Note: Descriptions are shown in the official language in which they were submitted.
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MODULATORS OF TNF-ALPHA AND IL-1 CELL SURFACE RECEPTOR ACTIVITY
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. provisional patent application
no. 60/407,396, the
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and reagents for modulating
cell surface receptor
activity. The invention also relates to assays for identification of agents
that modulate receptor
activity and compositions containing such agents. The invention finds
application in the fields of
biology and medicine.
BACKGROUND OF THE INVENTION
[0003] Signaling molecules such as neurotransmitters, protein hormones,
cytokines and growth
factors bind to specific receptors on the surface of the taxget cells they
influence. These signaling
molecules (ligands) bind the cell surface receptors, resulting in one or more
intracellular signals that
alter the behavior of target cells.
[0004] In some cases, receptor signaling results in undesirable effects in the
subject. For
example, activation of receptors by proinflammatory cytokines such as tumor
necrosis factor-alpha
(TNF-alpha) and interleukin-1 (IL-1) play key roles in several disease
conditions. In such cases, it
may be desirable to antagonize the effects of these ligands.
[0005] Thus, methods for modulating the activity of receptors and for
identifying receptor-
modulating agents will be of significant medical benefit.
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention provides a method for modulating the
activity of the
interleukin 1 (IL-1) receptor. The IL receptor is referred to as "IL-1RI (IL-
1R-alpha)." In a related
aspect, the invention provides a method for modulating the activity of the TNF-
alpha receptor. The
TNF-alpha receptor is referred to as "TNF-RI (p55)". In an aspect, the
invention provides a method
for modulating the activity of IL-1RI (IL-1R-alpha) by contacting the receptor
with an exogenous
compound that binds in the activation domain, wherein the activation domain
comprises a sequence
identical to, or substantially similar to, SEQ ID N0:2. In an embodiment, the
IL-1RI (IL-1R-alpha)
is human. In an embodiment, receptor activity is decreased. In an embodiment,
the contacting takes
place in vitro. In an embodiment, the contacting occurs in the presence of IL-
1. In an embodiment,
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the exogenous compound is an oligopeptide. In an embodiment, the
oligopeptidehas at least about 8
contiguous residues of a sequence substantially similar to, or identical to,
SEQ ID N0:2.
[0007] In an aspect, the invention provides a method for screening candidate
agents for the
ability to modulate activity of a IL-1RI (IL-1R-alpha) by determining the
binding of the agent to a
receptor activation domain sequence having a sequence of SEQ ID N0:2
[0008] In a related aspect, the invention provides a method for screening
candidate' agents for the
ability to modulate activity of an IL-1RI (IL-1R-alpha) having an activation
domain comprising a
sequence substantially similar to SEQ ID N0:2 by a) contacting the receptor
with a candidate agent
and an activation-domain binding oligopeptide comprising a sequence
substantially similar to SEQ ID
N0:2; and b) determining the binding of the agent or the oligopeptide to the
activation domain,
wherein the binding of the agent to the activation domain or a reduction in
the binding of the
oligopeptide to the activation domain identifies a candidate agent for
modulating cell-surface receptor
activity. In an embodiment, the method includes the step of contacting the
receptor with receptor
ligand. In an embodiment, the candidate agent or oligopeptide is labeled.
[0009] In a related aspect, the invention provides a method for screening
candidate agents for the
ability to modulate activity of an IL-1RI (IL-1R-alpha) by identifying a
compound that binds in a
receptor activation domain of sequence SEQ ID N0:2, and assaying the ability
of the compound to
modulate activity of the cell surface receptor. In an embodiment, the ability
of the agent to reduce
ligand-induced receptor activation is assayed. In an embodiment, the ability
of the agent to increase,
or enhance, ligand-induced receptor activation is assayed.
[0010) In an aspect, the invention provides an isolated oligopeptide that
binds the IL-1RI (IL-
1R-alpha) activation domain. In an embodiment, the oligopeptide comprises at
least 8 amino acids of
SEQ ID N0:2. In an embodiment, the oligopeptide antagonizes IL-1 induced
activation of a IL-1 RI
(IL-1R-alpha). The invention also provides a pharmaceutical composition
comprising an
aforementioned oligopeptide in a sterile form and a pharmaceutically
acceptable excipient.
