCLVKNQQPLGATIKRH
1
EMTGDILVARIIHGGLAERSGLLYAGDKLVEVNG
VSVEGLDPEQVIHILAMSRGTIMFKVVPVSDPPVN
SS
DLG6, spliceAB05330 1 PTSPEIQELRQMLQAPHFKGATIfCRHEMTGDILVA
variant 3 RIIHGGLAERSGLLYAGDKLVEVNGVSVEGLDPE
2
QVIHILAMSRGTIMFKVVPVSDPPVNSS
DVL1 2291005 1 LNIVTVTLNMERHHFLGIS1VGQSNDRGDGGIYIG
SIMKGGAVAADGRIEPGDMLLQVNDVNFENMSN
DDAVRVLREIVSQTGPISLTVAKCW
DVL2 2291007 1 LNIITVTLNMEKYNFLGISIVGQSNERGDGG1YIGS
IMKGGAVAADGRIEPGDMLLQ V NDMNFENMSN
DDAVRVLRDIVHKPGPIVLTVAKCWDPSPQNS
DVL3 6806886 1 IITVTLNMEKYNFLGISIVGQSNERGDGGIYIGS1M
KGGAVAADGRIEPGDMLLQVNElNFENMSNDDA
VRVLREIVHKPGPITLTVAKCWDPSP
ELFIN 1 2957144 1 TTQQIDLQGPGPWGFRLVGRKDFEQPLAISRVTPG
SKAALANLCIGDVITAIDGENTSNMTHLEAQNRIK
GCTDNLTLTV ARSEHKV W SPLV
ENIGMA 561636 1 IFMDSFKVVLEGPAPWGFRLQGGKDFNVPLSISRL
TPGGKAAQAGVAVGDWVLSTDGENAGSLTHIEA
QNKIRACGERLSLGLSRAQPV
ERBIN 8923908 1 QGHELAKQEIRVRVEKDPELGFSLSGGVGGRGNP
FRPDDDGIFVTRVQPEGPASKLLQPGDKIIQANGY
SF1NIEHGQAVSLLKTFQNTVELIIVREVSS
EZR1N 3220018 1 ILCCLEKGPNGYGFHLHGEKGKLGQYIRLVEPGSP
Binding AEKAGLLAGDRLVEVNGENVEKETHQQVVSRIR
Protein AALNAVRLLVVDPEFIVTD
50
EZRIN 3220018 2 IRLCTMKKGPSGYGFNLHSDICSICPGQFIRSVDPDS
Binding PAEASGLRAQDRIVEVNGVCMEGKQHGDVVSAI
Protein RAGGDETKLLVVDRETDEFFMNSS
50
FLJ00011 104403521 KNPSGELKTVTLSKMKQSLGISISGGIESKVQPMV
KIEKIFPGGAAFLSGALQAGFELVAVDGENLEQV
THQRAVDTIRRAYRNKAREPMELV VRVPGPSPRP
SPSD
FLJ11215 114363651 EGHSHPRVVELPKTEEGLGFNIMGGKEQNSPIYIS
RIIPGGIADRHGGLKRGDQLLS VNGV S VEGEHHE
KAVELLKAAQGKVKLV VRYTPI~V LEEME
FLJ12428 BC01204 1 PGAPYARKTFTIVGDAVGWGFVVRGSKPCHIQAV
0 DPSGPAAAAGMKVCQFVVSVNGLNVLHVDYRT
V SNLILTGPRTIVME VMEELEC
FLJ12615 104342091 GQYGGETVKIVRIEKARDTPLGATVRNEMDSVIIS
R1VKGGAAEKSGLLHEGDE V L:E l NGIEIRGKD
VNE
VFDLLSDMHGTLTFVLIPSQQ IKPPPA
FLJ20075 7019938 1 ILAHVKGIEKEVNVYKSEDSLGLTITDNGVGYAFI
KRIKDGGVIDSVKT1CVGDHIESINGENIVGWRHY
114
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
DVAKKLKELKKEELFTMKLIEPKKAFEI
FLJ21687 104378361 KPSQASGHFSVELVRGYAGFGLTLGGGRDVAGD
TPLAVRGLLKDGPAQRCGRLEVGDLVLHINGEST
QGLTHAQAVERIRAGGPQLHLVIRRPLETHPGKP
RGV
FLJ31349 AK055911 PVMSQCACLEEVHLPN1KPGEGLGMYIKSTYDGL
1 HVITGTTENSPADRSQKLHAGDEVIQVNQQTVVG
WQLKNLVKKLRENPTGV VLLL I~KRPTGSFNFTPE
FIVTD
FLJ32798 AK057361 LDDEEDSVKIIRLVKNREPLGATIKKDEQTGAIIVA
0 R1MRGGAADRSGLIHVGDELREVNGIPVEDKRPE
EIIQILAQSQGAITFKIIPGSKEETPSNSS
GRIP 1 45390831 VVELMKKEGTTLGLTVSGGIDKDGKPRVSNLRQ
GGIAARSDQLDVGDYIKAVNG1NLAKFRHDEIISL
LKNVGERVVLEVEYE
GRIP 1 45390832 RSSVIFRTVEVTLHKEGNTFGFVIRGGAHDDRNKS
RPVVITCVRPGGPADREGTIKPGDRLLSVDGIRLL
GTTHAEAMSILKQCGQEAALLIEYDVSVMDSVAT
ASGNSS
GRIP 1 45390833 HVATASGPLLVEVAKTPGASLGVALTTSMCCNK
AVIV IDKIKSASIADRCGALH V G D HI LSmGTSMEY
CTLAEATQFLANTTDQVKLEILPHHQTRLALKGP
NSS
GRIP 1 45390834 TETTEVVLTADPVTGFGIQLQGSVFATETLSSPPLI
SYIEADSPAERCGVLQIGDRVMAINGIPTEDSTFEE
ASQLLRDSSITSKVTLEIEFDVAES
GRIP 1 45390835 AESVIPSSGTFHVKLPKKHNVELGIT1SSPSSRKPG
DPLVISDIKKGSVAHRTGTLELGDKLLAIDNIRLD
NCSMEDAVQILQQCEDLVKLKIRKDEDNSD
GRIP 1 45390836 IYTVELKRYGGPLGITISGTEEPFDPIIISSLTKGGL
AERTGAIHIGDRILAIN S S SLKGKPLSEAIHLLQMA
GETVTLKIKKQTDAQSA
GRIP 1 45390837 IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEK
GVYVKNIRPAGPGDLGGLKPYDRLLQVNHVRTR
DFDCCLV VPLIAESGNKLDLVISRNPLA
