U.S. patent application number 12/538665 was filed with the patent office on 2010-06-17 for assays for detecting inhibitors of binding between cox-2 and pdz proteins.
This patent application is currently assigned to Arbor Vita Corporation. Invention is credited to Michael P. Belmares, Jonathan David Garman, Peter S. Lu.
Application Number | 20100152294 12/538665 |
Document ID | / |
Family ID | 37595453 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100152294 |
Kind Code |
A1 |
Belmares; Michael P. ; et
al. |
June 17, 2010 |
Assays For Detecting Inhibitors Of Binding Between COX-2 And PDZ
Proteins
Abstract
The invention provides an assay for determining whether a test
agent is a COX modulator. In general terms, the assay includes:
determining whether a test agent modulates binding of a
PDZ-containing polypeptide to a COX PL-containing polypeptide. The
PDZ-containing polypeptide may contain the PDZ domain of PDZ domain
of MAGI1, TIP-1, MAST2, PSD95, or SHANK. The assays may be done in
a cell-free environment or in a cellular environment, particularly
using a neuronal cell. The invention finds use in a variety of
therapeutic applications, including for identifying agents for use
in treating cancer, pain, inflammation and neuronal conditions
caused by acute insult, e.g., stroke.
Inventors: |
Belmares; Michael P.;
(Sunnyvale, CA) ; Garman; Jonathan David;
(Sunnyvale, CA) ; Lu; Peter S.; (Sunnyvale,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Arbor Vita Corporation
Sunnyvale
CA
|
Family ID: |
37595453 |
Appl. No.: |
12/538665 |
Filed: |
August 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11426282 |
Jun 23, 2006 |
7588911 |
|
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12538665 |
|
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60693988 |
Jun 23, 2005 |
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Current U.S.
Class: |
514/569 ;
435/325; 435/7.21; 436/501; 514/570 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 2500/00 20130101; C12Q 1/26 20130101; G01N 33/6896 20130101;
G01N 33/88 20130101 |
Class at
Publication: |
514/569 ;
435/7.21; 435/325; 514/570; 436/501 |
International
Class: |
A61K 31/192 20060101
A61K031/192; G01N 33/567 20060101 G01N033/567; C12N 5/079 20100101
C12N005/079; G01N 33/566 20060101 G01N033/566; A61P 35/00 20060101
A61P035/00 |
Claims
1. An assay for detecting a COX modulator, comprising: determining
whether a test agent modulates binding of a PDZ-containing
polypeptide to a COX PDZ ligand-containing polypeptide.
2. The assay of claim 1, wherein said COX PDZ ligand-containing
polypeptide is a COX-2 PDZ ligand-containing polypeptide.
3. The assay of claim 1, wherein said PDZ-containing polypeptide
contains a PDZ domain of MAGI1, TIP-1, MAST2, PSD95, or SHANK.
4. The assay of claim 3, wherein said SHANK PDZ-containing
polypeptide comprises a PDZ domain of SHANK1, SHANK2 or
SHANK-3.
5-6. (canceled)
7. The assay of claim 1, wherein said assay is a cell-free
assay.
8. The assay of claim 1, wherein said assay is a cellular
assay.
9-10. (canceled)
11. The assay of claim 8, wherein said assay is performed using
neuronal cells that contain said PDZ domain-containing polypeptide
and said COX PDZ ligand-containing polypeptide.
12. The assay of claim 1, wherein said assay further comprises
testing said agent for COX-2 cycloxygenase inhibitory activity.
13. The assay of claim 1, wherein said test agent is an inhibitor
of a cycloxygenase activity of COX-2.
14. The assay of claim 1, wherein said test agent is PDZ domain
analog.
15. The assay of claim 1, further comprises testing said compound
in a neuronal cell.
16. The assay of claim 15, further comprising subjecting said
neuronal cell to insult.
17. The assay of claim 15, wherein said insult is hypoxia or
ischemia.
18. A method of reducing binding between COX-2 and a PDZ-containing
polypeptide in a cell, comprising: administering to said cell a PDZ
domain analog or a compound which competes with the binding of
COX-2 to said PDZ-containing polypeptide; and maintaining said cell
under conditions suitable for said PDZ domain analog or compound to
reduce said binding.
19-23. (canceled)
24. The method of claim 18, wherein said cell is an insulted
neuronal cell.
25. The method of claim 24, wherein said neuronal cell is a hypoxic
or ischemic neuronal cell.
26. The method of claim 24, wherein said method results in reduced
NMDA receptor activation.
27. The method of claim 18, wherein said administration comprises
administering a compound selected from the group consisting of:
sulindac sulphide, fenoprofen, derivatives thereof, analogs
thereof, and combinations thereof.
28. The method of claim 18, wherein said reduction in binding
further results in anti-tumor and/or anti-cellular proliferate
properties when administered in vivo.
29. The method of claim 28, wherein said PDZ domain analog or said
compound which competes with the binding of COX-2 to said
PDZ-containing polypeptide is administered to a subject suffer from
cancer, and said anti-tumor and/or anti-cellular proliferate
properties results in treatment of said cancer.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of the priority date
under 35 U.S.C. .sctn.119(e) of U.S. Provisional Application No.
60/693,988, filed Jun. 23, 2005, the contents of which are herein
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The prostaglandins are a potent class of biologically active
lipid derivatives that play a crucial role in the inflammatory
response. The inflammatory response is a localized tissue response
to injury or other trauma characterized by pain, heat, redness and
swelling. Prostaglandins mediate this response by inhibiting
platelet aggregation, increasing vascular permeability, increasing
vascular dilation, inducing smooth-muscle contraction and causing
the induction of neutrophil chemotaxis. Because of their central
role in mediating the inflammatory response, significant efforts
have been directed toward elucidating compositions that are capable
of inhibiting the biosynthesis of prostaglandins.
[0003] Toward that end, prostaglandin biosynthesis has been
extensively characterized. Prostaglandins are a group of oxygenated
fatty acids that are generally derived from arachidonic acid. The
biosynthesis of prostaglandins from arachidonic acid occurs in a
three step process that includes 1) hydrolysis of arachidonic acid
from phospholipid precursors catalyzed by a phospholipase A.sub.2;
2) cyclooxygenase ("COX") catalyzed oxygenation of arachidonic acid
to prostaglandin G2 ("PGG2"). This COX catalyzed reaction is the
first committed and rate limiting step in prostaglandin synthesis;
and 3) conversion of prostaglandin G2 to the biologically active
end product, prostaglandin, catalyzed by a series of synthases and
reductases. Upon their synthesis, prostaglandins exit the cell and
act in a hormone-like manner by affecting the target cell via G
protein linked membrane receptors.
[0004] Inactivation of the COX enzyme is a natural target as a
means to inhibit prostaglandin production due to this enzyme's
pivotal role in the prostaglandin biosynthetic pathway. It is now
known that two gene products possessing COX enzyme activity are
expressed, termed COX-1 and COX-2. COX-1 was the first discovered
isoform and is constitutively expressed in most tissue types.
Because it is constitutively expressed, COX-1 is available to
participate in activities requiring a rapid physiological response
and causes the production of prostaglandins involved in
"house-keeping" functions. For example, COX-1 is responsible for
acute production of prostaglandins that regulate vascular
homeostasis, maintain gastrointestinal integrity, and maintain
kidney function. Thus, COX-1 activity is responsible for the
synthesis of prostaglandins required for the maintenance of several
cell types.
[0005] COX-2, on the other hand, is a recently discovered isoform
that is inducibly expressed in response to numerous stimuli such as
bacterial lipopolysaccharides, growth factors, cytokines, and
phorbol esters. In addition, COX-2 is only expressed in a limited
number of cell types including monocytes, macrophages, neutrophils,
fibroblasts and endothelial cells. COX-2 expression, but not COX-1
expression, has been shown to increase in rheumatoid synovial
tissue. Contrastingly, COX-2 expression is inhibited in response to
glucocorticoids and by anti-inflammatory cytokines. Thus, based
upon these observations, COX-2 has been shown to be the isoform
responsible for mediating the production of prostaglandins that
participate in the inflammatory response and inflammatory related
disorders. In addition, COX-2 has also been shown to participate in
certain cancers, Alzheimer's disease, atherosclerosis, and central
nervous system damage resulting from stroke, ischemia and
trauma.
[0006] Nonsteroidal anti-inflammatory drugs (NSAIDs) are also
utilized as a means to reduce effects associated with the
inflammatory response. The principal pharmaceutical effects of
NSAIDs are due to their ability to prevent COX activity resulting
in the inhibition of prostaglandin synthesis. Inhibition of
prostaglandin synthesis by NSAIDs is anti-pyretic, analgesic,
anti-inflammatory, and anti-thrombogenic. However, administration
of NSAIDs may also result in severe side effects such as
gastrointestinal bleeding, ulcers and incidence of renal
problems.
[0007] There is a great need for new drugs that modulate COX
activity, as well as assays to facilitate the discovery of such
drugs. This invention meets this need.
BRIEF SUMMARY OF THE INVENTION
[0008] In certain aspects, the invention provides an assay for
determining whether a test agent is a COX modulator. In general
terms, the assay includes: determining whether a test agent
modulates binding of a PDZ-containing polypeptide to a COX
PL-containing polypeptide. The PDZ-containing polypeptide may
contain the PDZ domain of PDZ domain of MAGI1, TIP-1, MAST2, PSD95,
or SHANK. The assays may be done in a cell-free environment or in a
cellular environment, particularly using a neuronal cell. The
invention finds use in a variety of therapeutic applications,
including for identifying agents for use in treating pain, cancer,
inflammation and neuronal conditions caused by acute insult, e.g.,
stroke.
[0009] The invention is based on the discovery that COX-2 contains
a PDZ ligand (i.e., an amino acid sequence that binds to PDZ
proteins; or "PL" for short) at its C-terminus, and the further
discovery of the cellular PDZ-containing proteins to which COX-2's
PDZ ligand binds.
[0010] In another aspect of the invention, it has also been found
that COX-1 contains a PDZ ligand (i.e., and amino sequence that
binds to PDZ proteins; or "PL" for short) at its C-terminus.
[0011] The discovery of the cellular proteins to which COX-2 binds
allows assays to be performed in order to identify COX-2 modulatory
agents. The COX-2 modulatory agents may, in certain embodiments,
inhibit binding between COX-2 and the subject PDZ-domain containing
binding proteins. In other embodiments, inhibitors of cycloxygenase
activity of COX-2 may be tested in the subject binding assays to
identify inhibitors that do or do not modulate binding of COX-2 to
the subject PDZ domain-containing proteins.
[0012] In certain aspects, COX-2 binds to all three members of the
SHANK family (which includes SHANK1, SHANK2 and SHANK3). Proteins
of the SHANK family are known to interact with components of the
postsynaptic membrane, including NMDA receptors, metabotropic
glutamate receptors and the actin-based cytoskeleton. For example,
SHANK1 is known to be expressed in neuronal tissues and modulates
synaptic responses by interaction with inhibitory G-proteins in
pre- and post-synaptic compartments. Further, SHANK1 is known to
act as scaffold in the post-synaptic density (PSD), crosslinking
NMDA receptor/PSD95 complexes and coupling them to cytoskeleton
regulators. SHANK1 also crosslink Horner/PSD95 complexes, and
mediates mGluR and NMDA receptor signaling. SHANK2 is expressed
only in the brain, and SHANK3 is expressed mainly in the cerebral
cortex and is highly enriched in the PSD/excitatory synapses.
[0013] Accordingly, in accordance with certain aspects of the
invention, COX-2, as well as having a cycloxygenase activity that
is involved in the production of prostaglandins, may have a binding
activity that is involved in NMDA receptor activation in brain
tissue. Inhibitors of binding between COX-2 and PDZ-containing
proteins, in certain embodiments, may be employed to treat acute
insults to nerve tissue, such as ischemic events (including stroke
or cardiac arrest), hypoxic events and trauma, as well as other
neuron-related conditions and cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A shows SHANK1, SHANK2, SHANK3 and MAST2 PDZ domain
and COX-2 PDZ ligand sequence that may be employed herein.
Minimally-lengthed PDZ domains are shown, as well as exemplary PDZ
domains of longer length.
[0015] FIG. 1B shows a sequence alignment of SHANK1, SHANK2, SHANK3
and MAST2 PDZ domains indicating amino acids that substitutions may
be made.
[0016] FIG. 2 shows an amino acid sequence alignment between the
PDZ domains of the SHANK1, SHANK2 and SHANK3.
[0017] FIG. 3A-3B shows exemplary results identifying SHANK1,
SHANK2, SHANK3 and MAST2 as COX-2 binding proteins.
[0018] FIG. 4A-4C shows further exemplary results identifying
SHANK1, SHANK2, SHANK3 and MAST2 as COX-2 binding proteins.
[0019] FIGS. 5A and 5B shows the sequences of polypeptides that can
bind to the PDZ domain at least one SHANK polypeptide (FIG. 5A) and
the PDZ domain of MAST2 (FIG. 5B).
[0020] FIG. 6 shows amino acid sequences for MAGI1 d1, TIP-1, PSD95
d1, PSD95 d2, and PSD95 d3 PDZ domain sequences that may be
employed in accordance with certain embodiments of the present
invention.
[0021] FIGS. 7A-6G show exemplary results from assays screening
exemplary candidate small molecule drug therapeutics in accordance
with various embodiments of the invention.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention pertains. The
following references provide one of skill with a general definition
of many of the terms used in this invention: Singleton et al.,
Dictionary of Microbiology and Molecular Biology (2d Ed. 1994); The
Cambridge Dictionary of Science and Technology (Walker Ed., 1988);
and Hale & Marham, The Harper Collins Dictionary of Biology
(1991). Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are
described. The following definitions are provided to assist the
reader in the practice of the invention.
[0023] The term "modulation" as used herein refers to both
upregulation, (i.e., activation or stimulation) for example by
agonizing, and downregulation (i.e., inhibition or suppression) for
example by antagonizing, of a bioactivity (e.g., a binding
activity). As used herein, the term "COX PDZ ligand binding
modulator" refers to an agent that is able to alter binding of the
PDZ-ligand (i.e., "PL") of COX (e.g., COX-1 or COX-2 or both) with
the PDZ domain of, e.g., MAGI1 (including MAGI1 d1), TIP-1, SHANK
(including SHANK1, SHANK2, and SHANK3), PSD95 (including PSD95 d1,
PSD95 d2, and PSD95 d3) or MAST2. Modulators include, but are not
limited to, both activators and inhibitors. An inhibitor may cause
partial or complete inhibition of binding.
[0024] A "COX PDZ ligand binding modulator" generally reduces
binding between COX-2 and a PDZ polypeptide by at least 20%, e.g.,
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, up to about 99% or
100%, as compared to controls that do not include the test
compound. In general, agents of interest are those which exhibit
IC.sub.50s in a particular assay in the range of about 1 mM or
less. Compounds which exhibit lower IC.sub.50s, for example, in the
range of about 100 .mu.M, 10 .mu.M, 1 .mu.M, 100 nM, 10 nM, 1 nM,
or even lower, are particularly useful for as therapeutics or
prophylactics to treat or prevent COX-mediated disorders.
Equivalent definitions will apply for COX-2 PDZ ligand binding
modulators and COX-1 PDZ ligand binding modulators.
[0025] By "COX-inhibitory," "COX-1 inhibitory," or
"COX-2-inhibitory", as in the context of a "COX-2-inhibitory
compound", is meant having an activity that inhibits any activity
of COX, COX-2, or COX-2, respectively, including, e.g., a
cycloxygenase (i.e., the prostaglandin-producing) activity of
COX-2, a binding activity of COX-2, or an ability of COX-2 to
increase or decrease activation of the NMDA receptor.
[0026] A "COX-mediated disorder," "COX-1 mediated disorder," or
"COX-2 mediated disorder" is any disorder that may be mediated by
an activity of COX, COX-2, or COX-2, respectively. For example,
many COX-2-mediated disorders involve inflammation and pain.
COX-2-mediated disorders also include certain types of cancer,
Alzheimer's disease, atherosclerosis, and central nervous system
damage resulting from stroke, ischemia or trauma, for example.