[0011] In an aspect, the invention provides a method for treating a condition
in a patient
characterized by an undesired level of IL-1 receptor activation by
administering to a patient an
exogenous bioactive compound that binds the IL-1 receptor activation domain
and which decreases
receptor activity when contacted with the receptor.
[0012] In an aspect, the invention provides a method for treating a condition
in a patient
characterized by a deficiency of IL-1 by administering to a patient an
exogenous bioactive compound
that binds in a receptor activation domain and which increases receptor
activity.
[0013] In an aspect, the invention provides a method for modulating the
activity of TNF-RI (p55)
by contacting the receptor with an exogenous compound that binds in the
activation domain, wherein
the activation domain comprises a sequence identical to, or substantially
similar to, SEQ ID NO:1. In
an embodiment, the TNF-RI (p55) is human. In an embodiment, receptor activity
is decreased. In an
embodiment, the contacting takes place in vitro. In an embodiment, the
contacting occurs in the
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presence of TNF-alpha. In an embodiment, the exogenous compound is an
oligopeptide, e.g., an
oligopeptide that comprises at least about 8 residues of a sequence
substantially similar to, or identical
to, SEQ ID NO:1.
[0014] In an aspect, the invention provides a method for screening candidate
agents for the
ability to modulate activity of a TNF-RI (p55) by determining the binding of
the agent to a receptor
activation domain sequence having a sequence of SEQ ID NO:1.
[0015] In a related aspect, the invention provides a method for screening
candidate agents for the
ability to modulate activity of a TNF-RI (p55) having an activation domain
comprising a sequence
substantially similar to SEQ ID NO:1 by a) contacting the receptor with a
candidate agent and an
activation-domain binding oligopeptide comprising a sequence substantially
similar to SEQ ID NO:1;
and b) determining the binding of the agent or the oligopeptide to the
activation domain, wherein the
binding of the agent to the activation domain or a reduction in the binding of
the oligopeptide to the
activation domain identifies a candidate agent for modulating cell-surface
receptor activity. In an
embodiment, the method includes the step of contacting the receptor with
receptor ligand. In an
embodiment, the candidate agent or oligopeptide is labeled.
[0016] In related aspect, the invention provides a method for screening
candidate agents for the
ability to modulate activity of a TNF-RI (p55) by identifying a compound that
binds in a receptor',
activation domain having the sequence SEQ ID NO:1 and assaying the ability of
the compound to
modulate activity of the cell surface receptor. In an embodiment, the ability
of the agent to reduce
ligand-induced receptor activation is assayed. In an embodiment, the ability
of the agent to increase,
or enhance, ligand-induced receptor activation is assayed.
(0017] In an aspect, the invention provides an isolated oligopeptide that
binds the TNF-RI (p55)
activation domain. In an embodiment, the oligopeptide comprises at least 8
amino acids of SEQ ID
NO:1. In an embodiment, the oligopeptide antagonizes TNF-alpha induced
activation of a TNF-RI
(p55).
[0018] In an aspect, the invention provides a pharmaceutical composition
comprising an
aforementioned oligopeptide in a sterile form and a pharmaceutically
acceptable excipient.
[0019] In an aspect, the invention provides a method for treating a condition
in a patient
characterized by an undesired level of TNF-RI (p55) activation by
administering to a patient an
exogenous bioactive compound that binds in the TNF-alpha receptor activation
domain and which
decreases receptor activity when contacted with the receptor.
[0020] In an aspect, the invention provides a method for treating a condition
in a patient
characterized by an undesired level of TNF-RI (p55) activation by
administering to a patient an
exogenous bioactive compound that binds in the TNF-RI (p55) activation domain
and which increases
receptor activity.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 is a digital image of a Western Blot showing the antagonistic
effect of a peptide
having the sequence of SEQ ID NO:1 (TNF-al peptide) on TNF-RI (p55) activity,
as measured by
the inhibition of TNF-a induced phosphorylation of p38 and ERK kinases in HT-
29 cells.
[0022] Figure 2 is a digital image of a Western Blot that showing the
antagonistic effect of TNF-
al peptide on TNF-RI (p55) activity, as measured by the inhibition of TNF-a
induced
phosphorylation of ERK kinase in mouse bone marrow macrophages.