GTPase 23890081 SRGCETRELALPRDGQGRLGFEVDAEGFVTHVER
Activating FTFAETAGLRPGARLLRVCGQTLPSLRPEAAAQL
Enzyme LRSAPKVCVTVLPPDESGRP
Guanine 66507651 AKAKWRQVVLQKASRESPLQFSLNGGSEKGFGIF
Exchange VEGVEPGSKAADSGLI{RGDQIlVI:EVNGQNFENITF
Factor MKAV EILRNNTHLALT VKTNIF V FKEL
HEMBA 104363671 LENVIAKSLLIKSNEGSYGFGLEDI~NKVPIIKLVEK
1000505 GSNAEMAGMEVGK1CIFAlNG.DLVFMRPFNEVDC
FLKSCLNSRKPLRVLVSTKP
HEMBA 104363672 PRETVKIPDSADGLGFQIRGFGPSVVHAVGRGTV
1000505 AAAAGLHPGQCIIICVNG1NVSK:E'l'HASV1AHVTA
115
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or DomSequence fused to GST Construct
Acc# ain#
CRKYRRPTKQDSIQ
HEMBA 7022001 1 EDFCYVFTVELERGPSGLGMGLI:DGMHTHLGAPG
1003117 LYIQTLLPGSPAAADGRLSLGDRLLLVNGSSLLGL
GYLRAVDLIRHGGKKMRFLVAKSD VETAKICI
HTRA3 AY04009 1 LTEFQDKQIKDWKKRFIGIRMRTTTPSLVDELKAS
4 NPDFPEVSSGIYVQEVAPNSPSQRGGIQDGDIIVK
VNGRPLVDSSELQEAVLTESPLLLEVRRGNDDLL
FSNSS
HTRA4 AL57644 1 HKKYLGLQMLSLTVPLSEELKMHYPDFPDVSSGV
4 YVCKVVEGTAAQSSGLRDHDVIVNTNGKPITTTT
DV VKALDSDSLSMAVLRGKDNLLLTVNSS
INADL 2370148 1 IWQIEYIDIERPSTGGLGFSVVALRSQNLGKVDIFV
KDVQPGSVADRDQRLKENDQILAINHTPLDQNIS
HQQAIALLQQTTGSLRLIVAREPVHTKSSTSSSE
INADL 2370148 2 PGHVEEVELINDGSGLGFGIVGGKTSGVVVRTIVP
GGLADRDGRLQTGDHILKIGGTNVQGMTSEQVA
QVLRNCGNSS
INADL 2370148 3 PGSDSSLFETYNVELVRKDGQSLGIRIVGYVGTSH
TGEASGIYVKSIIPGSAAYHNGHIQVNDKIVAVDG
VNIQGFANHDVVEVLRNAGQVVHLTLVRRKTSS
STSRIHRD
iNADL 2370148 4 NSDDAELQKYSKLLPIHTLRLGVEVDSFDGHHY(S
SIVSGGPVDTLGLLQPEDELLEVNGMQLYGKSRR
EAVSFLKEVPPPFTLVCCRRLFDDEAS
1NADL 2370148 5 LSSPEVKIVELVKDCKGLGFSILDYQDPLDPTRSVI
VIRSLVADGVAERSGGLLPGDRLVS VNEYCLDNT
SLAEAVEILKAVPPGLVHLGICI<PLVEFIVTD
INADL 2370148 6 PNFSHWGPPRTVEIFREPNVSLGISIVVGQTVIKRL
KNGEELKGIFIKQVLEDSPAGKrCNALKTGDKILEV
SGVDLQNASHSEAVEA1KNAGNPVVFIVQSLSSTP
RVIPNVHNKANS S
1NADL 2370148 7 PGELHIIELEKDKNGLGLSLAGNKDRSRMSIFVVG
INPEGPAAADGRMRIGDELLEINNQ1LYGRSHQNA
SAIIKTAPSKVKLVFIRNEDAVNQMANSS
INADL 2370148 8 PATCPIVPGQEMIIEISKGRSGLGLSIVGGKDTPLN
AIVIHEVYEEGAAARDGRLWAGDQILEVNGVDL
RNSSHEEAITALRQTPQKVRLV V Y
KIAA0147 1469875 1 ILTLTILRQTGGLGISIAGGKGSTPYKGDDEGIFISR
VSEEGPAARAGVRVGDKLLEVNGVALQGAEHHE
AVEALRGAGTAVQMRVWRERMVEPENAEFIVTD
KIAA0147 1469875 2 PLRQRHVACLARSERGLGFSTAGGKGSTPYRAGD
AGIFV SRIAEGGAAHRAGTLQ V G D R V LS
TNG VD V
TEARHDHAVSLLTAASPTIA LLLEREAGG
KIAA0147 1469875 3 LEGPYPVEEIRLPRAGGPLGLSIVGGSDHSSHPFG
I
VQEPGVFISKVLPRGLAARSGLRVGDRILAVNGQ
DVRDATHQEAVSALLRPCLELS LLVRRDPAEFIVT
116
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
D
KIAA0147 14698754 RELCIQKAPGERLGISIRGGARGHAGNPRDPTDEG
IFISKVSPTGAAGRDGRLRVG LRLLEVNQQSLLGL
THGEAVQLLRSVGDTLTVLVCDGFEASTDAALEV
S
KIAA0303 22245461 PHQPIVIHSSGKNYGFTIRAIRVYVGDSDIYTVHHI
VWNVEEGSPACQAGLKAGDLITH1NGEPVHGLV
HTEVIELLLKSGNKVSITTTPF
KIAA0313 76572601 ILACAAKAItRRLMTLTKPSREAPLPFILLGGSEKG
FGIFVDSVDSGSKATEAGLKRGDQILEVNGQNFE
NIQLSKAMEILRNNTHLS IT VKTNLF VFKELLTN
S S
KIAA0316 66831231 IPPAPRKVEMRRDPVLGFGFVAGSEKPVVVRSVT
PGGPSEGKLIPGDQIVM1NDEPVSAAPRERVIDLV
RSCKESILLTVIQPYPSPK
KIAA0340 22246201 LNKRTTMPKDSGALLGLKVVGGKMTDLGRL~AF
ITKVKKGSLAD V V GHLRAGDEVLEWNGKPLPGA
TNEEVYNIILESKSEPQVEIIV SRPIGDIPRIHRD
KIAA0380 22247001 QRCVIIQKDQHGFGFTVSGDRIVLVQSVRPGGAA
MKAGVKEGDRIIKVNGTMVTNS SHLEV VKLIKSG
AYVALTLLGSS