[0027] As used herein, the term "acute insult to the central
nervous system" includes short-term events that pose a substantial
threat of neuronal damage mediated by glutamate excitotoxicity.
These include ischemic events (which involve inadequate blood flow,
such as a stroke or cardiac arrest), hypoxic events (involving
inadequate oxygen supply, such as drowning, suffocation, or carbon
monoxide poisoning), trauma to the brain or spinal cord (in the
form of mechanical or similar injury), certain types of food
poisoning which involve an excitotoxic poison such as domoic acid,
and seizure-mediated neuronal degeneration, which includes certain
types of severe epileptic seizures. It can also include trauma that
occurs to another part of the body, if that trauma leads to
sufficient blood loss to jeopardize blood flow to the brain (for
example, as might occur following a shooting, stabbing, or
automobile accident).
[0028] The term "agent" includes any substance, molecule, element,
compound, entity, or a combination thereof. It includes, but is not
limited to, e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, and the like. It can be a natural
product, a synthetic compound, or a chemical compound, or a
combination of two or more substances. Unless otherwise specified,
the terms "agent", "substance", and "compound" can be used
interchangeably. Further, a "test agent" or "candidate agent" is
generally a subject agent for use in an assay of the invention for
investigation as a potential COX-2 PDZ ligand binding
modulator.
[0029] The term "analog" is used herein to refer to a molecule that
structurally resembles a molecule of interest but which has been
modified in a targeted and controlled manner, by replacing a
specific substituent of the reference molecule with an alternate
substituent. Compared to the starting molecule, an analog may
exhibit the same, similar, or improved utility. Synthesis and
screening of analogs, to identify variants of known compounds
having improved traits (such as higher binding affinity, or higher
selectivity of binding to a target and lower activity levels to
non-target molecules) is an approach that is well known in
pharmaceutical chemistry.
[0030] As used herein, "contacting: has its normal meaning and
refers to combining two or more agents (e.g., two proteins, a
polynucleotide and a cell, etc.). Contacting can occur in vitro
(e.g., two or more agents, such as a test compound and a cell
lysate, are combined in a test tube or other container) or in situ
(e.g., two polypeptides can be contacted in a cell by coexpression
in the cell, of recombinant polynucleotides encoding the two
polypeptides), in the presence or absence of a cell lysate.
[0031] A "biopolymer" is a polymer of one or more types of
repeating units, regardless of the source. Biopolymers may be found
in biological systems and particularly include polypeptides and
polynucleotides, as well as such compounds containing amino acids,
nucleotides, or analogs thereof. The term "polynucleotide" refers
to a polymer of nucleotides, or analogs thereof, of any length,
including oligonucleotides that range from 10-100 nucleotides in
length and polynucleotides of greater than 100 nucleotides in
length. The term "polypeptide" refers to a polymer of amino acids
of any length, including peptides that range from 6-50 amino acids
in length and polypeptides that are greater than about 50 amino
acids in length.
[0032] In most embodiments, the terms "polypeptide" and "protein"
are used interchangeably. The term "polypeptide" includes
polypeptides in which the conventional backbone has been replaced
with non-naturally occurring or synthetic backbones, and peptides
in which one or more of the conventional amino acids have been
replaced with one or more non-naturally occurring or synthetic
amino acids. The term "fusion protein" or grammatical equivalents
thereof references a protein composed of a plurality of polypeptide
components, that while not attached in their native state, are
joined by their respective amino and carboxyl termini through a
peptide linkage to form a single continuous polypeptide. Fusion
proteins may be a combination of two, three or even four or more
different proteins. The term polypeptide includes fusion proteins,
including, but not limited to, fusion proteins with a heterologous
amino acid sequence, fusions with heterologous and homologous
leader sequences, with or without N-terminal methionine residues;
immunologically tagged proteins; fusion proteins with detectable
fusion partners, e.g., fusion proteins including as a fusion
partner a fluorescent protein, .beta.-galactosidase, luciferase,
and the like.
[0033] In general, polypeptides may be of any length, e.g., greater
than 2 amino acids, greater than 4 amino acids, greater than about
10 amino acids, greater than about 20 amino acids, greater than
about 50 amino acids, greater than about 100 amino acids, greater
than about 300 amino acids, usually up to about 500 or 1000 or more
amino acids. "Peptides" are generally greater than 2 amino acids,
greater than 4 amino acids, greater than about 10 amino acids,
greater than about 20 amino acids, usually up to about 50 amino
acids. In some embodiments, peptides are between 5 and 30 amino
acids in length.
[0034] In certain embodiments, variants of amino acid and nucleic
acid sequences include "conservatively modified variants." With
respect to particular nucleic acid sequences, conservatively
modified variants may refer to those nucleic acids which encode
identical or essentially identical amino acid sequences, or where
the nucleic acid does not encode an amino acid sequence, to
essentially identical sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given protein.
[0035] For instance, the codons GCA, GCC, GCG and GCU all encode
the amino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded
polypeptide.
[0036] Such nucleic acid variations are "silent variations," which
are one species of conservatively modified variations. Every
nucleic acid sequence herein which encodes a polypeptide also
describes every possible silent variation of the nucleic acid. One
of skill will recognize that each codon in a nucleic acid (except
AUG, which is ordinarily the only codon for methionine, and TGG,
which is ordinarily the only codon for tryptophan) can be modified
to yield a functionally identical molecule. Accordingly, each
silent variation of a nucleic acid which encodes a polypeptide is
implicit in each described sequence.
[0037] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0038] By way of example, the following eight groups each contain
amino acids that are conservative substitutions for one
another:
[0039] 1) Alanine (A), Glycine (G);
[0040] 2) Aspartic acid (D), Glutamic acid (E);
[0041] 3) Asparagine (N), Glutamine (Q);
[0042] 4) Arginine (R), Lysine (K);
[0043] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V);
[0044] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (V);
[0045] 7) Serine (S), Threonine (T); and
[0046] 8) Cysteine (C), Methionine (M)
[0047] (see, e.g., Creighton, Proteins (1984)).
[0048] As recognized by those of skill in the art, macromolecular
structures such as polypeptide structures can be described in terms
of various levels of organization. For a general discussion of this
organization, see, e.g., Alberts et al., Molecular Biology of the
Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry
Part L' The Conformation of Biological Macromolecules (1980).
"Primary structure" refers to the amino acid sequence of a
particular peptide. "Secondary structure" refers to locally
ordered, three dimensional structures within a polypeptide. These
structures are commonly known as domains.
[0049] Domains are portions of a polypeptide that form a compact
unit of the polypeptide and are typically 50 to 350 amino acids
long. Typical domains are made up of sections of lesser
organization such as stretches of .beta.-sheet and .alpha.-helices.
"Tertiary structure" refers to the complete three dimensional
structure of a polypeptide monomer. "Quaternary structure" refers
to the three dimensional structure formed by the non-covalent
association of independent tertiary units. Anisotropic terms are
also known as energy terms.
[0050] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, may refer to
two or more sequences or subsequences or domains that are the same
or have a specified percentage of amino acid residues or
nucleotides that are the same (i.e., 50% identity, optionally 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher identity over a
specified region), when compared and aligned for maximum
correspondence over a comparison window, or designated region as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. Such sequences are
then said to be "substantially identical." This definition also
refers to the compliment of a test sequence. Optionally, the
identity exists over a region that is at least about 50 amino acids
or nucleotides in length, or more preferably over a region that is
75-100 amino acids or nucleotides in length.
[0051] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. By way of
example, when using a sequence comparison algorithm, test and
reference sequences are entered into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. Default program parameters can
be used, as described below for the BLASTN and BLASTP programs, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0052] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0053] A preferred example of an algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 21 S:403-410 (1990), respectively. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSLTM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0054] Another example of a useful algorithm is PILEUP. PILEUP
creates a multiple sequence alignment from a group of related
sequences using progressive, pairwise alignments to show
relationship and percent sequence identity. It also plots a tree or
dendogram showing the clustering relationships used to create the
alignment. PILEUP uses a simplification of the progressive
alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360
(1987). The method used is similar to the method described by
Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align
up to 300 sequences, each of a maximum length of 5,000 nucleotides
or amino acids. The multiple alignment procedure begins with the
pairwise alignment of the two most similar sequences, producing a
cluster of two aligned sequences: This cluster is then aligned to
the next most related sequence or cluster of aligned sequences. Two
clusters of sequences are aligned by a simple extension of the
pairwise alignment of two individual sequences. The final alignment
is achieved by a series of progressive, pairwise alignments. The
program is run by designating specific sequences and their amino
acid or nucleotide coordinates for regions of sequence comparison
and by designating the program parameters. Using PILEUP, a
reference sequence is compared to other test sequences to determine
the percent sequence identity relationship using the following
parameters: default gap weight (3.00), default gap length weight
(0.10), and weighty end gaps. PILEUP can be obtained from the GCG
sequence analysis software package, e.g., version 7.0 (Devereaux et
al., Nuc. Acids Res. 12:387-395 (1984)).
[0055] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0056] The phrase "selectively (or specifically) hybridizes to" may
refer to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0057] The phrase "stringent hybridization conditions" may refer to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology-Hybridisation with
Nucleic Probes, "Overview of principles of hybridization and the
strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the.
thermal melting point (Tm) for the specific sequence at a defined
ionic strength pH. The T.sub.m is the temperature (under defined
ionic strength, pH, and nucleic concentration) at which 50% of the
probes complementary to the target hybridize to the target sequence
at equilibrium (as the target sequences are present in excess, at
T.sub.m, 50% of the probes are occupied at equilibrium). Stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, Sx SSC, and 1% SDS,
incubating at 42.degree. C., or, Sx SSC, 1% SDS, incubating at
65.degree. C., with wash in 0.2.times.SSC, and 0.1% SDS at
65.degree. C. Such hybridizations and wash steps can be carried out
for, e.g., 1, 2, 5, 10, 15, 30, 60; or more minutes.
[0058] Nucleic acids that do not hybridize to each other under
stringent conditions may still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code.' In
such cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. Such hybridizations and wash steps can
be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes.
A positive hybridization is at least twice background. Those of
ordinary skill will readily recognize that alternative
hybridization and wash conditions can be utilized to provide
conditions of similar stringency.
[0059] The term "capture agent" refers to an agent that binds an
analyte through an interaction that is sufficient to permit the
agent to bind and concentrate the analyte from a homogeneous
mixture of different analytes. The binding interaction may be
mediated by an affinity region of the capture agent. Representative
capture agents include polypeptides and polynucleotides, for
example antibodies, peptides or fragments of single stranded or
double stranded DNA may employed. Capture agents usually
"specifically bind" one or more analytes.
[0060] Accordingly, the term "capture agent" refers to a molecule
or a multi-molecular complex which can specifically bind an
analyte, e.g., specifically bind an analyte for the capture agent,
with a dissociation constant (K.sub.D) of less than about 10.sup.-6
M.
[0061] The term "specific binding" refers to the ability of a
capture agent to preferentially bind to a particular analyte that
is present in a homogeneous mixture of different analytes. In
certain embodiments, a specific binding interaction will
discriminate between desirable and undesirable analytes in a
sample, in some embodiments more than about 10 to 100-fold or more
(e.g., more than about 1000- or 10,000-fold). In certain
embodiments, the affinity between a capture agent and analyte when
they are specifically bound in a capture agent/analyte complex is
characterized by a K.sub.D (dissociation constant) of less than
10.sup.-6M, less than 10.sup.-7 M, less than 10.sup.-8M, less than
10.sup.-9 M, usually less than about 10.sup.-10 M.
[0062] The term "capture agent/analyte complex" is a complex that
results from the specific binding of a capture agent with an
analyte, i.e., a "binding partner pair". A capture agent and an
analyte for the capture agent specifically bind to each other under
"conditions suitable for specific binding", where such conditions
are those conditions (in terms of salt concentration, pH,
detergent, protein concentration, temperature, etc.) which allow
for binding to occur between capture agents and analytes to bind in
solution. Such conditions, particularly with respect to proteins,
are well known in the art (see, e.g., Harlow and Lane (Antibodies:
A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989)). Conditions suitable for specific binding
typically permit capture agents and target pairs that have a
dissociation constant (K.sub.D) of less than about 10.sup.-6 M to
bind to each other, but not with other capture agents or
targets.
[0063] As used herein, "binding partners" and equivalents refer to
pairs of molecules that can be found in a capture agent/analyte
complex, i.e., exhibit specific binding with each other.
[0064] The phrase "surface-bound capture agent" refers to a capture
agent that is immobilized on a surface of a solid substrate, where
the substrate can have a variety of configurations, e.g., a sheet,
bead, or other structure, such as a plate with wells. In certain
embodiments, the collections of capture agents employed herein are
present on a surface of the same support, e.g., in the form of an
array.
[0065] "Isolated" or "purified" generally refers to isolation of a
substance (compound, polynucleotide, protein, polypeptide,
polypeptide composition) such that the substance comprises a
significant percent (e.g., greater than 2%, greater than 5%,
greater than 10%, greater than 20%, greater than 50%, or more,
usually up to about 90%-100%) of the sample in which it resides. In
certain embodiments, a substantially purified component comprises
at least 50%, 80%-85%, or 90-95% of the sample. Techniques for
purifying polynucleotides and polypeptides of interest are
well-known in the art and include, for example, ion-exchange
chromatography, affinity chromatography and sedimentation according
to density. Generally, a substance is purified when it exists in a
sample in an amount, relative to other components of the sample,
that is not found naturally.
[0066] The term "fusion protein" or grammatical equivalents thereof
is meant a protein composed of a plurality of polypeptide
components, that while typically unjoined in their native state,
typically are joined by their respective amino and carboxyl termini
through a peptide linkage to form a single continuous polypeptide.
Fusion proteins may be a combination of two, three or even four or
more different proteins. The term polypeptide includes fusion
proteins, including, but not limited to, fusion proteins with a
heterologous amino acid sequence, fusions with heterologous and
homologous leader sequences, with or without N-terminal methionine
residues; immunologically tagged proteins; fusion proteins with
detectable fusion partners, e.g., fusion proteins including as a
fusion partner a fluorescent protein, .beta.-galactosidase,
luciferase, etc.; and the like.
[0067] The term "assessing" includes any form of measurement, and
includes determining if an element is present or not. The terms
"determining", "measuring", "evaluating", "assessing" and
"assaying" are used interchangeably and may include quantitative
and/or qualitative determinations. Assessing may be relative or
absolute. "Assessing binding" includes determining the amount of
binding, and/or determining whether binding has occurred (i.e.,
whether binding is present or absent).
[0068] The terms "treatment", "treating", "treat", and the like,
refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment", as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease and/or relieving
one or more disease symptoms. "Treatment" is also meant to
encompass delivery of an agent in order to provide for a
pharmacologic effect, even in the absence of a disease or
condition. For example, "treatment" encompasses delivery of a COX-2
modulator that can provide for enhanced or desirable effects in the
subject (e.g., beneficial increase in a physiological parameter of
the subject, reduction of disease symptoms, decreased pain
sensation or decreased inflammation, decreased side effects of
another COX-2 inhibitor, etc.).
[0069] "Subject", "individual," "host" and "patient" are used
interchangeably herein, to refer to an animal, human or non-human,
susceptible to or having a COX-2 amenable to therapy according to
the methods of the invention. Generally, the subject is a mammalian
subject. Exemplary subjects include, but are not necessarily
limited to, humans, non-human primates, mice, rats, cattle, sheep,
goats, pigs, dogs, cats, and horses, with humans being of
particular interest.
[0070] Various biochemical and molecular biology methods referred
to herein are well known in the art, and are described in, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, N.Y. Second (1989) and Third (2000)
Editions, and Current Protocols in Molecular Biology, (Ausubel, F.
M. et al., eds.) John Wiley & Sons, Inc., New York
(1987-1999).