[0023] Figure 3 is a digital image of a Western Blot that demonstrates the
antagonistic effect of a
peptide having the sequence of SEQ ID N0:2 (IL-1RI peptide) on IL-1RI (IL-1R-
alpha) activity, as
measured by dose-responsive inhibition of p38, TRAF6, and IRAK phosphorylation
in HepG2 cells.
[0024] Figure 4 shows the effect on TNF-a-induced IL-8 production in Normal
Human Dermal
Fibroblasts (NHDF) cells of a small molecule antagonist and agonist that
compete with TNRal
peptide for binding to the TNF receptor activation site.
[0025] Figure 5 shows the effect of small molecule compounds on TNFa-induced
TNF-RI
downstream substrate phosphorylation (p38 protein) in Normal Human Dermal
Fibroblasts
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In one aspect of the invention, methods and compositions for modulating
cell surface
receptor activity are provided, as well as methods for identification of
agents that modulate cell
surface receptor activity. As used herein, modulation can be an enhancement or
increase in receptor
activation (i.e., in the presence of an agent acting as a "receptor agonist")
or a decrease in ligand-
induced receptor activation (i.e., in the presence of an agent acting as a
"receptor antagonist"). The
modulatory agents, sometimes referred to herein as "exogenous bioactive
compounds" are compounds
that bind an extracellular portion of the cell surface receptor termed the
"activation domain."
[0027] Activation domains are described generally in U.S. patent no.
6,333,031, which is
incorporated herein by reference. The activation domain of a receptor occupies
a site distinct from the
ligand binding site. Thus, the exogenous bioactive compounds used in the
practice of the present
invention do not compete with the natural ligand for binding to the receptor.
Further, binding to the
activation domain of the receptor usually does not substantially change the KD
of the binding of the
natural receptor ligand to the ligand binding site.
[0028] Table 1, infra, provides the sequences of activation domains of
receptors for TNF-alpha
(TNF-RI (p55)) and IL-1 IL-1R-alpha. The activation domain sequences for these
receptors were
identified generally according to the methods described in U.S. pat. no.
6,333,031 and by cross-
species sequence comparisons between human receptors and non-human homologs.
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Table 1
Receptor Activation Domain SequenceSEQ
ID
Receptor & NO:
(Sequence of Exemplary Receptor
Derived Peptide)
GQDTDCRECESGSFTASENHLRHCL
1
TNF-RI (p55) (TNF-al peptide)
KDDSKTPVSTEQASRIHQHKEKLWF
IL-1RI (IL-1R-alpha)
(IL-1RI peptide)
[0029] The TNF-alpha) and IL-1) receptors are known, and the amino acid
sequences, activities
and other characteristics of the human receptors and homologs from other
animals are well known in
the art. Receptor sequences are published in databases, e.g. a human TNF-RI
(p55) is described as
Swissprot accession number P19438, and a human IL-1RI (IL-1R-alpha) is
described as PIR
accession number P14778. The receptors used in methods of the invention
generally are mammalian,
e.g., human, primates (including nonhuman primates), rodents (mice, rats,
hamsters, guinea pigs),
cows, sheep, pigs, horses, and others. Receptors that may be used include
those with the complete
sequence of a naturally occurring receptor (including naturally occurring
alleles and variants, e.g.,
naturally occurring mammalian or human alleles) as well as recombinantly
expressed variants, and
portions of receptors (e.g., a receptor extracellular domain, or an activation-
domain containing
fragment comprising at least about 50 or at least about 100 amino acid
residues). Suitable receptors
for use in the methods of the invention include isolated receptor proteins and
activation domain-
containing fragments (e.g., for use in binding assays) and receptors expressed
on the surface of cells
(e.g., often for activity assays). Suitable cells include those that normally
expressing a TNF-RI (p55)
and/or IL-1RI (IL-1R-alpha) (e.g., without limitation HT-29, HepG2 cells, and
NHDF (normal
human dermal fibroblasts; Clonetics Inc.) as well as cells in which the
receptors are recombinantly
expressed. It will be understood that cells that express endogenous receptor
can also be engineered to
recombinantly express (e.g., overexpress) the same or a different receptor.