KIAA0382 76620871 ILVQRCVIIQKDDNGFGLTVSGDNPVFVQSVKED
GAAMRAGVQTGDRIIKVNGTLVTHSNHLEV VKLI
KSGSYVALTVQGRPPGNSS
KIAA0440 26621601 SVEMTLRRNGLGQLGFHVNYEGIVADVEPYGYA
WQAGLRQGSRLVEICKVAVATLSHEQMIDLLRTS
VTVKVVIIPPHD
KIAA0545 147628501 LKVMTSGWETVDMTLRRNGLGQLGFHVKYDGT
VAEVEDYGFAWQAGLRQGSRLVEICKVA V VTLT
HDQMIDLLRTSVTVKVVIIPPFEDGTPRRGW
KIAA0559 30436411 HYIFPHARIK1TRDSKDHTVSGNGLGIRIVGGKEIP
GHSGEIGAYIAKILPGGSAEQTGKLMEGMQVLEW
NGIPLTSKTYEEVQSIISQQSGEAEICVRLDLNML
KIAA0561 30436451 LCGSLRPPIVIHSSGKKYGFSLRAIRVYMGDSDVY
TVHHVVWSVEDGSPAQEAGLRAGDLITH1NGESV
LGLVHMDVVELLLKSGNKISLRTTALENTSIKVG
KIAA0613 33270391 SYSVTLTGPGPWGFRLQGGKDFNMPLTISRITPGS
KAAQSQLS QGDLV V AIDG VNTDTMTHLEAQNKI
KSASYNLSLTLQKSKNSS
KIAA0751 127341651 ISRDSGAMLGLKV V GGKMTES GRLCAF1TKV
KKG
SLADTVGHLRPGDEVLEWNGRLLQGATFEEVYNI
ILESKPEPQVELV VSRPIAIHRD
KIAA0807 38823341 ISALGSMRPPITTHRAGKKYGFTLRATRVYMGDSD
VYTVHHMVWHVEDGGPASEAG LRQGDLITHVN
GEPVHGLVHTEV VELILKSGNKVAISTTPLENSS
KIAA0858 42402041 FSDMRISINQTPGKSLDFGFTIKWDIPGIFVASVEA
GSPAEFSQLQVDDEIIAINNTKFSYNDSKEWEEAM
11'7
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to-GST Construct
Acc# ain#
AKAQETGHLVMDVRRYGKAGSPE
KIAA0902 42402921 QSAHLEVIQLANIKPSEGLGMYIItSTYDGLHVITG
TTENSPADRCI{KIHAGDEVIQVNHQTVVGWQLK
NLVNALREDPSGVILTLKKRPQS MLTSAPA
KIAA0967 45895771 ILTQTLIPVRHTVKIDKDTLLQDYGFHISESLPLTV
VAVTAGGSAHGKLFPGDQILQMNNEPAEDLS WE
RAVDILREAEDSLSITVVRCTSGVPKSSNSS
KIAA0973 45895891 GLRSPITIQRSGKKYGFTLRAIRVYMGDTDVYSV
HHIV WHVEEGGPAQEAGLCAGDLITHVNGEPVH
GMVHPEVVELILKSGNKVAVTTTPFE
KIAA1095 58895261 QGEETKSLTLVLHRDSGSLGFNIIGGRPSVDNHDG
SSSEGIFVSKIVDSGPAAICEGGLQIHDRIIEVNGRD
LSRATHDQAV EAFKTAKEPI V V Q V LRRTP
RTKMF
TP
KIAA1095 58895262 QEMDREELELEEVDLYRMNSQDKLGLTVCYRTD
DEDDIGIYISEIDPNSIAAKDGRIREGDRIIQINGIEV
QNREEAVALLTSEENKNFSLLIARPELQLD
KIAA1202 63304211 RSFQYVPVQLQGGAPWGFTLKGGLEHCEPLTVSK
IEDGGKAALSQKMRTGDELVNINGTPLYGSRQEA
LILIKGSFRILKLIVRRRNAP V S
KIAA1222 63306101 ILEKLELFPVELEKDEDGLGISIIGMGVGADAGLE
KLGIFVKTVTEGGAAQRDGRIQVNDQIVEVDGISL
VGVTQNFAATVLRNTKGNVRFVIGREKPGQVS
KIAA1284 63313691 KDVNVYVNPKKLTVIKAKEQLKLLEVLVGIIHQT
KWSWRRTGKQGDGERLVVHGLLPGGSAMKSGQ
VLIGDVLVAVNDVDVTTENIERVLSCIPGPMQVK
LTFENAYDVKRET
KIAA1389 72431581 TRGCETVEMTLRRNGLGQLGFI-IVNFEGIVADVEP
FGFAWKAGLRQGSRLVEICKVAVATLTHEQMIDL
LRTSVTVKVVIIQPHDDGSPRR
KIAA1415 72432101 VENILAKRLLILPQEEDYGFDIEEI{NKAVVVKSVQ
RGSLAEVAGLQVGRKIYSINEDLVFLRPFSEVESIL
NQSFCSRRPLRLLVATKAKEIIKIP
KIAA1526 58171661 PDSAGPGEVRLVSLRRAKAHEGLGFSIRGGSEHG
VGIYVSLVEPGSLAEKEGLRV GDQILRVNDKSLA
RVTHAEAVKALKGSKKLVLSVYSAGRIPGGYVT
NH
KIAA1526 58171662 LQGGDEKKVNLVLGDGRSLGLTIRGGAEYGLGIY
ITGVDPGSEAEGSGLKVGDQILEVNWRSFLNILHD
EAVRLLKSSRHLILTVKDVGRLPHARTTVDE
KIAA1526 58171663 WTSGAHVHSGPCEEKCGHPGHRQPLPRIVTIQRG
GSAHNCGQLKVGHV I LEVNG LT LRGKEHREAARI
IAEAFKTKDRDY IDFLDSL
KIAA1620 100473161 ELRRAELVEIIVETEAQTGVSGINVAGGGK:EGIFV
RELREDSPAARSLS LQEGDQLLSARVFFENFKYED
ALRLLQCAEPYKVSFCLKRTVPTGDLALRP
118
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
KIAA1634 100473441 PSQLKGVLVRASLKKSTMGFGFTIIGGDRPDEFLQ
VKNVLKDGPAAQDGKIAPGDVIVDINGNCVLGH
THADVVQMFQLVPVNQYVNLTLCRGYPLPDDSE
D
KIAA1634 100473442 ASSGSSQPELVTIPLIKGPKGFGFAIADSPTGQKVK
MILDSQWCQGLQKGDIIKEIYHQNVQNLTHIJQVV