[0071] The invention provides an assay for determining whether a
test agent is a COX modulator. In general terms, the assay
includes: determining whether a test agent modulates binding of a
PDZ-containing polypeptide to a PL-containing polypeptide, e.g., a
COX-2 PL containing polypeptide. The PDZ-containing polypeptide may
contain the PDZ domain of, e.g., MAGI1 (including MAGI1 d1), TIP-1,
SHANK (including SHANK1, SHANK2, and SHANK3), PSD95 (including
PSD95 d1, PSD95 d2, and PSD95 d3) or MAST2. The assays may be done
in a cell-free environment or in a cellular environment,
particularly using a neuronal cell. The invention finds use in a
variety of therapeutic applications, including for identifying
agents for use in treating pain, cancer, inflammation and neuronal
conditions caused by acute insult, e.g., stroke.
[0072] Before the present invention is described in such detail,
however, it is to be understood that this invention is not limited
to particular variations set forth and may, of course, vary.
Various changes may be made to the invention described and
equivalents may be substituted without departing from the true
spirit and scope of the invention. In addition, many modifications
may be made to adapt a particular situation, material, composition
of matter, process, process act(s) or step(s), to the objective(s),
spirit or scope of the present invention. All such modifications
are intended to be within the scope of the claims made herein.
[0073] Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as the
recited order of events. Furthermore, where a range of values is
provided, it is understood that every intervening value, between
the upper and lower limit of that range and any other stated or
intervening value in that stated range is encompassed within the
invention. Also, it is contemplated that any optional feature of
the inventive variations described may be set forth and claimed
independently, or in combination with any one or more of the
features described herein.
[0074] The referenced items are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the present
invention is not entitled to antedate such material by virtue of
prior invention.
[0075] Reference to a singular item includes the possibility that
there are plural of the same items present. More specifically, as
used herein and in the appended claims, the singular forms "a,"
"an," "said" and "the" include plural referents unless the context
clearly dictates otherwise. It is further noted that the claims may
be drafted to exclude any optional element. As such, this statement
is intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the
recitation of claim elements, or use of a "negative"
limitation.
II. Assays, Modulators, and Methods
[0076] As noted above, the invention provides a variety of assays
for identifying modulators of COX-2 and PDZ ligand binding
modulators, e.g., COX-2 PDZ ligand binding modulators. In general,
the methods involve testing binding of a PDZ ligand polypeptide,
including COX-2 PDZ ligand polypeptides, to a polypeptide having a
PDZ domain in the presence of a test agent (i.e., candidate drug
compound). A test agent that modulates binding between the PDZ
ligand polypeptide and a polypeptide having a PDZ domain modulates
(i.e., increases or decreases, including abolishes) binding between
the two proteins. As will be described below, binding between the
two polypeptides may be assessed using a variety of means. Also as
will be described in greater detail below, the assay may be
performed in a cell-free environment (i.e., "in vitro") using
isolated polypeptides. In certain embodiments, the assay may be a
cellular assay in which binding of the polypeptides within a cell,
in the presence of a test agent, is evaluated. A wide variety of
assay platforms are therefore available.
[0077] Binding of the polypeptides may be assayed using methods
that are well known in the art. For example, binding may be assayed
biochemically, or, in other embodiments, the two proteins may be
assayed by detecting a signal that is only produced when the
proteins are bound together. In testing candidate agents, such a
signal can be evaluated in order to assess binding between the two
proteins. For example, as used in the subject assays, the
polypeptides may form a fluorescence resonance energy transfer
(FRET) system, bioluminescence resonance energy transfer (BRET)
system, or colorimetric signal producing system that can be
assayed.
[0078] The assay, whether it is performing in vitro or in a
cellular environment, generally involves a) a polypeptide including
the PDZ ligand and b) a polypeptide including a PDZ domain from,
e.g., MAGI1 (including MAGI1 d1), TIP-1, SHANK (including SHANK1,
SHANK2, and SHANK3), PSD95 (including PSD95 d1, PSD95 d2, and PSD95
d3) or MAST2. In many embodiments, at least one of the polypeptides
may be a fusion protein that facilitates detection of binding
between the polypeptides. Accordingly, one of the polypeptides may
contain, for example, an affinity tag domain or an optically
detectable reporter domain.
[0079] Suitable affinity tags include any amino acid sequence that
may be specifically bound to another moiety, usually another
polypeptide, most usually an antibody. Suitable affinity tags
include epitope tags, for example, the V5 tag, the FLAG tag, the HA
tag (from hemagglutinin influenza virus), the myc tag, etc.
Suitable affinity tags also include domains for which, binding
substrates are known, e.g., HIS, GST and MBP tags, etc., and
domains from other proteins for which specific binding partners,
e.g., antibodies, particularly monoclonal antibodies, are
available. Suitable affinity tags also include any protein-protein
interaction domain, such as a IgG Fc region, which may be
specifically bound and detected using a suitable binding partner,
e.g., the IgG Fc receptor.
[0080] Suitable reporter domains include any domain that can
optically report the presence of a polypeptide, e.g., by emitting
light or generating a color. Suitable light emitting reporter
domains include luciferase (from, e.g., firefly, Vargula, Renilla
reniformis or Renilla muelleri), or light emitting variants
thereof. Other suitable reporter domains include fluorescent
proteins, (from e.g., jellyfish, corals and other coelenterates as
such those from Aequoria, Renilla, Ptilosarcus, Stylatula species),
or light emitting variants thereof. Light emitting variants of
these reporter proteins are very well known in the art and may be
brighter, dimmer, or have different excitation and/or emission
spectra, as compared to a native reporter protein. For example,
some variants are altered such that they no longer appear green,
and may appear blue, cyan, yellow, enhanced yellow red (termed BFP,
CFP, YFP eYFP and RFP, respectively) or have other emission
spectra, as is known in the art. Other suitable reporter domains
include domains that can report the presence of a polypeptide
through a biochemical or color change, such as
.beta.-galactosidase, .beta.-glucuronidase, chloramphenicol acetyl
transferase, and secreted embryonic alkaline phosphatase. In some
preferred embodiments, the reporter domain is Renilla luciferase
(e.g., pRLCMV; Promega, catalog number E2661).
[0081] Also as is known in the art, an affinity tag or a reporter
domain may be present at any position in a polypeptide of interest.
However, in certain embodiments, they are present at the C- or
N-terminal end of a polypeptide.
[0082] In particular embodiments, one or both of the polypeptides
may contain a tag or reporter. For example, if FRET or BRET methods
are employed, the polypeptides may both be tagged using different
autofluorescent polypeptides.
[0083] In certain embodiments, the PDZ domain-containing
polypeptide includes at least the PDZ domain from SHANK1, SHANK2 or
SHANK3, which PDZ domains each bind to the PDZ ligand of COX-2. The
SHANK PDZ domain may contain the PDZ domain of a "wild-type" SHANK
polypeptide, or a variant thereof that retains ability to bind to
the PDZ ligand of COX-2. The sequence of the PDZ domains for
wild-type SHANK1, SHANK2 or SHANK3 are illustrated in FIG. 1A. Any
length of PDZ domain, including the minimum, intermediate and +10
lengths shown in FIG. 1A, may be employed herein.
[0084] The SHANK1 and SHANK2 and SHANK3 PDZ domain polypeptides and
encoding cDNAs are deposited in the GenBank database as GID NOS:
7025450 and 6049185, respectively, whereas the coding sequence for
SHANK3 is encoded by GenBank accession no. XM.sub.--037493 (GI:
51476100).
[0085] In other embodiments, the PDZ domain-containing polypeptide
may include at least the PDZ domain from MAST2, which PDZ domain
generally binds to the PDZ ligand of COX-2. The MAST2 PDZ domain
may contain the PDZ domain of a "wild-type" MAST2 polypeptide, or a
variant thereof that retains ability to bind to the PDZ ligand of
COX-2. The MAST2 PDZ domain polypeptide and encoding cDNA are
deposited in the GenBank database as accession no. AB047005.
[0086] In other embodiments, the PDZ domain-containing polypeptide
may include at least the PDZ domain from MAGI1, including MAGI1 d1,
which PDZ domains generally bind to the PDZ ligand of COX-2. The
MAGI1 PDZ domains may contain the PDZ domain of a "wild-type" MAGI1
d1 polypeptide, or a variant thereof that retains ability to bind
to the PDZ ligand of COX-2. The MAGI1 d1 PDZ domain polypeptide and
encoding cDNA are deposited in the GenBank database as accession
no. Q96QZ7
[0087] In other embodiments, the PDZ domain-containing polypeptide
may include at least the PDZ domain from TIP-1, which PDZ domain
generally binds to the PDZ ligand of COX-2. The TIP-1 PDZ domain
may contain the PDZ domain of a "wild-type" TIP-1 polypeptide, or a
variant thereof that retains ability to bind to the PDZ ligand of
COX-2. The TIP-1 PDZ domain polypeptide and encoding cDNA are
deposited in the GenBank database as accession no. AF028823.
[0088] In other embodiments, the PDZ domain-containing polypeptide
may include at least the PDZ domain from PSD95, including PSD95 d1,
PSD95 d2, and PSD95 d3, which PDZ domains generally bind to the PDZ
ligand of COX-2. The PSD95 PDZ domains may contain the PDZ domain
of a "wild-type" PSD95 polypeptide, or a variant thereof that
retains ability to bind to the PDZ ligand of COX-2. The PSD95 PDZ
domain polypeptides and encoding cDNA are deposited in the GenBank
database as accession no. AAC52113.
[0089] In certain embodiments, the COX-2 PDZ ligand-containing
polypeptide contains at least the PDZ ligand of COX-2, or a variant
or fragment thereof. The COX-2 PDZ ligand may contain the PDZ
ligand of a "wild-type" COX-2 polypeptide, or a variant or fragment
thereof that retains ability to bind to a PDZ domain, e.g., a
domain of a MAGI1 (including MAGI1d1), TIP-1, SHANK (including
SHANK1, SHANK2, and SHANK3), PSD95 (including PSD95 d1, PSD95 d2,
and PSD95 d3) or MAST2 polypeptide.
[0090] The sequence of a wild-type COX-2 PDZ ligand is illustrated
in FIG. 1A, including several variants thereof. Any combination of
the indicated variants are envisioned, as well as conservatively
modified variants thereof. For instance, the COX-2 PDZ ligand
polypeptides of the invention may comprise a PL region having at
least 50% identity, optionally at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95% or more identity to SEQ. ID NO.: 13.
Further, any length COX-2 PDZ ligand polypeptide may be used, which
retains its ability to bind a PDZ domain. For instance, COX-2 PDZ
polypeptides having a total length of at least about, e.g., 30, 28,
26, 25, etc., amino acids, and comprising a PL region having, e.g.,
a fragment of 5 contiguous amino acids, 6 contiguous amino acids, 7
contiguous amino acids, 8 contiguous amino acids or 9 contiguous
amino acids of SEQ. ID NO.: 13, or a variant thereof that retains
its ability to bind to a PDZ domain.
[0091] In another embodiment, the PDZ ligand-containing polypeptide
may including the putative PDZ ligand of COX-1. The COX-1 PDZ
ligand may contain the PDZ ligand of a "wild-type" COX-1
polypeptide, or a variant or fragment thereof that retains ability
to bind to a PDZ domain, e.g., of a MAGI1 (including MAGI1 d1),
TIP-1, SHANK (including SHANK1, SHANK2, and SHANK3), PSD95
(including PSD95 d1, PSD95 d2, and PSD95 d3) or MAST2 polypeptide.
The sequence of a "wild-type" COX-1 PDZ ligand is generally
AVERPSTEL (SEQ. ID NO.: 93), and may be employed herein.
Conservatively modified variants thereof and fragments are
envisioned as well. For instance, the COX-1 PDZ ligand polypeptides
of the invention may comprise a PL region having at least 50%
identity, optionally at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95% or more identity to SEQ. ID NO.: 93. Further, any
length COX-1 PDZ ligand polypeptide may be used, which retains its
ability to bind a PDZ domain. For instance, COX-1 PDZ polypeptides
having a total length of at least about, e.g., 30, 28, 26, 25,
etc., amino acids, and comprising a PL region having, e.g., a
fragment of 5 contiguous amino acids, 6 contiguous amino acids, 7
contiguous amino acids, 8 contiguous amino acids or 9 contiguous
amino acids of SEQ. ID NO.: 93, or a variant thereof that retains
its ability to bind to a PDZ domain.
[0092] Variant polypeptides are readily designed since the PDZ
domain is well characterized at the structural level. For example,
the three-dimensional structure of the PDZ domain is described and
discussed in great detail in Doyle (Cell 1996 95:1067-1076) and the
structure of, e.g., SHANK1 bound to the PDZ ligand domain of
guanylate kinase-associated protein (GKAP1a) has been determined by
crystallography. Variants are generally at least 80% identical, at
least 90% identical, at least 95% identical or, in certain
embodiments at least 98% or at least 99% identical to a wild-type
PDZ domain amino acid sequence. In other words, as employed in a
method described herein, a PDZ domain-containing polypeptide may
contain at least 1, 2, 3, 4, or 5 or more and in certain
embodiments up to 10 amino acid substitutions, as compared to a
wild-type sequence. A substitution may be conservative (i.e.,
replacing one amino acid with another within the following groups:
gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg;
and phe, tyr), or non-conservative. By way of example, since each
of the SHANK PDZ domains bind COX-2 and are highly similar in
sequence (the SHANK1 and SHANK2 PDZ domains are approximately 85%,
the SHANK1 and SHANK3 PDZ domains are approximately 79% identical
and the SHANK3 and SHANK3 PDZ domains are approximately 80%
identical), amino acids may be readily substituted from one
sequence to another without losing the ability to bind to COX-2.
Exemplary amino acid substitutions that could be made in the
subject polypeptides are illustrated in FIG. 1B and FIG. 2. In
other words, since all the polypeptides shown in FIG. 1B bind to
the same PDZ ligand, amino acids at the same position within each
of the polypeptides may be substituted without significant loss of
binding activity. The PDZ domain of the polypeptides employed in
the instant methods may be longer or shorter by up to 10 or more
amino acids than the polypeptides illustrated in FIG. 1B.
[0093] When a particular PDZ domain-containing polypeptide is
referenced herein, e.g., when a reference is made to a MAGI1,
TIP-1, PSD95, SHANK1, SHANK2, SHANK3 or MAST2 PDZ domain-containing
polypeptide, the reference is intended to encompass polypeptides
containing a wild-type PDZ domain, and variants or fragments
thereof that retain PDZ ligand binding activity, e.g., COX-1 or
COX-2 PL binding activity.
[0094] When a particular PDZ ligand-containing polypeptide is
referenced herein, e.g., when a reference is made to a COX-2 PDZ
ligand-containing polypeptide or COX-2 PDZ ligand-containing
polypeptide, the reference is intended to encompass polypeptides
containing a wild-type PDZ ligand, and variants and fragments
thereof that retain PDZ domain binding activity.
[0095] Such polypeptides may be made synthetically (i.e., using a
machine) or using recombinant means, as is known in the art.
Methods and conditions for expression of recombinant proteins are
well known in the art. See, e.g., Sambrook, supra, and Ausubel,
supra. Typically, polynucleotides encoding the polypeptides used in
the invention are expressed using expression vectors. Expression
vectors typically include transcriptional and/or translational
control signals (e.g., the promoter, ribosome-binding site, and ATG
initiation codon). In addition, the efficiency of expression can be
enhanced by the inclusion of enhancers appropriate to the cell
system in use. For example, the SV40 enhancer or CMV enhancer can
be used to increase expression in mammalian host cells. Typically,
DNA encoding a polypeptide of the invention is inserted into DNA
constructs capable of introduction into and expression in an in
vitro host cell, such as a bacterial (e.g., E. coli, Bacillus
subtilus), yeast (e.g., Saccharomyces), insect (e.g., Spodoptera
frugiperda), or mammalian cell culture systems. Mammalian cell
systems are preferred for many applications. Examples of mammalian
cell culture systems useful for expression and production of the
polypeptides of the present invention include human embryonic
kidney line (293; Graham et al., 1977, J. Gen. Virol. 36:59); CHO
(ATCC CCL 61 and CRL 9618); human cervical carcinoma cells (HeLa,
ATCC CCL 2); and others known in the art. The use of mammalian
tissue cell culture to express polypeptides is discussed generally
in Winnacker, From Genes to Clones (VCH Publishers, N.Y., N.Y.,
1987) and Ausubel, supra. In some embodiments, promoters from
mammalian genes or from mammalian viruses are used, e.g., for
expression in mammalian cell lines. Suitable promoters can be
constitutive, cell type-specific, stage-specific, and/or
modulatable or regulatable (e.g., by hormones such as
glucocorticoids). Useful promoters include, but are not limited to,
the metallothionein promoter, the constitutive adenovirus major
late promoter, the dexamethasone-inducible MMTV promoter, the SV40
promoter, and promoter-enhancer combinations known in the art.