Methods for recombinant
expression of polypeptides are well known in the art. See, e.g., (Sambrook and
Russet, 2001,
Molecular Cloning: A Laboratory Manual, third edition Cold Spring Harbor
Laboratory Press, Cold
Spring Harbor, NY. The polynucleotide sequences of TNF-RI (p55)and IL-1RI (IL-
1R-alpha) are
also'known (e.g., sequences for human receptors) or easily determinable. For
expression, typically,
polynucleotides encoding the receptor are used in expression vectors
containing typically include
transcriptional and/or translational control signals (e.g., transcriptional
regulatory element, promoter,
ribosome-binding site, and ATG initiation codon). DNA encoding the receptor or
receptor fragment
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 systems. Numerous examples of
mammalian cell culture
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systems are known (e.g., CHO cells, BaF3 cells). Useful human and nonhuman
cell lines are widely
available, e.g., from the American Type Culture Collection (ATCC), P.O. Box
1549, Manassas, VA
20108.
[0030] The TNF-RI (p55) and IL-1RI (IL-1R-alpha) act by transducing a signal.
Without
intending to be bound by a specific mechanism, it is believed that TNF-alpha
binding to the
extracellular domain of TNF-RI (p55) leads to homotrimerization. Once
aggregated, the~receptors
form a complex with other proteins (e.g., TRADD and TRADD-interacting
proteins) to form a
complex that activates at least two distinct signaling cascades, apoptosis and
NF-kappa B signaling.
Without intending to be bound by a specific mechanism, it is believed IL-1
signal transduction
pathway is initiated by the binding of IL-1 to (IL-1R-alpha), which then forms
a complex with IL-
lRAcP, resulting in the recruitment of MyD88 and IRAK. IRAK then dissociates
from the receptor
complex and interacts with TRAF6, which initiates kinase cascade leading to
the activation of JNK
and NF-kappaB.
[0031] In one aspect, the invention provides a method for modulating the
activity of a TNF-RI
(p55) or IL-1 cell surface receptor by contacting the receptor with an
exogenous bioactive compound
that binds in the activation domain of the receptor, where the activation
domain has a sequence
identical or substantially similar to SEQ ID.NO:l and 2, and where the binding
effects a response in
the signaling pathway of said receptor, i.e., modulates receptor activity. As
noted, modulation of
receptor activity can include an increase in receptor activation in the
presence or absence of the
natural ligand of the receptor (referred to as agonist activity). Conversely,
modulation of receptor
activity can include inhibition of receptor activation, or antagonist
activity. Thus, by "antagonist"
herein it is meant compounds that bind the receptor activation site but do not
activate the receptor, and
for which ligand-induced receptor activity is inhibited or blocked. The level
of inhibition of ligand-
induced receptor activity will vary depending on the concentrations of ligand,
receptor and exogenous
agent, but are often at least about a 5% decrease in receptor activity (e.g.,
as measured as shown in the
Examples, often at least about 25%, and frequently at least about 50% or more.
It has been
discovered, for example, that binding to the activation sequence of the TNF-RI
(p55) by a peptide
with the sequence SEQ ID NO:1 reduces TNF-RI (p55) activation (e.g., TNFa
induced activation).
Similarly, binding to the activation sequence of the IL-1RI (IL-1R-alpha) by a
peptide with SEQ ID
N0:2 reduces IL-1RI (IL-1R-alpha) activation (e.g., IL-1 induced activation).
By "binding in the
activation domain" is meant that the exogenous compound binds the receptor
through interaction with
some, but not necessarily all, amino acid residues in the activation domain.
[0032] Also shown in Table 1 are exemplary receptor derived peptide sequences
(exemplary
RDPSs). As is discussed below, the exemplary RDPSs, their homologs, fragments,
and variants, are
used to modulate activity of corresponding receptors and to identify still
other modulatory agents.
The RDPSs shown in Table 1 are "TNF-al peptide" having the sequence set forth
as SEQ ID NO:l,
and "IL-1R peptide " having the sequence set forth as SEQ ID N0:2.