EVLKQFPVGADV PLLILRGGPPSPTKTAI~!I
KIAA1634 100473443 LYEDKPPLTNTFLISNPRTTADPR1LYEDKPPNTKD
LDVFLRKQESGFGFRVLGGDGPDQSIYIGAIIPLGA
AEKDGRLRAADELMCIDG1P VKGKSHKQV LDLM
TTAARNGHVLLTVRRKIFYGEKQPEDDSGSPGIH
BELT
KIAA1634 100473444 PAPQEPYDVVLQRKENEGFGFVLLTSKNKPPPGVI
PHKIGRVIEGSPADRCGKLKVGDH1SAVNGQS1VE
LSHDNIVQLIKDAGVTVTLTV IAEEEHHGPPS
KIAA1634 100473445 QNLGCYPVELERGPRGFGFSLRGGKEYNMGLFIL
RLAEDGPAIKDGRIHVGDQIVEINGEPTQGITHTR
AIELIQAGGNKVLLLLRPGTGLIPDHGLA
KIAA1719 1267982 0 ITVVELIKKEGSTLGLTISGGTDKDGKPRVSNLRP
GGLAARSDLLNIGDYIRS VNGIHLTRLRHDEIITLL
KNVGERVVLEVEY
KIAA1719 1267982 1 ILDVSLYKEGNSFGFVLRGGAHEDGHKSRPLVLT
YVRPGGPADREGSLKVGDRLLSVDGIPLHGASHA
TALATLRQCSHEALFQVEYDVATP
KIAA1719 1267982 2 IHTVANASGPLMVEIVKTPGSALGISLTTTSLRNK
SVITIDRIKPASVVDRSGALHPGDI~ILS1DGTSMEH
CSLLEATKLLASISEKVRLEILPVPQSQRPL
KIAA1719 1267982 3 IQIVHTETTEVVLCGDPLSGFGLQLQGGIFATETLS
SPPLVCFIEPDSPAERCGLLQVGDRVLS1NGLATED
GTMEEANQLLRDAALAHKV V LEV EFDVAES V
KIAA1719 1267982 4 IQFDVAESVIPSSGTFHVKLPKI<RSVELGITISSASR
KRGEPLIISDIKKGSVAHRTGTLEPGDKLLAIDNhR
LDNCPMEDAV QILRQCEDLVICLKIRKDEDN
KIAA1719 1267982 5 IQTTGAVSYTVELKRYGGPLGlTISGTEEPFDPNIS
GLTKRGLAERTGAIHVGDRILAINNVSL1CGRPLSE
AIHLLQVAGETVTLKIKKQLDR
KIAA1719 1267982 6 ILEMEELLLPTPLEMHKVTLHICDPMRHDFGFSVS
DGLLEKGVYVHTVRPDGPAHRGGLQPFDRVLQV
NHVRTRDFDCCLAVPLLAEAGDVLELIISRICPH1,A
HSS
LIM Mystique127342501 MALTVDVAGPAPWGFRITGGRDFHTPIMVTKVA
ERGKAKDADLRPGDIIVAINGESAEGMLHA EAQS
KIRQSPSPLRLQLDRSQATSPGQT
LIM Protein3108092 1 SNYSVSLVGPAPWGFRLQGGK:DPNMPLTISSLKD
GGKAAQANVRIGDV V LSIDG LNAQGMTH LEAQN
KIKGCTGSLNMTLQRAS
119
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
LIMK1 45874981 TLVEHSKLYCGHCYYQTVVTPVIEQILPDSPGSHL
PHTVTLV SIPAS SHGKRGLS V STDPPHGPPGCGTEH
SHTVRVQGVDPGCMSPDVKNSIHVGDRILEINGT
PIRNVPLDEIDLLIQETSRLLQLTLEHD
LIMK2 18055931 PYSVTLISMPATTEGRRGFSVSVESACSNYATTVQ
VKEVNRMHISPNNRNAIHPGDRLLEINGTPVRTLR
VEEVEDAISQTSQTLQLLIEHD
LIM-RIL 10850211 IHSVTLRGPSPWGFRLVGRDFSAPLTISRVHAGSK
ASLAALCPGDLIQAINGESTELMTHLEAQNRIKGC
HDHLTLSVSRPE
LU-1 U52111 1 VCYRTDDEEDLGIYVGEVNPNSIAAKDGRIREGD
RIIQINGVDVQNREEAVAILSQEENTNTSLLV ARPE
SQLA
MAGI1 33709971 IQKKNHWTSRVHECTVKRGPQGELGVTVLGGAE
HGEFPYVGAVAAVEAAGLPGGGEGPRLGEGELL
LEVQGVRVSGLPRYDVLGV IDSCKEAVTFKAVRQ
GGR
MAGI1 33709972 PSELKGKFIHTKLRKSSRGFGFTVVGGDEPDEFLQ
IKSLVLDGPAALDGKMETGDVIVSVNDTCVLGHT
HAQVVKIFQSIPIGASVDLELCRGYPLPFDPDDPN
MAGI1 33709973 PATQPELITVHIVKGPMGFGFTIADSPGGGGQRVK
QIVDSPRCRGLKEGDLIVEVNKKNVQALTHNQVV
DMLVECPKGSEVTLLVQRGGNLS
MAGI1 33709974 PDYQEQDIFLWRKETGFGFRILGGNEPGEPIYIGI~I
VPLGAADTDGRL;RSGDELICVDGTPVIGKSHQLV
VQLMQQAAKQGHVNLTVRRKVVFAVPKTENSS
MAGI1 33709975 GVVSTVVQPYDVEIRRGENEGFGFVIVSSVSRPEA
GTTFAGNACVAMPHKIGRILEGSPADRCGKLKVG
DRILAVNGCSITNKSHSDTVNLIKEAGNTVTLRIIP
GDESSNA
MAGI1 33709976 QATQEQDFYTVELERGAKGhGFSLRGGREYNMD
LYVLRLAEDGPAERCGKMRLGD EILElNGETTKN
MKHSRAIELIKNGGRRVRLFLKRG
MGC5395 BC012471 PAK1VIEKEETTRELLLPNWQGSGSHGLTIAQRDDG
7 VFVQEVTQNSPAARTGVVKEGDQiVGATIYFDNL
QSGEVTQLLNTMGHHTVGLKLHRKGDRSPNS S
MINT1 26250241 SENCKdVFIEKQKGEILGVVTVESGWGSILPTVIIA
NMMHGGPAEKSGKLNIGDQ IMSINGTSLVGLPLS
TCQSIIKGLKNQSRVKLNIVRCPPVNSS
MINT1 26250242 LRCPPVTTVLIRRPDLRYQLGFSVQNGIICSLMRG
GIAERGGVRV GHRIIEINGQS V V ATPHEKIVHILSN