[0096] As noted above, the subject assay may be performed in vitro
(i.e., in which the polypeptides are present in a solution a not in
a cell) or in a cellular environment (in which the polypeptides are
present in a cell).
III. In Vitro Assays
[0097] In vitro assays may be performed using a wide variety of
platforms that are well known in the art. In certain embodiments,
the methods involve linking, either covalently or non-covalently, a
first polypeptide (either the PDZ domain polypeptide or the PDZ
ligand polypeptide) to a substrate, contacting the substrate-bound
polypeptide with the second polypeptide, and detecting the presence
of the second polypeptide. In other embodiments, the first and
second polypeptides are not substrate-bound, and the assay is
performed in solution. The method may be performed in the presence
of a test agent. In embodiments in which one of the polypeptides
are detectably labeled (e.g., as an optically-detectable fusion
protein), the presence of the labeled polypeptide is detected by
detecting the label.
[0098] A substrate contains a solid, semi-solid, or insoluble
support and is made from any material appropriate for linkage to a
polypeptide, and does not interfere with the detection method used.
As will be appreciated by those in the art, the number of possible
affinity substrates is very large. Possible substrates include, but
are not limited to, glass and modified or functionalized glass,
plastics (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, ceramics, and a variety of other polymers. In one
embodiment, the substrates allow optical detection and do not
themselves appreciably fluoresce or emit light. In addition, as is
known the art, the substrate may be coated with any number of
materials, including polymers, such as dextrans, acrylamides,
gelatins, agarose, biocompatible substances such as proteins
including bovine and other mammalian serum albumin.
[0099] In certain embodiments, the substrate is coated in an agent
that facilitates the specific binding (either directly or
indirectly) of a polypeptide to the substrate. For example, the
substrate is coated in streptavidin, and can bind a biotinylated
polypeptide with affinity to the polypeptide of interest. In
another example, the substrate is directly or indirectly (e.g.,
through protein A) coated with an antibody specific for the
polypeptide.
[0100] As mentioned above, after the first polypeptide is linked to
the substrate, the second polypeptide is contacted with the
substrate and maintained under conditions suitable for specific
binding of the second polypeptide to the first polypeptide,
typically in the presence of a test agent. The second polypeptide
is only detectable on the substrate only if the first and second
polypeptides form a complex. Detection of the second polypeptide
indicates that the first and second polypeptides form a complex.
Detection of the second polypeptide that is bound to the affinity
substrate is carried out directly (while the second polypeptide is
bound to the substrate), or indirectly (e.g., after elution of the
polypeptide from the substrate).
[0101] In embodiments where the second polypeptide contains a
reporter domain, the second polypeptide may be detected by
detecting reporter activity. Methods of determining reporter
activity, e.g., luciferase and GFP activity, are generally well
known in the art (e.g., Ramsay et al., Br. Pharmacology, 2001,
133:315-323), and need not be described any further. Detection of
the second polypeptide may also be accomplished using an antibody,
e.g., a labeled antibody. Methods for detecting polypeptides using
antibodies are also well known in the art (e.g., Ausubel et al.,
Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons,
1995; and Harlow et al., Antibodies: A Laboratory Manual, First
Edition 1988 Cold Spring Harbor, N.Y.) and need not be described in
more detail.
[0102] Fluorescence Resonance Energy Transfer (FRET) and
Bioluminescence Resonance Energy Transfer (BRET) systems may also
be employed, as generally understood by those skilled in the art.
Such systems are described in further detail below with reference
to cell based assays.
[0103] In order to determine whether a test agent modulates binding
between the subject polypeptides, the above assay may be performed
in the presence or absence of a test agent.
[0104] Two complementary assays, termed "A" and "G" (or a modified
"G" assay), 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). 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.
[0105] Details of the A and G assays are set forth in the Examples
section below, and in U.S. patent application Ser. No. 10/630,590,
filed Jul. 29, 2003 and published as US20040018487.
IV. Cellular assays
[0106] Cellular assays generally involve co-producing (i.e.,
producing in the same cell, regardless of the time at which they
are produced), the subject polypeptides using recombinant DNA.
Suitable cells for producing the subject polypeptides include
prokaryotic, e.g., bacterial cells, as well as eukaryotic cells
e.g., an animal cell (for example an insect, mammal, fish,
amphibian, bird or reptile cell), a plant cell (for example a maize
or Arabidopsis cell), or a fungal cell (for example a S. cerevisiae
cell). Any cell suitable for expression of subject
polypeptide-encoding nucleic acid may be used as a host cell.
Usually, an animal host cell line is used, examples of which are as
follows: monkey kidney cells (COS cells), monkey kidney CV1 cells
transformed by SV40 (COS-7, ATCC CRL 165 1); human embryonic kidney
cells (HEK-293, Graham et al., J. Gen Virol. 36:59 (1977));
HEK-293T cells; baby hamster kidney cells (BHK, ATCC CCL 10);
chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl.
Acad. Sci. (USA) 77:4216, (1980); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); african green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51);
TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1).
[0107] In particular embodiments, neuronal cells, e.g., SHSY5Y
(neuroblastoma cell line), hippocampal murine HT-22 cells, primary
cultures from astrocytes, cerebral cortical neuronal-astrocytic
co-cultures, mixed neuronal/glial hippocampal cultures, cerebellar
granular neuronal cell cultures or primary neuronal cultures
derived from rat cortex (E15-17) may be employed.
[0108] Additional cell lines will become apparent to those of
ordinary skill in the art. A wide variety of cell lines are
available from the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209.
[0109] Again, a wide variety of platforms may be employed to detect
binding between the subject polypeptides in a cell. For example,
so-called "two-hybrid" methods may be employed, or a wide variety
of fluorescence-based methods, e.g., FRET or BRET-based methods. In
general, these methods involve contacting a cell that produces the
subject polypeptides with a test agent, and determining if the test
agent has any effect on binding between the subject
polypeptides.
[0110] In one embodiment, the GAL4 system is used to screen agents
that modulate binding between the subject polypeptides. Such
methods may employ a vector (or vector system) encoding two
polypeptides: a DNA binding domain polypeptide that contains either
a PDZ domain or a PDZ ligand and DNA activation domain polypeptide
containing the region not in the DNA binding domain polypeptide.
The interaction between the PDZ domain and the PDZ ligand activates
the expression of a reporter gene or selectable marker. The levels
of .alpha.- or .beta.-galactosidase, .beta.-lactamase are measured
by quantifying their enzymatic activity using colorimetric
substrates, such as orthomethylphenylthiogalactoside (OMTP) or
X-gal; the levels of light, e.g., fluorescence, may be assessed
photometrically, e.g., fluorometrically. Pools of agents or
individual agents are added to cultures in wells and the levels of
inhibition or facilitation of the interaction by the agents are
determined from the levels of the reporter gene activity. Such
methods are very well known in the art.
[0111] In another exemplary embodiment, Fluorescence Resonance
Energy Transfer (FRET) may be used to detect binding between two
polypeptides in a cell. Fluorescent molecules having the proper
emission and excitation spectra that are brought into close
proximity with one another can exhibit FRET. The fluorescent
molecules are chosen such that the emission spectrum of one of the
molecules (the donor molecule) overlaps with the excitation
spectrum of the other molecule (the acceptor molecule). The donor
molecule is excited by light of appropriate intensity within the
donor's excitation spectrum. The donor then emits the absorbed
energy as fluorescent light. The fluorescent energy it produces is
quenched by the acceptor molecule. FRET can be manifested as a
reduction in the intensity of the fluorescent signal from the
donor, reduction in the lifetime of its excited state, and/or
re-emission of fluorescent light at the longer wavelengths (lower
energies) characteristic of the acceptor. When the fluorescent
proteins physically separate, FRET effects are diminished or
eliminated. (See, U.S. Pat. No. 5,981,200, the disclosure of which
is hereby incorporated by reference in its entirety.)
[0112] For example, a cyan fluorescent protein is excited by light
at roughly 425-450 nm wavelength and emits light in the range of
450-500 nm. Yellow fluorescent protein is excited by light at
roughly 500-525 nm and emits light at 525-500 nm. If these two
proteins are present in a cell but not in close proximity, the cyan
and yellow fluorescence may be separately visualized. However, if
these two proteins are forced into close proximity with each other,
the fluorescent properties will be altered by FRET. The bluish
light emitted by CFP will be absorbed by YFP and re-emitted as
yellow light. FRET is typically monitored by measuring the spectrum
of emitted light in response to stimulation with light in the
excitation range of the donor and calculating a ratio between the
donor-emitted light and the acceptor-emitted light. When the
donor:acceptor emission ratio is high, FRET is not occurring and
the two fluorescent proteins are not in close proximity. When the
donor:acceptor emission ratio is low, FRET is occurring and the two
fluorescent proteins are in close proximity. In this manner, the
interaction between a first and second polypeptide fused to a first
and second reactive module, wherein the first and second reactive
modules are donor and acceptor fluorescent molecules, respectively,
may be measured. As such, the two polypeptides may contain a system
that provides for FRET, e.g., one polypeptide contains GFP whereas
the other contains YFP.
[0113] In a further embodiment, the first and seconds provide a
Bioluminescence Resonance Energy Transfer (BRET) system. In such a
system, one polypeptide of interest produces (or destroys) a
fluorescent product (or substrate) and the other polypeptide of
interest is a fluorescent protein that undergoes resonant energy
transfer with the fluorescent product (or substrate). In one
embodiment, a BRET system comprises a luciferase from Renilla and a
GFP. Exemplary BRET methodologies are described in Kroeger et al.,
J Biol Chem. 2001 Apr. 20; 276(16):12736-43 and Xu et al., Proc
Natl Acad Sci USA. 1999 Jan. 5; 96(1):151-6.
[0114] A variety of colorimetric signal producing systems may also
be employed.
[0115] The test agents employed in the subject methods may be any
type of compound. 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 (i.e., under about 500 Da in
weight), 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. 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., 1991, Science 251:767-773;
Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature
354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et
al., 1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmcycr 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,
Biotechniques 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 Aug. 18,
1994. In vitro translation-based libraries include but are not
limited to those described in PCT Publication No. WO 91/05058 dated
Apr. 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).
[0116] In certain embodiments, the test agent may be a COX-2
selective inhibitor of prostaglandin synthesis, e.g., a
diarylheterocycle (including celecoxib and rofecoxib), an acidic
sulfonamide, an indomethacin analog, a zomepirac analog, a chromene
analog or a di-t-butylphenol. For example, COX-2 inhibitory
oxazoles are described in U.S. Pat. No. 5,380,738, COX-2 inhibitory
cyclopentenes are described in U.S. Pat. No. 5,344,991, COX-2
inhibitory Spiro are described in U.S. Pat. No. 5,393,790, COX-2
inhibitory thiophene and furan derivatives are described in
WO94/15932 and COX-2 inhibitory pyrazolyl sulfonamide derivatives
are described in U.S. WO95/15316. The subject method may find
particular use as a counterscreen to identify inhibitors of COX-2
(e.g., inhibitors that reduce prostaglandin synthesis) that do, or
do not, also modulate binding between COX-2 and the PDZ-containing
proteins.
[0117] In other embodiments, test agent may be a PDZ domain, or an
analog thereof, a COX-2 PDZ ligand or analog thereof, or a
non-COX-2 PDZ ligand that binds to the PDZ domain or, e.g., MAGI1,
TIP-1, PSD95, SHANK or MAST2 (e.g., as illustrated in FIGS. 5A and
5B).
[0118] Once identified as an agent that modulates binding of COX-2
to a PDZ-containing polypeptide, i.e., a COX-2 PDZ ligand binding
modulator, the agent may be tested in a variety of different
assays, including cell-free assays, cellular assays and assays that
employ animals or brain sections ("ex vivo" brain sections). For
example, the binding-modulatory agent may be tested to determine if
the agent modulates cycloxidase activity, prostaglandin synthesis,
NMDA receptor activation, iNOS induction, pain, inflammation, COX-2
induction, COX-2 activity or nitric oxide levels, anti-tumor
activity assays, anti-cellular proliferation assays, etc., in any
appropriate system.
[0119] In particular embodiments, a binding-modulatory agent is
tested to determine if it provides protection against acute insult
(e.g., hypoxia or ischemia) or aid cell recovery after an insult.
Such assays may be performed in a variety of platforms, including,
but not limited to: cultured neurons (see, e.g., Shibuta, J.
Neurol. Sci. 2003 215:31-6, and Lee Neurochem. Int. 2004
44:107-18), ex vivo brain slices, e.g., organotypic hippocampal
slice cultures (see e.g., Barth et al., Exp. Brain Res. 2005
161:351-7, and Saransaari, Neurochem. Res. 2004 29:1511-8), and
animals (see, e.g., Lee J., Neurosci. Res. 2004 77:892-900;
Vannucci, Ann. N.Y. Acad. Sci. 1997 835:234-49). Such assays are
generally well known in the art.
V. Binding-Modulatory Compounds
[0120] In addition to the assays set forth above, the invention
also provides a variety of modulatory compounds that may be used as
PDZ-inhibitors based on their general ability to bind PDZ domains,
and to disrupt PDZ/PL interactions relevant to various disease
conditions or disorders, as recognized in the art. For instance,
the modulatory compounds may be employed as inhibitors of binding
between COX-2 and a PDZ-containing polypeptide in a cell, both in
vitro and in vivo.
[0121] In certain embodiments, the inhibitory compounds are
structurally related to the PDZ domains of MAGI1 (including MAGI1
d1), TIP-1, SHANK (including SHANK1, SHANK2, and SHANK3), PSD95
(including PSD95 d1, PSD95 d2, and PSD95 d3) or MAST2, such as
those shown in FIGS. 1A and 2B, and either contain the wild-type
amino acid sequence of the PDZ domain or a variant thereof that
retains COX-2 binding activity. Such polypeptides may be employed
to compete with a full-length PDZ peptide, e.g., MAGI1 (including
MAGI1 d1), TIP-1, SHANK (including SHANK1, SHANK2, and SHANK3),
PSD95 (including PSD95 d1, PSD95 d2, and PSD95 d3) or MAST2
peptide, for binding to COX-2 in a cell.
[0122] In other embodiments, the inhibitory compounds are
structurally related to the PDZ ligand of COX-1 or COX-2, such as
those shown in FIG. 1A and SEQ. ID NO.: 93, and either contain the
wild-type amino acid sequence of the PL, or is a variant or
fragment thereof that retains it ability to bind to a PDZ domain,
e.g., MAGI1 (including MAGI1 d1), TIP-1, SHANK (including SHANK1,
SHANK2, and SHANK3), PSD95 (including PSD95 d1, PSD95 d2, and PSD95
d3) or MAST2 binding activity. In certain embodiments, the COX PLs
may includes a transporter peptide, such as but not limited to a
Tat transporter peptide sequence (e.g., YGRKKRRQRRR, SEQ. ID NO.:
94, from peptide 1956, SEQ. ID NO.: 15). Such transporter peptides
may act to facilitate transport into a cell following
administration in vivo, and optionally to enhance binding to the
PDZ domain. Further, such polypeptides may be employed to compete
with full-length COX-2 for binding to a PDZ peptide, e.g., MAGI1
(including MAGI1 d1), TIP-1, SHANK (including SHANK1, SHANK2, and
SHANK3), PSD95 (including PSD95 d1, PSD95 d2, and PSD95 d3) or
MAST2, in a cell.