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[0033] For clarity, each receptor activation sequence and each exemplary
receptor derived
peptide sequence (RDPS) listed in Table 1 is referred to as "corresponding
to," or being the "cognate"
of, the receptor from which its sequence is derived. In Table 1, cognate pairs
of receptors and
activation/peptide sequences presented in the same row. Thus, for example, (i)
the TNF-RI (p55)
activation sequence (SEQ ID NO:1) and (ii) the TNF-RI (p55) are a cognate
pair. Similarly, (i) a
receptor derived peptide that modulates the activity of the TNF-alpha receptor
and which has
sequence similarity to SEQ ID NO:1, and (ii) the TNF-alpha receptor are a
cognate pair.
Exogenous Bioactive Compounds
[0034] An noted above, receptor modulatory agents are sometimes referred to
herein as
"exogenous bioactive compounds." Specifically, "exogenous bioactive compound"
as used herein,
refers to a compound that (1) is not produced endogenously by the cell or
organism, i.e., it is
artificially introduced to the cell or organism; (2) binds amino acid residues
in the activation site of a
receptor (e.g., as determined by competition assays); and (3) modulates
receptor activity. Exogenous
bioactive compounds include oligopeptides described in detail herein, as well
as other compounds
described herein. Further, exemplary assays for identifying bioactive
compounds are described
hereinbelow. In an aspect, the invention provides a complex comprising a
receptor activation domain
and an exogenous bioactive compound (e.g., oliogpeptide) described herein.
[0035] A variety of different classes of molecules can act as exogenous
bioactive compounds,
including oligopeptides, other biomolecules (e.g., saccharides, fatty acids,
steroids, purines,
pyrimidines, derivatives, structural analogs) and small organic compounds
(e.g., having a molecular
weight of more that about 50 and less than about 10,000 daltons, often more
than about 100 and less
than about 2,500 daltons; most often between about 200 and about 600 daltons).
Usually candidate
agents comprise functional groups necessary for structural interaction with
proteins, particularly
hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl
or carboxyl group, and
often at least two of the functional chemical groups. The candidate agents
often comprise cyclical
carbon or heterocyclic structures and/or aromatic or polyaromatic structures
substituted with one or
more of the above functional groups. In one embodiment, the exogenous
bioactive compound is other
than a protein or peptide.
[0036] In one embodiment, the exogenous bioactive compound is an oligopeptide.
As used
herein, "oligopeptide" is used interchangeably with "peptide" and
"polypeptide" and refers to a
polymer of amino acids. In an embodiment, the oligopeptide comprises a
sequence identical or
substantially similar to SEQ ID NO:1 or SEQ ID N0:2, or to an activation
domain binding ,
subsequence thereof. For example, the oligopeptide compound that binds an
activation domain and
modulates receptor activity may comprises at least 8 contiguous residues of a
sequence in Table 1,
and often at least 10, at least 12, at least 15 or at least 20 residues.
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[0037] As used herein, the term "substantially similar" refers to oligopeptide
sequences that may
be identical to the receptor activation domain (e.g., SEQ ID NO:1 and 2) or
that may have a degree of
similarity to the receptor activation domain sufficient to allow binding of
the oligopeptide to the
receptor activation domain resulting in modulation of receptor activity. In
some embodiments, the
length of the peptide is at least 8 amino acids, usually at least about 12
amino acids, and more usually
at least about 18 amino acids. In some embodiments, the length of the peptide
is fewer than about 60
amino acids, more usually fewer than about 40 amino acids, more usually fewer
than 30 amino acids.
[0038] An oligopeptide that is substantially similar to a receptor activation
domain and which
modulates activity of a receptor may differ from the receptor activation
domain by amino acid
substitutions, insertions, or deletions as compared to the activation domain.
For example, the
oligopeptide substantially similar to SEQ. ID. NO:1 or 2 may include
additional residues, e.g., at the
5' or 3' terminus of SEQ. ID. NO:l-2 or an activation domain-binding
subsequence thereof. In
embodiments, the peptide will contain least 8 amino acids, at least about 12
amino acids, at least
about 15 amino acids, at least about 18 amino, at least about 21 amino acids,
or at least about 24
amino acids identical to a sequence of SEQ ID NO:1-2 and additional residues
that may not be
identical to SEQ ID NO:1 or 2. In some embodiments, a substantially similar
oligopeptide will have
an amino acid sequence at least about 60% identical to one of SEQ ID NO:1-2,
at least about 70%
identical, often at least about 80% identical, sometimes at least about 90%
identical. Sequence
identity between two peptide sequences can be easily determined by inspection.