AVGEIHMKTMPAAMYRLLNSS
MINT3 31698081 LSNSDNCREVHLEKRRGEGLGVALVESGWGSLLP
TAVIANLLHGGPAERSGALS1GDRLTAlNGTSLVG
LPLAACQAAVRETKSQTSVTLSIVHCPPVTTA1M
MINT3 31698082 LVHCPPVTTAIIHRPHAREQLGFCVEDGIICSLLRG
GIAERGGIRV GHRIIEII~I G QS V V A'hP
H A RIIELLTEA
120
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or DomSequence fused to GST Construct
Acc# ain#
YGEVHIKTMPAATYRLLTG
MPP1 189785 1 RKVRLIQFEKVTEEPMGiTLICLNEKQSCTVARILH
GGMIHRQGSLHVGDEILElNGTNVTNHS VDQLQK
AMKETKGMISLKV1PNQ
MPP2 939884 1 PVPPDAVRMVGIRKTAGEHLGVTFRVEGGELVIA
RILHGGMVAQQGLLHVGDLI KEVNGQPVGSDPRA
LQELLRNASGS VILKILPNYQ
MUPP1 2104784 1 QGRHVEVFELLKPPSGGLGFSVVGLRSENRGELGI
FVQEIQEGSVAHRDGRLKETDQILAINGQALDQTI
THQQAISILQKAKDTVQLVIARGSLPQLV
MUPP1 2104784 2 PVHWQHMETIELVNDGSGLGFGIIGGKATGVIVIC
TILPGGVADQlLGRLCSGDHILKIGDTDLAGMSSE
QVAQVLRQCGNRVKLMIARGAIEERTAPT
MUPP1 2104784 3 QESETFDVELTKNVQGLGITIAGYIGDItICLEPSGI.>~
VKSITKSSAVEHDGR1QIGDQIIA VDGTNLQGFTN
QQAVEVLRHTGQTVLLTLM.RRGMKQEA
MUPP1 2104784 4 LNYEIVVAHVSKFSENSGLG1SLEATVGHHFIRSV
LPEGPVGHSGKLFSGDELLEVNGITLLGENHQDV
VNILKELPIEVTMVCCRRTVPPT
MUPP1 2104784 5 WEAGIQHIELEKGSKGLGFSILDYQDPIDPASTVIII
RSLVPGGIAEKDGRLLPGDRLMFVNDVNLENSSL
EEAVEALKGAPSGTVRIGVAKPLPLSPEE
MUPP1 2104784 6 RNVSKESFERTINIAKGNSSLGMTVSANKDGLGM
IVRSIIHGGAISRDGRIAIGDCILS IN EE ST I
S V TNAQ
ARAMLRRHSLIGPDIKITYVPAEE-ILEE
MUPP1 2104784 7 LNWNQPRRVELWREPSKSLGIS1VGGRGMGSRLS
NGEVMRGIFIKHVLEDSPAGI~NGTLKPGDRIVEV
DGMDLRDASHEQAVEAIRKAGNPVVFMVQSIINR
PRKSPLPSLL
MUPP1 2104784 8 LTGELHMIELEKGHSGLGLSLAGNKDRSRIV1SVFI
VGIDPNGAAGICDGRLQTADELLEINGQILYGRSHQ
NASSIIKCAPSK VKIIFIRNKDAVNQ
MUPP1 2104784 9 LSSFKNVQHLELPKDQGGLGIAISEEDTLSGVIIKS
LTEHGVAATDGRLKVGDQILAV DD EIV VGYPIEI~
FISLLKTAKMTVKLTIHAENPDSQ
MUPPI 2104784 10 LPGCETTIEISKGRTGLGLSIVGGSDTLLGAIIIHEV
YEEGAACKDGRLWAGDQILEV NGIDLRKATHD E
AINVLRQTPQRVRLTLYRDEAPYKE
MUPP1 2104784 11 KEEEVCDTLTIELQKKPGKGLGLSTVGKRNDTGV
FVSDIVKGGIADADGRLMQGDQILMVNGEDVRN
ATQEAVAALLKCSLGTVTLEVGRTKAGPFHS
MUPP1 2104784 12 LQGLRTVEMKKGPTDSLGISIAGGVGSPLGDVPIFI
AMMHPTGVAAQTQKLRVGDRIVTICGTSTEGMT
HTQAVNLLKNASGSIEMQVVAGGDVSV
MUPP1 2104784 13 LGPPQCKSITLERGPDGLGFSIVGGYGSPHGDLPIY
VKTVFAKGAASEDGRLKRGDQIIAVNGQSLEGVT
121
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
HEEAVAILKRTKGTVTLMVLS
NeDLG 108639201 IQYEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFIT
KIIPGGAAAMDGRLGVNDCVLRVNEVEVSEVVH
SRAVEALKEAGPVVRLVVRRRQN
NeDLG 108639202 ITLLKGPKGLGFSIAGGIGNQHII'GDNSIYITKIIEG
GAAQKDGRLQIGDRLLAVNNTNLQDVRHEEAVA
SLKNTSDMVYLKVAKPGSLE
NeDLG 108639203 ILLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADL
SGELRRGDRILS VNGVNLRNATHEQAAAALKRA
GQSVTIVAQYRPEEYSRFESI~IDLREQMMNSSM
SSGSGSLRTSEKRSLE
Neurabin AJ4011891 CVERLELFPVELEKDSEGLGISIIGMGAGADMGLE
II
KLGIFVKTVTEGGAAHRDGRIQVNDLLVEVDGTS
LVGVTQSFAAS VLRNTKGRVRFMIGRERPGEQS E
VAQRIHRD
NOS 1 642525 1 IQPNVISVRLFK.RKVGGLGFLVKERVSKPPVIISDL
IRGGAAEQSGLIQAGDLILAVNGRPLVDLSYDSAL
EVLRGIASETHV VLILRGP
novel PDZ 72281771 QANSDESDIIHSVRVEKSPAGRLGFSVRGGSEHGL
gene GIFVSKVEEGSSAERAGLCVGDKITEVNGLSLEST
TMGSAVKVLTSSSRLHMMVRRMGRVPGIKFSKE
KNSS
novel PDZ 72281772 PSDTSSEDGVRRIVHLYTTSDDFCLGFNIRGGKEF
gene GLGIYVSKVDHGGLAEENGIKVGDQVLAANGVR
FDDISHSQAVEVLKGQTHIMLTIKETGRYPAYKE