[0123] In other embodiments, the inhibitory compounds are
structurally related to the PDZ ligand of other PDZ-ligand
containing polypeptides that bind to the PDZ domain of, e.g., MAGI1
(including MAGI1 d1), TIP-1, SHANK (including SHANK1, SHANK2, and
SHANK3), PSD95 (including PSD95 d1, PSD95 d2, and PSD95 d3) or
MAST2. The sequences of several exemplary PDZ ligands that bind to
such PDZ peptides are shown in FIGS. 5A and 5B, and either contain
the wild-type amino acid sequence of PDZ ligand or a variant
thereof that retains SHANK1, SHANK2, SHANK3 or MAST2 binding
activity. Such polypeptides may be employed to compete with
full-length COX-2 for binding to SHANK1, SHANK2, SHANK3 or MAST2 in
a cell. It is understood that for any peptide or mimetic thereof
based on the sequence of a PDZ ligand, the sequence at the extreme
C-terminus of the polypeptide may be any of the following
sequences: TEL, SEL, TRL, SRL, SAL, TKL, SKL, SKI, TKI, SR1, TR1,
SDL, SDI or TDI.
[0124] In particular embodiments, the inhibitor compound may be a
mimetic of a subject PDZ domain or PDZ ligand, i.e., a synthetic
chemical compound that has substantially the same structural and/or
functional characteristics as a subject PDZ domain or PDZ ligand.
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 inhibiting
binding between the subject polypeptides.
[0125] Mimetics can contain any combination of normatural
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.
[0126] 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 groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH.sub.2-- for
--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin
(CH.dbd.CH), ether (CH.sub.2--O), thioether (CH.sub.2--S),
tetrazole (CN.sub.4--), 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).
[0127] 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. Normatural residues are well
described in the scientific and patent literature; a few exemplary
normatural compositions useful as mimetics of natural amino acid
residues and guidelines are described below.
[0128] 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-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or
L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycinc;
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 normatural amino
acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,
benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic
rings.
[0129] 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.dbd.--N--C--N--R.dbd.) 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.
[0130] 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. Asparaginyl and glutaminyl
residues can be deaminated to the corresponding aspartyl or
glutamyl residues.
[0131] 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.
[0132] 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.
[0133] 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-chloromcrcuribenzoate; 2-chloromercuri-4
nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole.
[0134] 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.
[0135] 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.
[0136] Other mimetics include, e.g., those generated by
hydroxylation of prolinc 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.
[0137] An amino acid of a subject 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.
[0138] 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.
[0139] The subject compounds may be further modified to make the
compound more soluble or to facilitate its entry into a cell. For
example, the compound may be PEGylated at any position, or the
compound may contain a transmembrane transporter region.
[0140] 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 J. 18:411-419) and transportan (Pooga
et al., 1998, FASEB J. 12:67-77). In certain embodiments, a
truncated HIV tat peptide may be employed.
[0141] In yet other embodiments, the inhibitory compounds may be a
small molecule compound that inhibits the PDZ/PL interaction, e.g.,
though binding of a PDZ domain. Exemplary small molecule compounds
include COX-2 inhibitors, such as but not limited to, niflumic
acid, ibuprofen, naproxen sodium, diclofenac sodium salt,
acetylsalicyclic acid, salicyclic, flurbiprofen, sulindac sulphide,
sulindac, etodolac, indomethancin, ketorolac tris salt, ketoprofen,
mefenamic acid carprofen, baclofen, fenoprofen, and structural
analogs thereof.
VI. Anti-Cancer Therapeutics
[0142] In another aspect of the invention, the PDZ-binding
inhibitory compounds, e.g., COX-2 PDZ-binding inhibitory compounds,
identified in accordance with the present invention may be used to
treat various cancers, and their therapeutic effectiveness may be
increased by optimizing the COX inhibitor structures for binding of
the PDZ domains.
[0143] COX-2 inhibitors have been known to have anti-oncogenic
properties in various cancers. Further, COX-1, COX-2, and PDZ's
have been linked to various forms of cancer and tumor growth. The
following are some examples and references of work demonstrating
the link between COX and various cancers; (a) prostate and colon
cancer, M. Hughes-Fulford et. al., "Arachidonic acid, an omega-6
fatty acid, induces cytoplasmic phospholipase A2 in prostate
carcinoma cells", Carcinogenesis, 2005, 26(9): 1520-6; J. Y. Liou
et. al., "Mitochondrial localization of cyclooxygenase-2 and
calcium-independent phospholipase A2 in human cancer cells:
implication in apoptosis resistance", Exp. Cell Res. 2005, 306(1):
75-84; (b) ovarian cancer, T. Daikoku et. al., "Cyclooxygenase-1 is
a potential target for prevention and treatment of ovarian
epithelial cancer", Cancer Res. 2005, 65(9): 3735-44; (c) other
cancers may include lung cancer, and cervical cancer (see MAGI1 d1
inhibitor data in the examples, infra).
[0144] In accordance with certain aspects of the invention, without
being bound by theory, it is believed that the NSAIDS possess
anti-oncogenic properties by virtue of their ability to bind PDZ
proteins, such as MAGI1 d1, TIP-1, SHANK1, and PSD95. The
structures of some COX-2 inhibitors mimic the C-terminal region of
PDZ ligands, where a carboxylate group may hydrogen bond with the
GLGF loop of PDZ's, and a hydrophobic group may interact with the
PO hydrophobic pocket of PDZ's. As shown in the examples, infra,
certain COX-2 inhibitors have been found to have PDZ binding
properties, which are believed to have activity in cancer pathology
by virtue of their PDZ binding activity in accordance with certain
aspects of the invention.
[0145] Furthermore, modification of NSAID's for the purpose of
increasing their PDZ binding affinity and specificity may generate
drugs with higher anti-tumor activity with less undesirable side
effects, such as cardiac malfunction and interference
anti-coagulation treatment via use of Aspirin. More specifically,
COX inhibitors interfere with the binding pocket of aspirin.
Aspirin is an anti-coagulant, and interfering with it's effect may
lead to coagulation in people depending on aspirin for blood
thinning A downstream complication may be cardiac malfunction. In
sum, it has been discovered that COX inhibitors bind to PDZ's, and
have "side-effect" related to PDZ binding, as well as others not
related to COX-2/PDZ interaction (such as the downstream
anti-coagulation effects). Some of these side-effects may be
beneficial and may have to do with the underlying activity of these
inhibitors themselves.
[0146] In certain aspects, the experimental structures of PDZ's
(NMR or crystallographic, for example) may be used to aid in the
modification and design of COX inhibitors of higher anti-tumor
potency and fewer side effects. The following are examples of
biological experiments and readouts to determine the anti-tumor
efficacy of such the COX inhibitors or optimized COX
inhibitors:
[0147] Cellular proliferation assays may be used to determine the
anti-tumorigenic potency of the COX inhibitors or their optimized
variants. A reduction in cellular proliferation of cancer cells by
the presence of these compounds may be an indication that the
compound has a beneficial therapeutic effect. Such assays are
readily described in the literature (Lisa G. Horvath et. al.,
"Membranous Expression of Secreted Frizzled-Related Protein 4
Predicts for Good Prognosis in Localized Prostate Cancer and
Inhibits PC3 Cellular Proliferation in Vitro", Clinical Cancer
Research, Vol. 10, 621-625, Jan. 15, 2004). In this work, a
colorimetric method (Cell Titer 96 kit (Promega) and a manual cell
counting approach was used to measure proliferation.
[0148] Cellular migration assays may also be used to determine the
anti-tumorigenic potency of the COX inhibitors or their optimized
variants. A reduction in cellular migration of cancer cells by the
presence of these compounds may be an indication that the compound
has a beneficial therapeutic effect. Such assays are readily
described in the literature (Philippe Merle, et. al., "Functional
Consequences of Frizzled-7 Receptor Overexpression in Human
Hepatocellular Carcinoma", Gastroenterology (Clinical-Liver,
Pancreas, and Billiary Tract) 2004; Volume 127, pages 1110-1122).
In this example, a luminescence based assay is used to evaluate
cell migration and motility. Other migration approaches are
described elsewhere.
[0149] Colony formation assays may also be used to determine the
anti-tumorigenic potency of the COX inhibitors or their optimized
variants. A reduction in colony formation of cancer cells by the
presence of these compounds may be an indication that the compound
has a beneficial therapeutic effect. Such assays are readily
described in the literature (Kazutsugu Uematsu et al., "Wnt Pathway
Activation in Mesothelioma: Evidence of Dishevelled Overexpression
and Transcriptional Activity of .beta.-catenin", Cancer Research,
volume 63, pages 4547-4551, Aug. 1, 2003). Essentially, cells are
grown in soft agar, and colony formation is measured after days to
weeks (example: 4 weeks) by staining with a special dye
commercially available.
[0150] Apoptosis/cell death assays may also be used to determine
the anti-tumorigenic potency of the COX inhibitors or their
optimized variants. An enhancement of apoptosis/cell death of
cancer cells by the presence of these compounds may be an
indication that the compound has a beneficial therapeutic effect.
Such assays are readily described in the literature (Iwao Mikami et
al., "Efficacy of Wnt-1 monoclonal antibody in sarcoma cells", BMC
Cancer, volume 5:53, pages 1-7, 2005). The method as described in
the reference is based on analysis of Annexin V-FITC cell staining
by FACS (Fluorescence Activated Cell Sorter, Flow cytometry).
[0151] Sensitization towards apoptosis/dcath by NSAIDs. In this
experiment, NSAIDS or NSAID derivatives may be used to sensitize
tumor cells to apoptosis or death by chemotherapeutic agents such
as cis-platin.
VII. Therapeutic Utility
[0152] Compounds identified by the above methods generally find use
in modulating PDZ/PL interactions and/or binding between a COX,
e.g., COX-1 or COX-2, and a PDZ polypeptide in a cell. Such methods
generally involve contacting the cell with a compound or
combination of compounds for a time and under conditions sufficient
for binding of a PDZ/PL (or multiple PDZ/PLs) to be inhibited.
Without being limited by theory, in certain aspects of the
invention, as COX-1 and COX-2 have been found to have PDZ ligands,
inhibiting the COX-1/PDZ and/or COX-2/PDZ interactions may have
additive therapeutic effects.
[0153] In yet another aspect, it has been found that certain chiral
forms of COX inhibitors do not bind COX enzymatic pockets, but
still have observed anti-inflammatory effects. Without being
limited by theory, it is believed that the particular chiral form
of such COX inhibitors may bind PDZ's and exert their COX activity
via their interaction with PDZ's.
[0154] The compounds further find use in treating COX-2 mediated
conditions, which conditions, as discussed above, include pain,
cancer, inflammation and neurological disorders (including damage
from acute insult and recovery therefrom). In one embodiment, the
subject compounds may be administered to a subject suffering from
cancer, pain, and/or inflammation (e.g., arthritis or a similar
condition) or, in other embodiments, a subject at risk for or
having undergone a stroke or another acute insult-inducing
event.
[0155] In particular, the subject compounds may be employed to
decrease pain and/or inflammation, to decrease side effects of
known COX-2 inhibitors, or to improve or repair neuronal circuits
within impaired areas of patients with mild to severe traumatic
brain injury, including diffuse axonal injury, hypoxic-ischemic
encephalopathy and other forms of craniocerebral trauma. Further,
the subject compounds may be used to treat infections of the
nervous system, such as common bacterial meningitis, and to treat
strokes including those caused by ischemic infarction, embolism and
haemorrhage such as hypotensive haemorrhage or other causes.
Moreover, the compounds may also be useful for the treatment of
neurodegenerative diseases including Alzheimer's disease, Lewy Body
dementia, Parkinson's disease (PD), Huntington's disease (HD),
multiple sclerosis, motor neuron disease, muscular dystrophy,
peripheral neuropathies, metabolic disorders of the nervous system
including glycogen storage diseases, and other conditions where
neurons are damaged or destroyed. In other embodiments, the subject
compounds may be used to treat cancer or slow tumor growth. In
certain embodiments, the subject compounds may exert their
anti-cancer and anti-tumor activity with fewer undesirable side
effects, as compared to traditional treatments, such as cardiac
malfunction.
[0156] In particular embodiments, the subject compound may be
co-administered in conjunction with an inhibitor of prostaglandin
synthesis by COX-2 (which may be a non-specific or specific COX-2).
Such a compound may be a non-steroidal anti-inflammatory drug
(NSAID) of a category listed above. In particular embodiments, the
compound may be co-administered with, for example, aspirin,
indomethacin (Indocin), ibuprofen (Motrin), naproxen (Naprosyn),
piroxicam (Feldene), nabumetone (Relafen), rofecoxib (Vioxx),
celecoxib (celebrex) or valdecoxib (Bextra). Such COX-2 inhibitors
are well known.
EXAMPLES
[0157] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention. Efforts have been made to ensure accuracy with
respect to numbers used (e.g. amounts, temperature, etc.) but some
experimental errors and deviations should be accounted for. Unless
indicated otherwise, parts are parts by weight, molecular weight is
weight average molecular weight, temperature is in degrees
Centigrade, and pressure is at or near atmospheric.
Example 1
"A Assay" Detection of PDZ-Ligand Binding Using Immobilized PL
Peptide
[0158] The following describes 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 one embodiment, the PDZ-domain fusion protein is a
GST/PDZ fusion protein and the assay is carried out as follows:
[0159] (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
.mu.l, per well of 20 .mu.g/mL of avidin (Pierce) in phosphate
buffered saline without calcium and magnesium, pH 7.4 ("PBS",
GibcoBRL) at 4.degree. C. for 12 hours. The plate is then treated
to block nonspecific interactions by addition of 200 .mu.L per well
of PBS containing 2 g per 100 mL protease-free bovine serum albumin
("PBS/BSA") for 2 hours at 4.degree. C. The plate is then washed 3
times with PBS by repeatedly adding 200 .mu.L 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.
[0160] (2) Biotinylated PL peptides (or candidate PL peptides) are
immobilized on the surface of wells of the plate by addition of 50
uL per well of 0.4 uM peptide in PBS/BSA for 30 minutes at
4.degree. 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 GST/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.
[0161] (3) GST/PDZ-domain fusion protein (prepared as described
supra) is allowed to react with the surface by addition of 50 .mu.L
per well of a solution containing 5 .mu.g/mL GST/PDZ-domain fusion
protein in PBS/BSA for 2 hours at 4.degree. 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.
[0162] (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
uL per well of an anti-GST antibody in PBS/BSA (e.g., 2.5 .mu.g/mL
of polyclonal goat-anti-GST antibody, Pierce) is added to the plate
and allowed to react for 20 minutes at 4.degree. C. The plate is
washed 3 times with PBS and a second, detectably labeled antibody
is added. In one embodiment, 50 .mu.L per well of 2.5 .mu.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.degree. C. The plate is washed 5 times with 50
mM Tris pH 8.0 containing 0.2% Tween 20, and developed by addition
of 100 .mu.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 .mu.L per well of 1M
sulfuric acid and the absorbance (A) of each well of the plate is
read at 450 nm.
[0163] (5) Specific binding of a PL peptide and a PDZ-domain
polypeptide is detected by comparing the signal from the well(s) 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 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.
[0164] As noted, in an embodiment of the "A" assay, the signal from
binding of a GST/PDZ-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 B1
and/or mean B2.
Example 2
"G Assay"--Detection of PDZ-Ligand Binding Using Immobilized
PDZ-Domain Fusion Polypeptide
[0165] An assay in which a GST/PDZ fusion protein is immobilized on
a surface is described below. ("G" assay). The binding of labeled
PL peptide to this surface is then measured. The assay may be
carried out as follows:
[0166] (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 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: [0167] (a) 100 .mu.L per well of 5 .mu.g/mL goat
anti-GST polyclonal antibody (Pierce) in PBS is added to a
polystyrene 96-well plate (e.g., Nunc Polysorb) at 4.degree. C. for
12 hours. [0168] (b) The plate is blocked by addition of 200 .mu.L
per well of PBS/BSA for 2 hours at 4.degree. C. [0169] (c) The
plate is washed 3 times with PBS. [0170] (d) 50 .mu.l per well of 5
.mu.g/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.degree. C. [0171] (e) The plate is
again washed 3 times with PBS
[0172] (2) Biotinylated PL peptides are allowed to react with the
surface by addition of 50 .mu.L per well of 20 .mu.M solution of
the biotinylated peptide in PBS/BSA for 10 minutes at 4.degree. C.,
followed by an additional 20 minute incubation at 25.degree. C. The
plate is washed 3 times with ice cold PBS.