Alternatively,
algorithms such as the Best Fit sequence program described by Devereux et
a1,1984, Nucl. Acid Res.
12:387-95, with default settings preferred.
[0039] As noted, peptides suitable for use as exogenous bioactive compounds of
the invention
may have amino acid substitutions, insertions, or deletions as compared to
sequences of SEQ ID
NO:1-2. In one embodiment, amino acid substitutions are made. In one
embodiment the number of
changes will not be more than about 30%, sometimes not more than about 20%,
sometimes not more
than about 10 %, of the number of amino acids in the activation domain,
although in some instances
higher numbers of alterations may be made. In one embodiment, with reference
to the RDPSs shown
in Table 1, not more than about five, alternatively not more than about three
substitutions or deletions
will be made. In general, it is preferable that residues critical for
biological activity are either not
altered or are conservatively altered (e.g. to conserve charge). Examples of
conservative alterations
include the substitutions shown in Table 2. Critical residues may be
elucidated using known
mutagenesis techniques followed by activity or binding assays, e.g., using
scanning mutagenesis
techniques, wherein single amino acid residues within the oligopeptide are
modified by substitution
with an aliphatic amino acid, e.g., serine, alanine, glycine, valine, and the
like.
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Table 2
Original Residue Exemplary Substitutions
Ala Ser
Arg Lys
Asn Gln, His
Asp Glu . .
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His Asn, Gln
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe Met, Leu, Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp, Phe
Val Ile, Leu
[0040] Oligopeptide exogenous bioactive compounds can contain naturally
occurring and
synthetic amino acids, as well as amino acid analogs and amino acid mimetics
that function in a
manner similar to the naturally occurring amino acids. Naturally occurring
amino acids are those
encoded by the genetic code, as well as those amino acids that are later
modified, e.g., hydroxyproline
and O-phosphoserine. Amino acid analogs refers to compounds that have the same
basic chemical
structure as a naturally occurring amino acid, i.e., an alpha-carbon that is
bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide,
methionine methyl sulfonium. Such analogs have modified R groups (e.g.,
norleucine) or modified
peptide backbones, but retain the same basic chemical structure as a naturally
occurring amino acid.
Amino acid mimetics refers to chemical compounds that have a structure that is
different from the
general chemical structure of an amino acid, but that functions in a manner
similar to a naturally
occurring amino acid.
[0041] In one embodiment modifications that are made do not substantially
alter the biological
activity of the oligopeptide, i.e., the modification does not prevent binding
of the oligopeptide to its
cognate receptor and does not destroy the modulatory activity.
[0042] Alternatively, variants in which biological function has been modified
can be selected for.
The substitutions which in general are expected to produce the greatest
changes in oligopeptide
properties are those in which a nonconservative substitution is made in a
critical residue, e.g., (a) a
hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a
hydrophobic residue, e.g.,
leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any
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other residue; (c) a residue having an electropositive side chain, e.g.,
lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g., glutamyl or
aspartyl; or (d) a residue having a
bulky side chain, e.g., phenylalanine, is substituted for (or by) one not
having a side chain, e.g.,
glycine.
[0043] Oligopeptides can be made by chemical synthesis, through recombinant
means, or any
other synthesis method. Usually, the oligopeptides are prepared in accordance
with conventional
techniques, such as synthesis (for example, use of a Beckman Model 990 peptide
synthesizer or other
commercial synthesizer). Peptides may be produced directly by recombinant
methods (see Sambrook
et al. Molecular Cloning: A Laboratory Manual, CSHL Press, Cold Spring Harbor,
N.Y.,1989) or as a
fusion protein, for example, to a protein that is one of a specific binding
pair, allowing purification of
the fusion protein by means of affinity reagents, followed by proteolytic
cleavage, usually at a site
engineered to yield the desired peptide (see for example Driscoll et al.
(1993) J. Mol. Bio. 232:342-
350).