MNSS
Novel Serine16212431 KIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSS
Protease KAKELKDRHRDFPDVISGAYI1EV1PDTPAEAGGL
KENDVIISINGQSVVSANDVSDVIICRl:STLNIVIVVR
RGNEDIMITV
Numb BindingAK056821 PDGEITSIKINRVDPSESLSIRLVGGSETPLVHIIIQI
Protein 3 IYRDGVIARDGR LLPGD IILKVNG MDISNVPFINYA
VRLLRQPCQVLWLTVMREQKFRSRNSS
Numb BindingAK056822 HRPRDDSFHVLLNKSSPEEQLGIKLVRKVDEPGVF
Protein 3 IFNVLDGGVAYRHGQL:EENDRVLAINGHDLRYGS
PESAAHLIQASERRVHLVVSRQVRQRSPENSS
Numb BindingAK056823 PTITCHEKVVNIQKDPGESLGMTVAGGASHREWD
Protein 3 LPIYVISVEPGGVISRDGRIKTGDILLNVDGVELTE
VSRSEAVALLKRTSSSIVLKALEVKEYEPQEFIV
Numb BindingAK056824 PRCLYNCKDiVLRRNTAGSLGFCTVGGYEEYNGN
Protein 3 KPFFIKSIVEGTPAYNDGRIRCGDILLAVNGRSTSG
MIHACLARLLKELKGRITLTIV S WPGrhFL
Outer 70238251 LLTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGI
Membrane YVSRIKENGAAALDGRLQEGDK:I:LSVNGQDLKNL
LHQDAVDLFRNAGYA VSLRVQ HRLQVQNG I=HS
p55T 127333671 VDAIRILGIHKRAGEPLGVTFRVENNDLVIARILH
P
122
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or DomSequence fused to GST Construct
Acc# ain#
GGMIDRQGLLHVGDIIKEVNGHEVGNNPKELQEL
LKNISGSVTLKILPSYRDTITPQQ
PAR3 8037914 1 DDMVKLVEVPNDGGPLGIHVVPFSARGGRTLGLL
VKRLEKGGKAEHENLFRENDCIVRINDGDLRNRR
FEQAQHMFRQAMRTPIIWFHV VPAA
PAR3 8037914 2 GKRLNIQLKKGTEGLGFSITSRDVTIGGSAPIYVK
NILPRGAAIQDGRLKAGDRLIEVNG V DLV GKS
QE
EVVSLLRSTKMEGTVSLLVFRQEDA
PAR3 8037914 3 TPDGTREFLTFEVPLNDSGSAGLGVSVKGNRSKE
NHADLGIFVKSIINGGAASKDGRLRVNDQLIAVN
GESLLGKTNQDAMETLRRSMST EGNKRGMIQLIV
A
PARE 2613011 1 LPETHRRVRLHKHGSDRPLGFYIRDGMSVRVAPQ
GLERVPGIFISRLVRGGLAESTG LLA V SDEILEVNG
IEVAGKTLDQVTDMMVANSHNLI:VTVKPANQR
PARE 13 5371181 IDVDLVPETHRRVRLHRHGCEK PLGFY1RDGAS V
GAMMA RVTPHGLEKVPGIFISRMVPGGLAESTGLLAVNDE
VLEVNGIEVAGKTLDQVTDMMTANSHNLIVTVKP
ANQRNNVV
PDZ-73 5031978 1 RSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFIS
HLIKGGQADSVGLQVGDEIVR1NGYS.ISSCTHEEVI
NLIRTKKTVSIKVRHIGLIPVKSSPDEFH
PDZ-73 5031978 2 IPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGIF
ISHVKPGSLSAEVGLEIGDQIVEVNGVDFSNLDHK
EAVNVLKSSRSLTISIVAAAGRELFMTDEF
PDZ-73 5031978 3 PEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKV
VVSAVYERGAAERHGGIVKGDE IMA INGI~IVTDY
TLAEADAALQKAWNQGGD W ID LV VAVCPPKEY
DD
PDZKI 2944188 1 LTSTFNPRECKLSKQEGQNYGhFLRIEICDTEGI-1LV
RVVEKCSPAEKAGLQDGDRVLRINGVFVDKEEH
MQVVDLVRKSGNSVTLLVLDGDSYEI~AGSPGII-I
RD
PDZK1 2944188 2 RLCYLVKEGGSYGFSLKTVQGKKGVYMTDUfPQ
GVAMRAGVLADDHLIEVNGENVEDASHEEVVEK
VKKSGSRVMFLLVDKETDK_REF I VTD
PDZK1 2944188 3 QFKRETASLKLLPHQPRIVEMIOKGSNGYGFYLRA
GSEQKGQIIKDIDSGSPAEEAGLKNND LV VAVNG
ESVETLDHDS V V EMIRKGGDQTSLLV VDKETDN
MYRLAEFIVTD
PDZK1 2944188 4 PDTTEEVDHKPKLCRLAKGENGYGFHLNAIRGLP
GSFIKEVQKGGPADLAGLEDEDVIIEVNGVNVLD
EPYEKVVDRIQSSGKNVTLLVZGICNSS
PICK1 4678411 1 PTVPGKVTLQKDAQNLIGISIGGGAQYCPCLYIVQ
VFDNTPAALDGTVAAGDEITGVNGRSIKGKTKV E
VAKMIQEVKGEVTIHYNKLQ
123
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
PIST 983743301 SQGVGPIRKVLLLKEDHEGLGISITGGKEHGVPILI
SEIHPGQPADRCGGLHVGDAILAVNGVNLRDTKH
KEAVTILSQQRGEIEFEV VYVAPEVDSD
prILl6 14784921 IHVTILHKEEGAGLGFSLAGGADLENICVITVHRVF
PNGLASQEGTIQKGNEVLSINGKSLKGTTHHDAL
AILRQAREPRQAV IVTRKLTPEEF IVTD
prILl6 14784922 TAEATVCTVTLEKMSAGLGFSLEGGI{GSLHGDKP