[0173] (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 .mu.L per well of 0.5 .mu.g/mL streptavidin-horse radish
peroxidase (HRP) conjugate dissolved in BSA/PBS is added and
allowed to react for 20 minutes at 4.degree. 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 .mu.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 addition of
100 .mu.L per well of 1M sulfuric acid, and the absorbance of each
well of the plate is read at 450 nm.
[0174] (4) Specific binding of a PL peptide and a PDZ domain
polypeptide is determined by comparing the signal from the well(s)
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 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 "B1"). 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.
Example 3
Identification of PDZ Binding Partners OF COX-2
[0175] Polynucleotides encoding approximately 250 different PDZ
domains were cloned into the vector pGEX-3.times. and expressed
according to the methods set forth in U.S. patent application Ser.
Nos. 09/710,059, 09/724,553 and 09/688,017. Binding of those PDZ
domains was tested against the C-terminus of COX-2 (LLKERSTEL)
(SEQ. ID NO. 13) that contains a potential class 1 PDZ-binding
domain (PL) conforming to the consensus (S/T-X-V/L) (SEQ. ID. NO.
14). An ELISA-based assay was performed as described below.
Materials
[0176] Nunc Maxisorp 96 well Immuno-plate (Nunc cat # 62409-005)
[0177] (Maxisorp plates have been shown to have higher background
signal) [0178] PBS pH 7.4 (Gibco BRL cat #16777-148) or [0179] AVC
phosphate buffered saline, 8 gm NaCl, 0.29 gm KCl, 1.44 gm
Na.sub.2HPO4, 0.24 gm KH.sub.2PO4, add H.sub.2O to 1 L and pH 7.4;
0.2 micron filter [0180] 2% BSA/PBS (10 g of bovine serum albumin,
fraction V (ICN Biomedicals [0181] cat # IC15142983) into 500 ml
PBS [0182] Goat anti-GST mAb stock@5 mg/ml, store at 4.degree. C.,
(Amersham Pharmacia [0183] cat # 27-4577-01), dilute 1:1000 in PBS,
final concentration 5 ug/ml [0184] GST-PDZ fusion protein (stock
stored at -80.degree. C. in 35% glycerol, based on pGEX-3X vector),
diluted to 5 ug/ml in 2% BSA/PBS [0185] Peptide: 0.06 uM
N-terminally biotinylated Tat-COX-2 peptide in 2% BSA/PBS [0186]
Peptide 1956 sequence: YGRKKRRQRRRLLKERSTEL (SEQ. ID NO. 15)
(underlined sequence=wt COX-2 C-terminus, N-terminus is Tat Protein
transduction domain sequence for solubility) [0187] Peptide 1957
sequence: RRRSGLDDINPTVLLKERSTEL (SEQ. ID NO. 92) (underlined
sequence=COX-2 C-terminus) [0188] Wash Buffer, PBS, pH 7.4 [0189]
TMB (3,3',5,5', teramethylbensidine), tablets, Sigma cat. #T5525,
lot#: [0190] Per plate, dissolve 1 TMB tablet in 1 mL DMSO, add 9
mL Citrate Phosphate Buffer pH 5.4 and 2 uL H.sub.2O.sub.2 [0191]
0.18M H.sub.2SO.sub.4 (SIGMA cat. #S1526) [0192] Hamilton liquid
handler, MPH-96 [0193] 250 ml reagent reservoirs, [0194] 15 ml
polypropylene conical tubes [0195] RP-Streptavidin, 2.5 mg/2 mL
stock, Zymed cat #43-4323 [0196] Dilute 1:2000 in 2% BSA/PBS, Final
Concentration 0.5 ug/mL [0197] Skan Washer 300 version B w/
Stacker, Molecular Devices [0198] Molecular Devices microplate
reader (450 nm filters) [0199] oftmax Pro Software for microplate
reader
Methods
[0200] 1. Coat plate with 100 .mu.l of 5 pg/ml anti-GST Ab, O/N @
4.degree. C. 2. Wash Plate with Plate Washer 3.times.. 3.
Blocking--Add 200 .mu.l per well 2% BSA/PBS
4. Incubate for 2 hrs at RT
[0201] 5. Rinse off blocking buffer by washing 3 times with 350
.mu.l per well PBS 6. Add 50 .mu.l 5 .mu.g/ml GST-PDZ fusion
protein in 2% BSA/PBS (or GST alone as control). 7. Incubate at RT
for 2 hours 8. Rinse off excess protein by washing 3 times with 350
ul per well PBS. 9. Add 95 .mu.l of the N-terminally biotinylated
peptide 10. Incubate at RT for 30 minutes 11. Rinse off excess
peptide by washing 3 times with 350 .mu.l per well PBS. 12. Add 95
.mu.l per well 0.5 .mu.g/ml of HRP-Streptavidin, 20 minutes at RT
13. Rinse by washing 7 times with 350 .mu.l/well with PBS pH 7.4
14. Add 95 .mu.l per well TMB substrate 15. Incubate in dark at
room temp, checking plate periodically (5, 10, & 20 minutes)
16. Take early readings, if necessary, at 650 nm 17. At 30 minutes,
stop reaction with 95 .mu.l of 0.18M H.sub.2SO.sub.4, and take
final reading at 450 nm
Results
[0202] The PDZ domains of four proteins SHANK1, SHANK2, SHANK3 and
MAST2 were shown to interact with the PL of COX-2. The results are
shown in FIG. 3A-3B and FIG. 4A-4C.
[0203] Titrations of peptide 1956 involve the Tat transporter
peptide sequence (YGRKKRRQRRR, SEQ. ID. NO.: 94) coupled to the
wild type 9 C-terminal amino acid sequence of COX-2 (LLKERSTEL,
SEQ. ID. NO.: 13) which leads to a higher affinity (lower EC50)
towards the various PDZ's relative to peptide 1957, essentially the
wild type COX-2 sequence. In accordance with certain embodiments of
the invention, binding ehancement affinity of Tat peptide-PDZ
ligand/PDZ binding for PDZ ligands may optionally be observed. As
such, in certain embodiments, transporter peptides such as Tat may
enhance the PDZ binding affinity of PDZ ligands in addition to
facilitating the PL peptide entry into the cell.
[0204] The sequence of the PDZ domains of SHANK1, SHANK2, SHANK3
and MAST2 employed in these assays are set forth below:
TABLE-US-00001 SHANK1 (GID 7025450): (SEQ. ID NO. 16)
ILKEKTVLLQKKDSEGFGFVLRGAKAQTPIEEFTPTPAFPALQYLESVDE
GGVAWRAGLRMGDFLIEVNGQNVVKVGHRQVVNMIRQGGNTLMVKVVMVT RHPDMDEAVQNSS
SHANK2 (GID 6049185): (SEQ. ID NO. 17)
ILEEKTVVLQKKDNEGFGFVLRGAKADTPIEEFTPTPAFPALQYLESVDE
GGVAWQAGLRTGDFLIEVKNENVVKVGHRQVVNMIRQGGNHLVLKVVTVT RNLDPDDNSS
SHANK3 (XM_037493, GID: 51476100): (SEQ. ID NO. 18)
SDYVIDDKVAVLQKRDHEGFGFVLRGAKAETPIEEFTPTPAFPALQYLES
VDVEGVAWRAGLRTGDFLIEVNGVNVVKVGHKQVVALIRQGGNRLVMKVV SVTRKPEEDG MAST2
(Accession no. AB047005): (SEQ ID. NO. 19)
ISALGSMRPPIIIHRAGKKYGFTLRAIRVYMGDSDVYTVHHMVWHVEDGG
PASEAGLRQGDLITHVNGEPVHGLVHTEVVELILKSGNKVAISTTPLENS S
[0205] In a further experiment, the PDZ domains of SHANK1, SHANK2,
SHANK3 and MAST2 were used to identify further PDZ domains (other
than that of COX-2) that bind to SHANK1, SHANK2, SHANK3 and MAST2.
A list of PDZ ligands that bind to SHANK1, SHANK2 or SHANK3 is set
forth in FIG. 5A. A list of PDZ ligands that bind to MAST2 is set
forth in FIG. 5B.
[0206] Such polypeptides and their variants and analogs may also be
employed to inhibit binding between COX-2 and SHANK1, SHANK2,
SHANK3 and MAST2 in a cell.
[0207] The above results and discussion demonstrate new COX-2
interacting proteins. Knowledge of the interaction provides a means
for identifying drugs that can modulate the COX-2. Accordingly, the
subject methods represent a significant contribution to the
art.
Example 4
Drug Competition Assay--Matrix Elisa Modified G Assay
[0208] The assay described in this example may be used in
accordance with certain embodiments of the invention to determine
the efficacy of candidate inhibitory compounds in disrupting PDZ/PL
binding or PDZ/COX-2 binding. The complete protocol and list of
reagents/supplies is provided below.
Materials:
[0209] 1) Nunc Maxisorp 96 well Immuno-plates 2) PBS pH 7.4
(phosphate buffered saline, 8 g NaCl, 0.29 g KCl, 1.44 g
Na.sub.2HPO.sub.4, 0.24 g 3) KH.sub.2PO.sub.4, add H.sub.2O to 1 L
and pH 7.4; 0.2.mu. filter) 4) Assay Buffer: 2% BSA in PBS (20 g of
BSA per liter PBS), ICN Biomedicals 5) Goat anti-GST polyclonal
antibody, stock 5 mg/ml, stored at 4.degree. C., Amersham Pharmacia
6) Dilute 1:1000 in PBS, final concentration 5 .mu.g/ml 7)
HRP-Streptavidin, 2.5 mg/2 ml stock stored@4.degree. C., Zymed,
[0210] dilute 1:2000 into Assay buffer, final [0.5 .mu.g/ml] 8)
Biotinylated peptides (from Anaspec, stored in -20.degree. C.
freezer) 9) GST-PRISM proteins (stock stored@, -80.degree. C.,
after 1.sup.st thaw store in -10.degree. C. freezer) 10) TMB
(3,3',5,5', teramethylbensidine), ready to use
11) 0.18M H.sub.2SO.sub.4
[0211] 12) 12-w multichannel pipettor 13) 200 .mu.L LTS tips 14) 50
ml reagent reservoirs 15) 50 polypropylene conical tubes 16) 15 mL
polypropylene round-bottom tubes 17) 1.5 mL microtubes
18) Costar Transtar 96
19) Transtar 96 Cartridge
[0212] 20) Molecular Devices microplate reader (450 nm filters) 21)
SoftMax Pro software 22) Assay buffer (1.times.PBS, 0.01% Triton
X-100)
Methods:
[0213] 18-20 plates were coated with 100 .mu.l of 5 .mu.g/ml
anti-GST antibody in each well, and left overnight at 4.degree. C.
The plates were then emptied by inverting and tapped dry on paper
towels. 200 .mu.l of blocking buffer (1.times.PBS/2% BSA) was added
to each well and the plates were left for 1-2 hrs at room
temperature. The proteins were then diluted to the required
concentration in 1.times.PBS/2% BSA. The plates were then washed
using the automatic plate washer (3.times. with room temperature
1.times.PBS), ensuring that the plates did not dry out. Proteins
were added to the wells at 50 .mu.l per well and were incubated for
1-2 hours at 4.degree. C.
[0214] The peptides, drugs, and HRP were then prepared in Assay
Buffer as follows: [0215] Peptides were prepared in one-quarter
final volume at 4.times. final concentration. [0216] HRP was
diluted (1:500) in one-quarter final volume at 4.times. final
concentration. [0217] Peptides and HRP were then mixed together,
and incubated for 20 minutes at room temperature. [0218] Whilst the
peptide/HRP mix was incubating, the drug titrations were prepared
in half the final volume at 2.times. final concentration. [0219]
Immediately before adding the final mixture to the plate, the drug
titration was combined with the peptide-HRP solution (mixture
should now be correct total volume and final concentrations).
[0220] The following PDZ peptide/PL combinations were tested with
the following drugs (alternatively, a COX-2 PL sequence may be
used). Exemplary PDZ domain sequences are illustrated in FIG.
6.
TABLE-US-00002 PDZ PL Sequence MAGI1 d1 GRWTGRSMSSWKPTRRETEV (SEQ.
ID NO. 20) (AVC 88) (AVC 1857) TIP-1 QISPGGLEPPSEKHFRETEV (SEQ. ID
NO. 21) (AVC 54) (AVC AA56) SHANK1 YGRKKRRQRRRYIPEAQTRL (SEQ. ID
NO. 22) (AVC 235) (AVC 1965) PSD95 d1 YGRKKRRQRRRRISSIETDV (SEQ. ID
NO. 23) (AVC 143) (AVC 1912) PSD95 d2 YGRKKRRQRRRKLSSIESDV (SEQ. ID
NO. 24) (AVC 265) (AVC AA348) PSD95 d3 YGRKKRRQRRRTKNYKQTSV (SEQ.
ID NO. 25) (AVC 466) (AVC 1916)
[0221] Drugs tested: 1) Niflumic acid; 2) Ibuprofen; 3) Naproxen
sodium; 4) Diclofenac sodium salt; 5) Acetylsalicylic acid; 6)
Salicylic; 7) Flurbiprofen; 8) Sulindac sulphide; 9) Sulindac; 10)
Etodolac; 11) Indomethacin; 12) Ketorolac Tris salt; 13)
Ketoprofen; 14) Mefenamic acid; 15) Carprofen; 16) Baclofen; 17)
Fenoprofen; 18) Benztropine mesylate; 19) Amitriptyline HCl; 20)
Cromolyn sodium; 21) Desipramine HCl; 22) Clomipramine HCl; 23)
Nortriptyline HCl, as recognized by those skilled in the art, (1-17
are COX-2 inhibitors).
[0222] The plates were then washed using the automatic plate washer
(3.times. with room temperature 1.times.PBS). The peptide/HRP/drug
mixtures were then added to the plates at 50 .mu.l per well and the
time of addition of the mixture was recorded on each plate. The
plates were then incubated at room temperature, after the last
peptide had been added, for exactly 30 minutes.
[0223] The plate reader was turned on the computer files were
prepared during the incubation. The plates were then washed using
the automatic plate washer (7.times. with room temperature
1.times.PBS). TMB substrate was then added to the plates at 100
.mu.l per well and the time of TMB addition was written on each
plate. The plates were then incubated in the dark at room
temperature for a maximum of 30 minutes. The reaction was then
stopped using 100 .mu.l of 0.18M H.sub.2SO.sub.4 30 minutes after
adding TMB. The plates were then read at 450 nm immediately after
stopping the reaction.
Results:
[0224] Results are shown in FIGS. 7A-7G, where the drugs were
competing with biotinylated peptides for binding to the PDZ capture
proteins on the ELISA plate. A decrease in the base OD signal (for
the peptide-PDZ interaction) corresponds to an increase in drug-PDZ
binding and successful competition of the drug against the
biotinylated peptide.
[0225] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference. Genbank records referenced by GID or
accession number, particularly any polypeptide sequence,
polynucleotide sequences or annotation thereof, are incorporated by
reference herein. The citation of any publication is for its
disclosure prior to the filing date and should not be construed as
an admission that the present invention is not entitled to antedate
such publication by virtue of prior invention.