[0044] In addition to modifications within the peptides, they may also contain
additional
sequences. For example, the oligopeptides may be extended to: 1) provide
convenient linking sites,
e.g., cysteine or lysine; 2) to enhance stability; 3) to provide for ease of
purification, e.g., epitope or
purification (Hisb) tags; 4) to alter the physical characteristics, e.g.,
solubility, charge, etc.; or 5) to
stabilize the conformation. The oligopeptides may be joined to non-wild-type
flanking regions as
fused proteins, joined either by linking groups or covalently linked through
cysteine (disulfide) or
peptide linkages. The oligopeptide may be linked through a variety of
bifunctional agents, such as
maleimidobenzoic acid, methyidithioacetic acid, mercaptobenzoic acid, S-
pyridyl dithiopropionate,
and the like. The oligopeptides may be joined to a single amino acid at the N-
or C-terminus of a
chain of amino acids, or may be internally joined. For example, the subject
peptides may be
covalently linked to an immunogenic protein, such as keyhole limpet
hemocyanin, ovalbumin, and the
like, to facilitate antibody production to the subject oligopeptides.
[0045] As noted, the oligopeptides may be shorter than those depicted in Table
1, i.e., residues '
from either the N- or C-terminus of the oligopeptide may be deleted with the
retention of biological
activity, preferably full biological activity. In some cases, internal
residues may be removed from the
oligopeptide. Generally, this will be done by sequentially removing residues
and assaying for the
ability to bind to the activation domain of a receptor. Once binding has been
established, activation
may be evaluated.
[0046] Alternatively, the subject oligopeptides may be expressed in
conjunction with other
peptides or proteins, so as to be a portion of the chain, either internal, or
at the N- or C-terminus.
Various post-expression modifications may be achieved. For example, by
employing the appropriate
coding sequences, one may provide farnesylation or prenylation, such that the
subject peptide will be
bound to a lipid group at one terminus, and will be able to be inserted into a
lipid membrane, such as a
liposome.
CA 02496634 2005-02-24
WO 2004/020608 PCT/US2003/027381
[0047] The subject oligopeptides may also be modified by the addition of
chemical moieties or
groups. For example, the oligopeptides may be PEGylated, where the
polyethyleneoxy group
provides for enhanced lifetime in the blood stream. The subject oligopeptides
may also be combined
with other proteins, such as the Fc of an IgG isotype to enhance complement
binding, or with a toxin,
such as ricin, abrin, diphtheria toxin, or the like, particularly the A chain.
The oligopeptides may be
linked to antibodies for site directed action. For conjugation techniques,
see, e.g., U.S. Pat. Nos.
3,817,837; 3,853,914; 3,850,752; 3,905,654; 4,156,081; 4,069,105; and
4,043,989, which are
incorporated herein by reference. As outlined herein, the oligopeptides may be
labeled as well.
[0048] Oligopeptides of the invention can be modified to increase stability,
enhance
pharmacological properties (half life, absorption, potency, efficacy) and the
like. Exogenous
bioactive compounds also include nonpeptide compounds (peptide analogs)
structurally similar to the
RDPSs shown in Table 1, sometimes referred to known as "peptide mimetics" or
"peptidomimetics"
(Fauchere,1986, Adv. Drug Res. 15: 29; Veber and Freidinger,1985, TINS p.392;
and Evans et al.,
1987, J. Med. Chem 30: 1229). For example, useful peptidomimetics may be
structurally similar to a
oligopeptide, but have one or more peptide linkages optionally replaced by a
linkage selected from the
group consisting of --CH2NH--, --CH2S--, --CH2 --CH2 --, --CH=CH--(cis and
trans), --COCH2
--, --CH(OH)CH2 --, and --CH2 SO--, by methods known in the-art and further
described in the
following references: Spatola, A. F. in "Chemistry and Biochemistry of Amino
Acids, Peptides, and
Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);
Spatola, A. F., Vega Data
(March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general
review); Morley, J. S.,
Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., Int J
Pept Prot Res (1979)
14:177-185 (--CH2 NH--, CH2 CH2 --); Spatola, A. F. et al., Life Sci (1986)
38:1243-1249 (--CH2 --
S); Hann, M. M., J Chem Soc Perkin Trans I (1982) 307-314 (--CH--CH--, cis and
trans); Alinquist~
R. G. et al., J Med Chem (1980) 23:1392-1398 (-OCH2 --); Jennings-White, C. et
al., Tetrahedron
Lett (1982). 23:2533 (--OCH2 --); Szelke, M. et al., European Appin. EP 45665
(1982) CA: 97:39405
(1982) (--CH(OH)CH2 --); Holladay, M. W. et al., Tetrahedron Lett (1983)
24:4401-4404 (--
C(OH)CH2 --); and Hruby, V. J., Life Sci (1982) 31:189-199 (--CH2 --S--); each
of which is
incorporated herein by reference. One useful non-peptide linkage is --CH2 NH--
. Such peptide
mimetics may have significant advantages over polypeptide embodiments,
including, for example:
more economical production, greater chemical stability, enhanced
pharmacological properties (half
life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-
spectrum of biological
activities), reduced antigenicity, and others.