LT1NRIFKGAASEQSET V QPGDE LLQLGGTAMQGL
TRFEAWNIIKALPDGPVTIVIRRKSLQSK
PSD95 33186521 LEYEeITLERGNSGLGFSIAGGTDNPH IGDDPSIFIT
KIIPGGAAAQDGRLRVNDSTLFVNEV DVREVTHS
AAVEALKEAGSIVRLYVMRRKPPAENSS
PSD95 33186522 HVMRRKPPAEKVMEIKLIICGPKGLGFSIAGGVGN
QHIPGDNSIYVTKIIEGGAAI-IKDGRLQIGDKILAV
NSVGLEDVMHEDAVAALKNTI'DVVYLKVAKPS
NAYL
PSD95 33186523 REDIPREPRRIVIHRGSTGLGFNLVGGEDGEGIFISFI
LAGGPADLS GELRKGDQILS VNG V DLRNASHEQA
AIALKNAGQTVTIIAQYKPEF1VTD
PTN-3 179912 1 LIRITPDEDGKFGFNLKGGVDQKMPLVVSR1NPES
PADTCIPKLNEGDQ1VLINGRDISEHTHDQV VMFI
KASRESHSRELALVIRRR
PTN-4 190747 1 IRMKPDENGRFGFNVKGGYDQKMPV IVSRV APG
TPADLCVPRLNEGDQVVLINGRDIAEHTHDQVVL
FIKASCERHSGELMLLVRPNA
PTPL1 515030 1 PEREITLVNLKKDAKYGLGFQIIGGEICMGRLDLGI
FISSVAPGGPADFHGCLKPGDRL TSVNSVSLEGVS
HHAAIEILQNAPEDVTLVISQPI<EKIS IOVPS'CPVHL
PTPL1 515030 2 GDIFEVELAKNDNSLGISVTGGVNTSVRHGGTYV
KAVIPQGAAESDGRIHKGDRVLAVNGVSLEGATH
KQAVETLRNTGQV VTiLLLEKGQSPTS1O
PTPL1 515030 3 TEENTFEVKLFKNSSGLGFSFSREDNLIPEQINASl
VRVKKLFAGQPAAESGKIDVGDV1LKVNGASLKG
LSQQEVISALRGTAPE VFLLLCRP PPG V LP
EIDT
PTPL1 515030 4 ELEVELLITLIKSEKASLGFTVTKGNQRTGCYVND
VIQDPAKSDGRLKPGDRLTKVNDTDVTNMTI37'D
AVNLLRAASKTVRLVTGRVL ELPRCPMLPI-l
PTPL1 515030 5 MLPHLLPDITLTCNKEELGFSLCGGH DSLYQV V
YI
SDINPRSVAAIEGNLQLLDVIHYVNGVSTQGMTL
EEVNRALDMSLPSL V LKATRNDLP V
RGS12 32900151 RPSPPRVRSVEVARGRAGYGFTLSGQAPCVLSCV
MRGSPADFVGLRAGDQILAVNE1NVKKASHEDV
VKLIGKCSGVLHMVIAEGVGRFESCS
RGS3 186447351 LCSERRYRQTTIPRGKDGFGFTICCDSPVRVQAVD
SGGPAERAGLQQLDTVLQLNERPVEI-IWKCV.ELA
HEIRSCPSEIILLVWRMVPQVK:PGIEIRD
124
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom equence fused to GST Construct
S
Acc# ain#
Rhophilin-like142794081 SFSANKRWTPPRSIRFTAEEGDLGFTLRGNAPVQ
I
VHFLDPYCSAS VAGAREGDYIVSIQLVDCICWLTL
S EVMKLLKSFGEDEIEMKVVSLLDSTSSMHNKSA
T
Serine Protease2738914 1 RGEKKNSSSGISGSQRRYIGVMMLTLSPSILAELQ
LREPSFPDV QHG VLIHKVILG S PAHRAGLRPGD
VI
LAIGEQMVQNAEDVYEAVRTQSQLAVQTRRGRE
TLTLYV
Shank 1 6049185 1 EEKTVVLQKKDNEGFGFVL.RGAKADTPIEEFTPTP
AFPALQYLES V DEGG VA W QAGLRTGDFLTE
VNN
ENVVKVGHRQV VNMIRQGGNHLVLKVVTVTRN
LDPDDTARKKA
Shank 3 * 1 SDYVIDDKVAVLQKRDHEGFGFVLRGAItAETPLE
EFTPTPAFPALQYLESVDVEGVAWRAGLRTGDF.L
I EVNGVNVVKVGHKQV VALIRQGGN RLVMKV VS
V TRKPEED G
Shroom 186528581 YLEAFLEGGAPWGFTLKGGLEHGEPLIISKVEEG
I
GKADTLSSKLQAGDEVVHINEVTLSSSRKEAVSL
VKGSYKTLRLVVRRDVCTDPGH
SIP1 2047327 1 IRLCRLVRGEQGYGFHLHGEKGRRGQFIRRVEPG
SPAEAAALRAGDRLVEVNGVNVEGETHHQVVQR
IKAVEGQTRLLVVDQN
SIP1 2047327 2 IRHLRKGPQGYGFNLHSDKSRPGQYI:RSVDPGSPA
ARSGLRAQDRLIEVNGQNVEGLRHAE V V A SIKAR
EDEARLLVVDPETDE
SITAC-18 8886071 1 PGVREIHLCKDERGKTGLRLRKVDQGLFVQLVQ
ANTPASLVGLRFGDQLLQIDGRDCAGWSSHKAH
QVVKKASGDKIVVVVRDRPFQRTVTM
SITAC-I8 8886071 2 PFQRTVTMHKDSMGHVGFVIICKGKIVSLVKGSSA
ARNGLLTNHYVCEVDGQNV IGL;KDK1<IMEILA'CA
GNVVTLTIIPSV1YEHIVEFIV
SSTRIP 7025450 1 LKEKTVLLQK:ICDSEGFGFVLRGAKAQTPIEEFTPT
PAFPALQYLESVDEG G VA WRAGLIRM GDFL1E
V N
GQNVVKV GHRQV VNMIRQGGNTLM VKV VMVT
RHPDMDEAV Q
SYNTENIN 2795862 1 LEIKQGIREVILCKDQDGKIGLRLKSIDNGIFVQLV
QANSPASLV GLRFGDQVLQING:ENCAG WSSDKA
HKVLKQAFGEKITMRII-IRD
SYNTENIN 2795862 2 RDRPFERTITMHKDSTGHVGF1FKNGI~ITSTVKDSS
AARNGLLTEHN ICEING QN V l G LIDS QIAD:I:LS
TS G
NSS