[0226] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
94189PRTHomo sapiens 1Val Leu Leu Gln Lys Lys Asp Ser Glu Gly Phe
Gly Phe Val Leu Arg1 5 10 15Gly Ala Lys Ala Gln Thr Pro Ile Glu Glu
Phe Thr Pro Thr Pro Ala 20 25 30Phe Pro Ala Leu Gln Tyr Leu Glu Ser
Val Asp Glu Gly Gly Val Ala 35 40 45Trp Arg Ala Gly Leu Arg Met Gly
Asp Phe Leu Ile Glu Val Asn Gly 50 55 60Gln Asn Val Val Lys Val Gly
His Arg Gln Val Val Asn Met Ile Arg65 70 75 80Gln Gly Gly Asn Thr
Leu Met Val Lys 852109PRTHomo sapiens 2Gly Ser Asp Tyr Ile Ile Lys
Glu Lys Thr Val Leu Leu Gln Lys Lys1 5 10 15Asp Ser Glu Gly Phe Gly
Phe Val Leu Arg Gly Ala Lys Ala Gln Thr 20 25 30Pro Ile Glu Glu Phe
Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr 35 40 45Leu Glu Ser Val
Asp Glu Gly Gly Val Ala Trp Arg Ala Gly Leu Arg 50 55 60Met Gly Asp
Phe Leu Ile Glu Val Asn Gly Gln Asn Val Val Lys Val65 70 75 80Gly
His Arg Gln Val Val Asn Met Ile Arg Gln Gly Gly Asn Thr Leu 85 90
95Met Val Lys Val Val Met Val Thr Arg His Pro Asp Met 100
1053129PRTHomo sapiens 3Ala Pro Ser Leu Met Asp Gly Ile Gly Pro Gly
Ser Asp Tyr Ile Ile1 5 10 15Lys Glu Lys Thr Val Leu Leu Gln Lys Lys
Asp Ser Glu Gly Phe Gly 20 25 30Phe Val Leu Arg Gly Ala Lys Ala Gln
Thr Pro Ile Glu Glu Phe Thr 35 40 45Pro Thr Pro Ala Phe Pro Ala Leu
Gln Tyr Leu Glu Ser Val Asp Glu 50 55 60Gly Gly Val Ala Trp Arg Ala
Gly Leu Arg Met Gly Asp Phe Leu Ile65 70 75 80Glu Val Asn Gly Gln
Asn Val Val Lys Val Gly His Arg Gln Val Val 85 90 95Asn Met Ile Arg
Gln Gly Gly Asn Thr Leu Met Val Lys Val Val Met 100 105 110Val Thr
Arg His Pro Asp Met Asp Glu Ala Val His Lys Lys Ala Pro 115 120
125Gly489PRTHomo sapiens 4Val Val Leu Gln Lys Lys Asp Asn Glu Gly
Phe Gly Phe Val Leu Arg1 5 10 15Gly Ala Lys Ala Asp Thr Pro Ile Glu
Glu Phe Thr Pro Thr Pro Ala 20 25 30Phe Pro Ala Leu Gln Tyr Leu Glu
Ser Val Asp Glu Gly Gly Val Ala 35 40 45Trp Gln Ala Gly Leu Arg Thr
Gly Asp Phe Leu Ile Glu Val Asn Asn 50 55 60Glu Asn Val Val Lys Val
Gly His Arg Gln Val Val Asn Met Ile Arg65 70 75 80Gln Gly Gly Asn
His Leu Val Leu Lys 855109PRTHomo sapiens 5Tyr Ser Asp Cys Ile Ile
Glu Glu Lys Thr Val Val Leu Gln Lys Lys1 5 10 15Asp Asn Glu Gly Phe
Gly Phe Val Leu Arg Gly Ala Lys Ala Asp Thr 20 25 30Pro Ile Glu Glu
Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr 35 40 45Leu Glu Ser
Val Asp Glu Gly Gly Val Ala Trp Gln Ala Gly Leu Arg 50 55 60Thr Gly
Asp Phe Leu Ile Glu Val Asn Asn Glu Asn Val Val Lys Val65 70 75
80Gly His Arg Gln Val Val Asn Met Ile Arg Gln Gly Gly Asn His Leu
85 90 95Val Leu Lys Val Val Thr Val Thr Arg Asn Leu Asp Pro 100
1056129PRTHomo sapiens 6Asn Asn Gly Arg Cys Pro Arg Asn Ser Leu Tyr
Ser Asp Cys Ile Ile1 5 10 15Glu Glu Lys Thr Val Val Leu Gln Lys Lys
Asp Asn Glu Gly Phe Gly 20 25 30Phe Val Leu Arg Gly Ala Lys Ala Asp
Thr Pro Ile Glu Glu Phe Thr 35 40 45Pro Thr Pro Ala Phe Pro Ala Leu
Gln Tyr Leu Glu Ser Val Asp Glu 50 55 60Gly Gly Val Ala Trp Gln Ala
Gly Leu Arg Thr Gly Asp Phe Leu Ile65 70 75 80Glu Val Asn Asn Glu
Asn Val Val Lys Val Gly His Arg Gln Val Val 85 90 95Asn Met Ile Arg
Gln Gly Gly Asn His Leu Val Leu Lys Val Val Thr 100 105 110Val Thr
Arg Asn Leu Asp Pro Asp Asp Thr Ala Arg Lys Lys Ala Pro 115 120
125Pro789PRTHomo sapiens 7Ala Val Leu Gln Lys Arg Asp His Glu Gly
Phe Gly Phe Val Leu Arg1 5 10 15Gly Ala Lys Ala Glu Thr Pro Ile Glu
Glu Phe Thr Pro Thr Pro Ala 20 25 30Phe Pro Ala Leu Gln Tyr Leu Glu
Ser Val Asp Val Glu Gly Val Ala 35 40 45Trp Arg Ala Gly Leu Arg Thr
Gly Asp Phe Leu Ile Glu Val Asn Gly 50 55 60Val Asn Val Val Lys Val
Gly His Lys Gln Val Val Ala Leu Ile Arg65 70 75 80Gln Gly Gly Asn
Arg Leu Val Met Lys 858109PRTHomo sapiens 8His Ser Asp Tyr Val Ile
Asp Asp Lys Val Ala Val Leu Gln Lys Arg1 5 10 15Asp His Glu Gly Phe
Gly Phe Val Leu Arg Gly Ala Lys Ala Glu Thr 20 25 30Pro Ile Glu Glu
Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr 35 40 45Leu Glu Ser
Val Asp Val Glu Gly Val Ala Trp Arg Ala Gly Leu Arg 50 55 60Thr Gly
Asp Phe Leu Ile Glu Val Asn Gly Val Asn Val Val Lys Val65 70 75
80Gly His Lys Gln Val Val Ala Leu Ile Arg Gln Gly Gly Asn Arg Leu
85 90 95Val Met Lys Val Val Ser Val Thr Arg Lys Pro Glu Glu 100
1059129PRTHomo sapiens 9Thr Val Gly Ser Tyr Asp Ser Leu Thr Ser His
Ser Asp Tyr Val Ile1 5 10 15Asp Asp Lys Val Ala Val Leu Gln Lys Arg
Asp His Glu Gly Phe Gly 20 25 30Phe Val Leu Arg Gly Ala Lys Ala Glu
Thr Pro Ile Glu Glu Phe Thr 35 40 45Pro Thr Pro Ala Phe Pro Ala Leu
Gln Tyr Leu Glu Ser Val Asp Val 50 55 60Glu Gly Val Ala Trp Arg Ala
Gly Leu Arg Thr Gly Asp Phe Leu Ile65 70 75 80Glu Val Asn Gly Val
Asn Val Val Lys Val Gly His Lys Gln Val Val 85 90 95Ala Leu Ile Arg
Gln Gly Gly Asn Arg Leu Val Met Lys Val Val Ser 100 105 110Val Thr
Arg Lys Pro Glu Glu Asp Gly Ala Arg Arg Arg Ala Met Lys 115 120
125Lys1079PRTHomo sapiens 10Ile His Arg Ala Gly Lys Lys Tyr Gly Phe
Thr Leu Arg Ala Ile Arg1 5 10 15Val Tyr Met Gly Asp Ser Asp Val Tyr
Thr Val His His Met Val Trp 20 25 30His Val Glu Asp Gly Gly Pro Ala
Ser Glu Ala Gly Leu Arg Gln Gly 35 40 45Asp Leu Ile Thr His Val Asn
Gly Glu Pro Val His Gly Leu Val His 50 55 60Thr Glu Val Val Glu Leu
Ile Leu Lys Ser Gly Asn Lys Val Ala65 70 751199PRTHomo sapiens
11Ala Leu Gly Ser Met Arg Pro Pro Ile Ile Ile His Arg Ala Gly Lys1
5 10 15Lys Tyr Gly Phe Thr Leu Arg Ala Ile Arg Val Tyr Met Gly Asp
Ser 20 25 30Asp Val Tyr Thr Val His His Met Val Trp His Val Glu Asp
Gly Gly 35 40 45Pro Ala Ser Glu Ala Gly Leu Arg Gln Gly Asp Leu Ile
Thr His Val 50 55 60Asn Gly Glu Pro Val His Gly Leu Val His Thr Glu
Val Val Glu Leu65 70 75 80Ile Leu Lys Ser Gly Asn Lys Val Ala Ile
Ser Thr Thr Pro Leu Glu 85 90 95Asn Thr Ser12119PRTHomo sapiens
12Asp Ser Ser Pro Ser Arg Asp Phe Leu Pro Ala Leu Gly Ser Met Arg1
5 10 15Pro Pro Ile Ile Ile His Arg Ala Gly Lys Lys Tyr Gly Phe Thr
Leu 20 25 30Arg Ala Ile Arg Val Tyr Met Gly Asp Ser Asp Val Tyr Thr
Val His 35 40 45His Met Val Trp His Val Glu Asp Gly Gly Pro Ala Ser
Glu Ala Gly 50 55 60Leu Arg Gln Gly Asp Leu Ile Thr His Val Asn Gly
Glu Pro Val His65 70 75 80Gly Leu Val His Thr Glu Val Val Glu Leu
Ile Leu Lys Ser Gly Asn 85 90 95Lys Val Ala Ile Ser Thr Thr Pro Leu
Glu Asn Thr Ser Ile Lys Val 100 105 110Gly Pro Ala Arg Lys Gly Ser
115139PRTHomo sapiens 13Leu Leu Lys Glu Arg Ser Thr Glu Leu1
5143PRTHomo sapiensMOD_RES(1)Ser or Thr 14Xaa Xaa Xaa11520PRTHomo
sapiens 15Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Leu Lys
Glu Arg1 5 10 15Ser Thr Glu Leu 2016113PRTHomo sapiens 16Ile Leu
Lys Glu Lys Thr Val Leu Leu Gln Lys Lys Asp Ser Glu Gly1 5 10 15Phe
Gly Phe Val Leu Arg Gly Ala Lys Ala Gln Thr Pro Ile Glu Glu 20 25
30Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr Leu Glu Ser Val
35 40 45Asp Glu Gly Gly Val Ala Trp Arg Ala Gly Leu Arg Met Gly Asp
Phe 50 55 60Leu Ile Glu Val Asn Gly Gln Asn Val Val Lys Val Gly His
Arg Gln65 70 75 80Val Val Asn Met Ile Arg Gln Gly Gly Asn Thr Leu
Met Val Lys Val 85 90 95Val Met Val Thr Arg His Pro Asp Met Asp Glu
Ala Val Gln Asn Ser 100 105 110Ser17110PRTHomo sapiens 17Ile Leu
Glu Glu Lys Thr Val Val Leu Gln Lys Lys Asp Asn Glu Gly1 5 10 15Phe
Gly Phe Val Leu Arg Gly Ala Lys Ala Asp Thr Pro Ile Glu Glu 20 25
30Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr Leu Glu Ser Val
35 40 45Asp Glu Gly Gly Val Ala Trp Gln Ala Gly Leu Arg Thr Gly Asp
Phe 50 55 60Leu Ile Glu Val Asn Asn Glu Asn Val Val Lys Val Gly His
Arg Gln65 70 75 80Val Val Asn Met Ile Arg Gln Gly Gly Asn His Leu
Val Leu Lys Val 85 90 95Val Thr Val Thr Arg Asn Leu Asp Pro Asp Asp
Asn Ser Ser 100 105 11018110PRTHomo sapiens 18Ser Asp Tyr Val Ile
Asp Asp Lys Val Ala Val Leu Gln Lys Arg Asp1 5 10 15His Glu Gly Phe
Gly Phe Val Leu Arg Gly Ala Lys Ala Glu Thr Pro 20 25 30Ile Glu Glu
Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr Leu 35 40 45Glu Ser
Val Asp Val Glu Gly Val Ala Trp Arg Ala Gly Leu Arg Thr 50 55 60Gly
Asp Phe Leu Ile Glu Val Asn Gly Val Asn Val Val Lys Val Gly65 70 75
80His Lys Gln Val Val Ala Leu Ile Arg Gln Gly Gly Asn Arg Leu Val
85 90 95Met Lys Val Val Ser Val Thr Arg Lys Pro Glu Glu Asp Gly 100
105 11019101PRTHomo sapiens 19Ile Ser Ala Leu Gly Ser Met Arg Pro
Pro Ile Ile Ile His Arg Ala1 5 10 15Gly Lys Lys Tyr Gly Phe Thr Leu
Arg Ala Ile Arg Val Tyr Met Gly 20 25 30Asp Ser Asp Val Tyr Thr Val
His His Met Val Trp His Val Glu Asp 35 40 45Gly Gly Pro Ala Ser Glu
Ala Gly Leu Arg Gln Gly Asp Leu Ile Thr 50 55 60His Val Asn Gly Glu
Pro Val His Gly Leu Val His Thr Glu Val Val65 70 75 80Glu Leu Ile
Leu Lys Ser Gly Asn Lys Val Ala Ile Ser Thr Thr Pro 85 90 95Leu Glu
Asn Ser Ser 1002020PRTHomo sapiens 20Gly Arg Trp Thr Gly Arg Ser
Met Ser Ser Trp Lys Pro Thr Arg Arg1 5 10 15Glu Thr Glu Val
202120PRTHomo sapiens 21Gln Ile Ser Pro Gly Gly Leu Glu Pro Pro Ser
Glu Lys His Phe Arg1 5 10 15Glu Thr Glu Val 202220PRTHomo sapiens
22Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Tyr Ile Pro Glu Ala1
5 10 15Gln Thr Arg Leu 202320PRTHomo sapiens 23Tyr Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg Arg Ile Ser Ser Ile1 5 10 15Glu Thr Asp Val
202420PRTHomo sapiens 24Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Lys Leu Ser Ser Ile1 5 10 15Glu Ser Asp Val 202520PRTHomo sapiens
25Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Thr Lys Asn Tyr Lys1
5 10 15Gln Thr Ser Val 202620PRTArtificial SequenceSynthetic
peptide 26Pro Ile Pro Ala Gly Gly Cys Thr Phe Ser Gly Ile Phe Pro
Thr Leu1 5 10 15Thr Ser Pro Leu 202720PRTArtificial
SequenceSynthetic peptide 27Leu Gln Phe His Arg Gly Ser Arg Ala Gln
Ser Phe Leu Gln Thr Glu1 5 10 15Thr Ser Val Ile 202820PRTArtificial
SequenceSynthetic peptide 28Thr Arg Glu Asp Ile Tyr Val Asn Tyr Pro
Thr Phe Ser Arg Arg Pro1 5 10 15Lys Thr Arg Val 202920PRTArtificial
SequenceSynthetic peptide 29Lys Glu Asn Asp Tyr Glu Ser Ile Ser Asp
Leu Gln Gln Gly Arg Asp1 5 10 15Ile Thr Arg Leu 203019PRTArtificial
SequenceSynthetic peptide 30Ala Trp Asp Asp Ser Ala Arg Ala Ala Gly
Gly Gln Gly Leu His Val1 5 10 15Thr Ala Leu3119PRTArtificial
SequenceSynthetic peptide 31Lys Asp Ser Arg Pro Ser Phe Val Gly Ser
Ser Ser Gly His Thr Ser1 5 10 15Thr Thr Leu3220PRTArtificial
SequenceSynthetic peptide 32Lys Pro Gln Ile Ala Ala Leu Lys Glu Glu
Thr Glu Glu Glu Val Gln1 5 10 15Asp Thr Arg Leu 203320PRTArtificial
SequenceSynthetic peptide 33Pro Ser Trp Arg Arg Ser Ser Leu Ser Glu
Ser Glu Asn Ala Thr Ser1 5 10 15Leu Thr Thr Phe 203420PRTArtificial
SequenceSynthetic peptide 34Ala Ala Gly Gly Arg Ser Ala Arg Gly Gly
Arg Leu Gln Gly Arg Arg1 5 10 15Glu Thr Ala Leu 203520PRTArtificial
SequenceSynthetic peptide 35Pro Pro Ala Thr Pro Ser Pro Arg Leu Ala
Leu Pro Ala His His Asn1 5 10 15Ala Thr Arg Leu 203618PRTArtificial
SequenceSynthetic peptide 36Thr Phe Ala Ala Gly Phe Asn Ser Thr Gly
Leu Pro His Ser Thr Thr1 5 10 15Arg Val3720PRTArtificial
SequenceSynthetic peptide 37Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp
Glu Asp Gln His Thr Gln1 5 10 15Ile Thr Lys Val 203820PRTArtificial
SequenceSynthetic peptide 38Gln Asp Phe Arg Arg Ala Phe Arg Arg Ile
Leu Ala Arg Pro Trp Thr1 5 10 15Gln Thr Ala Trp 203919PRTArtificial
SequenceSynthetic peptide 39Ser Ser Lys Ser Lys Ser Ser Glu Glu Ser
Gln Thr Phe Phe Gly Leu1 5 10 15Tyr Lys Leu4020PRTArtificial
SequenceSynthetic peptide 40Glu Ala Leu Gln Pro Glu Pro Gly Arg Lys
Arg Ile Pro Leu Thr Arg1 5 10 15Thr Thr Thr Phe 204120PRTArtificial
SequenceSynthetic peptide 41Leu Ala Ser Lys Ser Ala Glu Glu Gly Lys
Gln Ile Pro Asp Ser Leu1 5 10 15Ser Thr Asp Leu 204220PRTArtificial
SequenceSynthetic peptide 42Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg Pro Ser Arg Lys Leu1 5 10 15Asn Thr Glu Ile 204320PRTArtificial
SequenceSynthetic peptide 43Lys His Ser Arg Lys Ser Ser Ser Tyr Ser
Ser Ser Ser Thr Thr Val1 5 10 15Lys Thr Ser Tyr 204420PRTArtificial
SequenceSynthetic peptide 44Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp
Glu Asp Gln His Thr Gln1 5 10 15Ile Thr Lys Val 204520PRTArtificial
SequenceSynthetic peptide 45Ser Ala Thr Glu Ser Ala Glu Ser Ile Glu
Ile Tyr Ile Pro Glu Ala1 5 10 15Gln Thr Arg Leu 204620PRTArtificial
SequenceSynthetic peptide 46Asn Ser Tyr Val Arg Asp Asp Ala Ile Phe
Ile Lys Ala Ile Val Asp1 5 10 15Leu Thr Gly
Leu 204719PRTArtificial SequenceSynthetic peptide 47Ala Trp Asp Asp
Ser Ala Arg Ala Ala Gly Gly Gln Gly Leu His Val1 5 10 15Thr Ala
Leu4820PRTArtificial SequenceSynthetic peptide 48Arg Glu Leu Val
Asp Arg Gly Glu Val Arg Gln Phe Thr Leu Arg His1 5 10 15Trp Leu Lys
Val 204920PRTArtificial SequenceSynthetic peptide 49Gln Pro Thr Pro
Thr Leu Gly Leu Asn Leu Gly Asn Asp Pro Asp Arg1 5 10 15Gly Thr Ser
Ile 205020PRTArtificial SequenceSynthetic peptide 50Asp Ser Asp Pro
Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln1 5 10 15Ile Thr Lys
Val 205119PRTArtificial SequenceSynthetic peptide 51Lys Lys Gly Thr
Tyr Leu Thr Asp Glu Thr His Arg Glu Val Lys Phe1 5 10 15Thr Ser
Leu5220PRTArtificial SequenceSynthetic peptide 52Gln Asp Phe Arg
Arg Ala Phe Arg Arg Ile Leu Ala Arg Pro Trp Thr1 5 10 15Gln Thr Ala
Trp 205320PRTArtificial SequenceSynthetic peptide 53Val Gly Thr Leu
Leu Leu Glu Arg Val Ile Phe Pro Ser Val Lys Ile1 5 10 15Ala Thr Leu
Val 205419PRTArtificial SequenceSynthetic peptide 54Lys Asp Ser Arg
Pro Ser Phe Val Gly Ser Ser Ser Gly His Thr Ser1 5 10 15Thr Thr
Leu5518PRTArtificial SequenceSynthetic peptide 55Thr Phe Ala Ala
Gly Phe Asn Ser Thr Gly Leu Pro His Ser Thr Thr1 5 10 15Arg
Val5620PRTArtificial SequenceSynthetic peptide 56Thr Arg Glu Asp
Ile Tyr Val Asn Tyr Pro Thr Phe Ser Arg Arg Pro1 5 10 15Lys Thr Arg
Val 205720PRTArtificial SequenceSynthetic peptide 57Pro Ser Trp Arg
Arg Ser Ser Leu Ser Glu Ser Glu Asn Ala Thr Ser1 5 10 15Leu Thr Thr
Phe 205820PRTArtificial SequenceSynthetic peptide 58Leu Ala Ser Lys
Ser Ala Glu Glu Gly Lys Gln Ile Pro Asp Ser Leu1 5 10 15Ser Thr Asp
Leu 205920PRTArtificial SequenceSynthetic peptide 59Pro Ile Pro Ala
Gly Gly Cys Thr Phe Ser Gly Ile Phe Pro Thr Leu1 5 10 15Thr Ser Pro
Leu 206020PRTArtificial SequenceSynthetic peptide 60Ala Thr Asp Tyr
Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His1 5 10 15Gln Phe Tyr
Ile 206120PRTArtificial SequenceSynthetic peptide 61Thr Gly Ser Ala
Leu Gln Ala Trp Arg His Thr Ser Arg Gln Ala Thr1 5 10 15Glu Ser Thr
Val 206220PRTArtificial SequenceSynthetic peptide 62Lys Glu Asn Asp
Tyr Glu Ser Ile Ser Asp Leu Gln Gln Gly Arg Asp1 5 10 15Ile Thr Arg
Leu 206319PRTArtificial SequenceSynthetic peptide 63Ser Ser Lys Ser
Lys Ser Ser Glu Glu Ser Gln Thr Phe Phe Gly Leu1 5 10 15Tyr Lys
Leu6420PRTArtificial SequenceSynthetic peptide 64Ala Val Gly Gly
Arg Pro Ala Arg Gly Gly Arg Leu Gln Gly Arg Arg1 5 10 15Gln Thr Gln
Val 206520PRTArtificial SequenceSynthetic peptide 65Ala Ala Gly Gly
Arg Ser Ala Arg Gly Gly Arg Leu Gln Gly Arg Arg1 5 10 15Glu Thr Ala
Leu 206620PRTArtificial SequenceSynthetic peptide 66Val Pro Gly Ala
Leu Asp Tyr Ala Ala Phe Ser Ser Ala Leu Tyr Gly1 5 10 15Glu Ser Asp
Leu 206720PRTArtificial SequenceSynthetic peptide 67Thr Gln Gly Phe
Pro Gly Pro Ala Thr Trp Arg Arg Ile Ser Ser Leu1 5 10 15Glu Ser Glu
Val 206820PRTArtificial SequenceSynthetic peptide 68Pro Pro Ala Thr
Pro Ser Pro Arg Leu Ala Leu Pro Ala His His Asn1 5 10 15Ala Thr Arg
Leu 206920PRTArtificial SequenceSynthetic peptide 69Gly Arg Trp Thr
Gly Arg Ala Met Ser Ala Trp Lys Pro Thr Arg Arg1 5 10 15Glu Thr Glu
Val 207020PRTArtificial SequenceSynthetic peptide 70Ala Gly Ala Val
Arg Thr Pro Leu Ser Gln Val Asn Lys Val Trp Asp1 5 10 15Gln Ser Ser
Val 207120PRTArtificial SequenceSynthetic peptide 71Pro Tyr Ser Glu
Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro1 5 10 15Asp Ser Trp
Val 207220PRTArtificial SequenceSynthetic peptide 72Pro Ile Pro Ala
Gly Gly Cys Thr Phe Ser Gly Ile Phe Pro Thr Leu1 5 10 15Thr Ser Pro
Leu 207320PRTArtificial SequenceSynthetic peptide 73Val His Asp Ala
Glu Ser Ser Asp Glu Asp Gly Tyr Asp Trp Gly Pro1 5 10 15Ala Thr Asp
Leu 207420PRTArtificial SequenceSynthetic peptide 74Leu Asn Ser Cys
Ser Asn Arg Arg Val Tyr Lys Lys Met Pro Ser Ile1 5 10 15Glu Ser Asp
Val 207520PRTArtificial SequenceSynthetic peptide 75Gln Ala Thr Ser
Arg Asn Gly His Ser Ala Arg Gln His Val Val Ala1 5 10 15Asp Thr Glu
Leu 207620PRTArtificial SequenceSynthetic peptide 76Lys Thr Met Pro
Ala Ala Met Tyr Arg Leu Leu Thr Ala Gln Glu Gln1 5 10 15Pro Val Tyr
Ile 207720PRTArtificial SequenceSynthetic peptide 77Asp Thr Leu Leu
Leu Thr Glu Asn Glu Gly Asp Lys Thr Glu Glu Gln1 5 10 15Val Ser Tyr
Val 207820PRTArtificial SequenceSynthetic peptide 78His His Leu Val
Ala Gln Arg Asp Ile Arg Gln Phe Gln Leu Gln His1 5 10 15Trp Leu Ala
Ile 207920PRTArtificial SequenceSynthetic peptide 79Gly Thr Ser Asp
Met Lys Asp Leu Val Gly Asn Ile Glu Gln Asn Glu1 5 10 15His Ser Val
Ile 208020PRTArtificial SequenceSynthetic peptide 80Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Lys Leu Ser Ser Ile1 5 10 15Glu Ser Asp
Val 208120PRTArtificial SequenceSynthetic peptide 81Leu Gln Phe His
Arg Gly Ser Arg Ala Gln Ser Phe Leu Gln Thr Glu1 5 10 15Thr Ser Val
Ile 208220PRTArtificial SequenceSynthetic peptide 82Gln Ile Ser Pro
Gly Gly Leu Glu Pro Pro Ser Glu Lys His Phe Arg1 5 10 15Glu Thr Glu
Val 208320PRTArtificial SequenceSynthetic peptide 83Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Pro Ser Arg Lys Leu1 5 10 15Asn Thr Glu
Ile 208419PRTArtificial SequenceSynthetic peptide 84Thr Thr Asn Asn
Asn Pro Asn Ser Ala Val Asn Ile Lys Lys Ile Phe1 5 10 15Thr Asp
Val8520PRTArtificial SequenceSynthetic peptide 85Lys Pro Gln Ile
Ala Ala Leu Lys Glu Glu Thr Glu Glu Glu Val Gln1 5 10 15Asp Thr Arg
Leu 208696PRTHomo sapiens 86Leu Glu Tyr Glu Glu Ile Thr Leu Glu Arg
Gly Asn Ser Gly Leu Gly1 5 10 15Phe Ser Ile Ala Gly Gly Thr Asp Asn
Pro His Ile Gly Asp Asp Pro 20 25 30Ser Ile Phe Ile Thr Lys Ile Ile
Pro Gly Gly Ala Ala Ala Gln Asp 35 40 45Gly Arg Leu Arg Val Asn Asp
Ser Ile Leu Phe Val Asn Glu Val Asp 50 55 60Val Arg Glu Val Thr His
Ser Ala Ala Val Glu Ala Leu Lys Glu Ala65 70 75 80Gly Ser Ile Val
Arg Leu Tyr Val Met Arg Arg Lys Pro Pro Ala Glu 85 90
9587107PRTHomo sapiens 87Gly Ile His Val Met Arg Arg Lys Pro Pro
Ala Glu Lys Val Met Glu1 5 10 15Ile Lys Leu Ile Lys Gly Pro Lys Gly
Leu Gly Phe Ser Ile Ala Gly 20 25 30Gly Val Gly Asn Gln His Ile Pro
Gly Asp Asn Ser Ile Tyr Val Thr 35 40 45Lys Ile Ile Glu Gly Gly Ala
Ala His Lys Asp Gly Arg Leu Gln Ile 50 55 60Gly Asp Lys Ile Leu Ala
Val Asn Ser Val Gly Leu Glu Asp Val Met65 70 75 80His Glu Asp Ala
Val Ala Ala Leu Lys Asn Thr Tyr Asp Val Val Tyr 85 90 95Leu Lys Val
Ala Lys Pro Ser Asn Ala Tyr Leu 100 10588120PRTHomo sapiens 88Pro
Val Ala Lys Asp Leu Leu Gly Glu Glu Asp Ile Pro Arg Glu Pro1 5 10
15Arg Arg Ile Val Ile His Arg Gly Ser Thr Gly Leu Gly Phe Asn Ile
20 25 30Val Gly Gly Glu Asp Gly Glu Gly Ile Phe Ile Ser Phe Ile Leu
Ala 35 40 45Gly Gly Pro Ala Asp Leu Ser Gly Glu Leu Arg Lys Gly Asp
Gln Ile 50 55 60Leu Ser Val Asn Gly Val Asp Leu Arg Asn Ala Ser His
Glu Gln Ala65 70 75 80Ala Ile Ala Leu Lys Asn Ala Gly Gln Thr Val
Thr Ile Ile Ala Gln 85 90 95Tyr Lys Pro Glu Glu Tyr Ser Arg Phe Glu
Ala Lys Ile His Asp Leu 100 105 110Arg Glu Gln Leu Met Asn Ser Ser
115 12089101PRTHomo sapiens 89Pro Ser Glu Leu Lys Gly Lys Phe Ile
His Thr Lys Leu Arg Lys Ser1 5 10 15Ser Arg Gly Phe Gly Phe Thr Val
Val Gly Gly Asp Glu Pro Asp Glu 20 25 30Phe Leu Gln Ile Lys Ser Leu
Val Leu Asp Gly Pro Ala Ala Leu Asp 35 40 45Gly Lys Met Glu Thr Gly
Asp Val Ile Val Ser Val Asn Asp Thr Cys 50 55 60Val Leu Gly His Thr
His Ala Gln Val Val Lys Ile Phe Gln Ser Ile65 70 75 80Pro Ile Gly
Ala Ser Val Asp Leu Glu Leu Cys Arg Gly Tyr Pro Leu 85 90 95Pro Phe
Asp Pro Asp 10090103PRTHomo sapiens 90Gln Arg Val Glu Ile His Lys
Leu Arg Gln Gly Glu Asn Leu Ile Leu1 5 10 15Gly Phe Ser Ile Gly Gly
Gly Ile Asp Gln Asp Pro Ser Gln Asn Pro 20 25 30Phe Ser Glu Asp Lys
Thr Asp Lys Gly Ile Tyr Val Thr Arg Val Ser 35 40 45Glu Gly Gly Pro
Ala Glu Ile Ala Gly Leu Gln Ile Gly Asp Lys Ile 50 55 60Met Gln Val
Asn Gly Trp Asp Met Thr Met Val Thr His Asp Gln Ala65 70 75 80Arg
Lys Arg Leu Thr Lys Arg Ser Glu Glu Val Val Arg Leu Leu Val 85 90
95Thr Arg Gln Ser Leu Gln Lys 1009120PRTHomo sapiens 91Leu Asn Ser
Ser Ser Asn Arg Arg Val Tyr Lys Lys Met Pro Ser Ile1 5 10 15Glu Ser
Asp Val 209222PRTHomo sapiens 92Arg Arg Arg Ser Gly Leu Asp Asp Ile
Asn Pro Thr Val Leu Leu Lys1 5 10 15Glu Arg Ser Thr Glu Leu
20939PRTHomo sapiens 93Ala Val Glu Arg Pro Ser Thr Glu Leu1
59411PRTHomo sapiens 94Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1
5 10
* * * * *
References