Syntrophin 1145727 1 QRRRVTVRKADAGGLGISI1CGGRENI<MPIL1SKIE
1
alpha KGLAADQTEALFVGDAILSVNGEDLSSATHDEAV
QVLKKTGKEV V LEVKYMKDV SPYFK
Syntrophin 476700 1 IRVVKQEAGGLGISIKGGRENRMP1LISKIFPGLAA
beta 2 DQSRALRLG.DAILSVNGTDLRQATHDQAVQALIC
RAGKEVLLEVKFIREFIV TD
125
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
Syntrophin 95071621 EPFYSGERTVTLRRQTVGGFGLSIKGGAEHNIPVV
gamma 1 VSKISKEQRAELSGLLFIGDAILQINGINVRKCRHE
EVVQVLRNAGEEVTLTVSFLKRAPAFLKLP
Syntrophin 95071641 SHQGRNRRTVTLRRQPVGGLGLSIKGGSEHNVPV
gamma 2 VISKIFEDQAADQTGMLFVGDAVLQVNGIHVENA
THEEVVHLLRNAGDEVTITVEYLREAPAFL1C
TAX2-like 32531161 RGETKEVEVTKTEDALGLTITDNGAGYAFIKRIKE
protein GSIINRIEAVCVGDSIEAlNDHSIVGCRHYEVAKM
LRELPKSQPFTLRLVQPKRAF
TIAM 1 45075001 HSIHIEKSDTAADTYGFSLSSVEEDGLRRLYVNSV
KETGLASKKGLKAGDEILE1NNRAADA:LNS SM LK
DFLSQPSLGLLVRTYPELE
TIAM 2 69127031 PLNVYDVQLTKTGSVCDFGFAVTAQVDERQHLS
RIFISDVLPDGLAYGEGLRKGNE fMTLNGEA VSDL
DLKQMEALFSEKSVGLTLIARPPDTKATL
TIP1 26130011 QRVEIHKLRQGENLILGFSIGGGIDQDPSQNPISED
KTDKGIYVTRVSEGGPAEIAGLQIGD1CLMQVNGW
DMTMVTHDQARKRLTKRSEEV V RLLVTRQS LQK
TIl'2 26130031 RKEVEVFKSEDALGLTITDNGAGYAFI:ICRIICEGSV
IDHIHLISVGDMIEA1NGQSLLGCRHYE VARLLKE
LPRGRTFTLKLTEPRK
TIP33 26130071 HSHPRVVELPKTDEGLGFNVMGGKEQNSPIYISRI
IPGGVAERHGGLKRGDQL LS VNG V S VEGEHI~EIC
AVELLKAAKDSVKLVVRYTPKVL
TIP43 26130111 ISNQKRGVKVLKQELGGLGISIKGGKENKMPILIS
KIFKGLAADQTQALYVGDAILS VNGADLRDATH
DEAVQALKRAGKEVLLEVKYMREATPYV
X-11 beta 30055591 IHFSNSENCKELQLEKHKGEILGVVVVESGWGSIL
PTVILANMMNGGPAARSGKLSlGDQLMS1NGTSLV
GLPLATCQGIIKGLKNQTQV KLN 1 VSCPPVTTVLI
KRNS S
X-11 beta 30055592 IPPVTTVLIlCRPDLKYQLGFSVQNGIICSLMRGGIA
ERGGVRV GHRI1 E1NGQS V V ATA EfEK1 V
QA LSNS V
GEIHMKTMPAAMFRLLTGQENS S
ZO-1 292937 1 IWEQHTVTLHRAPGFGFGIAISGGRDNPHFQSGET
SIVISDVLKGGPAEGQLQENDRVAMVNGVSMDN
VEHAFAVQQLRKSGKNAK STIR RIUQ<VQIPNSS
ZO-1 292937 2 ISSQPAKPTKVTLVKSR1CNEEYGLRLASHIFVKEIS
QDSLAARDGNLQEGDVVLKINGTVTENMSLTDA
KTLIERSKGKLKM V V QRD RATLLNS S
ZO-1 292937 3 IRM.KLVKF'RKGDSVGLRLAGGNDVGfFVAGVLE
DSPAAKEGLEEGDQILRVNNVDFTNIIREEAV LFL
LDLPKGEEVTILAQKKKDVFSN
ZO-2 127347631 LIWEQYTVTLQKDSKRGFGIAVSGGRDNPHFENG
ETSIVISDVLPGGPADGLLQENDRVVMVNGTPME
DVLHSFAVQQLRKSGKVAAIV VK_RPR.KV
126
CA 02505479 2005-05-10
WO 2004/045535 PCT/US2003/036698
Gene Name GI or Dom Sequence fused to GST Construct
Acc# ain#
ZO-2 127347632 RVLLMKSRANEEYGLRLGSQIFVKEMTRTGLATK
DGNLHEGD l ILKINGTVTENMS LTDARKLIEKSRG
KLQLVVLRDS
ZO-2 127347633 HAPNTKMVRFKKGDSVGLRLAGGNDVGIFVAGI
QEGTSAEQEGLQEGDQILKVNTQDFRGLVREDAV
LYLLEIPKGEMVTILAQSRADVY
ZO-3 100926901 IPGNSTIWEQHTATLSKDPRRGFGIAISGGRDRPG
GSMVVSDVVPGGPAEGRLQTGDHIVMVNGVSME
NATSAFAIQILKTCTKMANITVKRPRRIHLPAEFIV
TD
ZO-3 100926902 QDVQMKPVKSVLVKRRDSEEFGVKLGSQLFIKHIT
DSGLAARHRGLQEGDLILQINGV SSQNLSLNDTR
RLIEKSEGKLSLLV LRDRGQFLV NIPNS S
ZO-3 100926903 RGYSPDTRVVRFLKGKSIGLRLAGGNDVGIFVSG
VQAGSPADGQGIQEGDQILQVNDVPFQNLTREEA
VQFLLGLPPGEEMELVTQRKQ:DI1~ WKMVQSCFIV
TD
*: No GI
number
for this
PDZ domain
containing
protein
- it was
computer
cloned
by
J.S. using
rat Shank3
seq against
human genomic
clone AC000036.
In silico
spliced
together
nt6400-6496,
6985-7109,
7211-7400
to create
hypothetical
human Shanl<3.
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