[0049] In one aspect, the invention provides a complex of an IL-1 receptor
activation domain.
and an exogenous agent, where, when bound to the activation domain of the IL-1
receptor on
an IL-1 receptor-expressing cell, the exogenous agent modulates activation of
the IL-1
receptor (and where the agent is not an MHC protein or protein comprising the
sequence of
11
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WO 2004/020608 PCT/US2003/027381
an MHC protein, or another molecule of the IL-1 receptor). In an embodiment
the agent is
not a oligopeptide or polypeptide. In an embodiment, the agent is a small
molecule with a
molecular weight of between about 100 and about 1000 daltons. In one
embodiment, the
complex is of an IL-1 receptor activation domain on an IL-1 receptor-
expressing cell and the
exogenous agent.
[0050] In one aspect, the invention provides a complex of a TNFa receptor
activation
domain and an exogenous agent, where, when bound to the activation domain of
the TNFa
receptor on an TNFa receptor-expressing cell, the exogenous agent modulates
activation of
theTNFa receptor (and where the agent is not an MHC protein or protein
comprising the
sequence of an MHC protein, or another molecule of the TNFa receptor). In an
embodiment
the agent is not a oligopeptide or polypeptide. In an embodiment, the agent is
a small,
molecule with a molecular weight of between about 100 and about 1000 daltons.
In one
embodiment, the complex is of an TNFa receptor activation domain on an TNFa
receptor-
expressing cell and the exogenous agent.
Binding arad Activity Assays
[0051] Identification of receptor activation domain sequences, as well as
determination of the
effect of binding of agents to activation domains (see Examples), permits the
design of screening
assays for agents that bind the activation domain sequences and modulate
receptor activity
(exogenous bioactive compounds). Initial screening or validation may be
carried out using binding
assays, with subsequent determination of the effect of the agent on receptor
activity. Alternatively,
assays that determine the effect of the agent on receptor activity can be
carned out without antecedent
binding assays.
[0052] It will be appreciated that assays for activation domain binding and
modulatory activity
are useful in at least two different, but related, contexts. In one context,
screening assays are carried
out in which a plurality of assay mixtures are run in parallel with different
candidate agents (e.g., high
throughput screening assays). Usually candidate agents are assayed at
different concentrations to
obtain a differential response to the various concentrations. Such assays can
be used to identify new
exogenous bioactive compounds. Candidate agents are obtained from a wide
variety of sources
including libraries of synthetic or natural compounds. For example, numerous
means are available for
random and directed synthesis of a wide variety of organic compounds and
biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries of natural
compounds in the form
of bacterial, fungal, plant and animal extracts are available or readily
produced. Additionally, natural
or synthetically produced libraries and compounds are readily modified through
conventional
chemical, physical and biochemical means. Known pharmacological agents may be
subjected to
12
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directed or random chemical modifications, such as acylation, alkylation,
esterification, amidification
to produce structural analogs.
[0053] In a second, related, context, the assays can be used to assess
activity of particular
variants of compounds known to bind a receptor activation sequence and
modulate receptor activity,
such as the receptor derived peptides described in the Examples, infra. For
example, the effect of
amino acid substitutions, insertions, or deletions in SEQ ID NO:1 on the
antagonistic activity of the
peptide on TNF-RI (p55) activity can be readily assessed. Variants for
example, may be selected to
develop exogenous bioactive compounds with enhanced half life or other
desirable properties. Useful
assays will be apparent to those of skill in the art based on the instant
disclosure, and include assays
described below as well as those described in U.S. Pat. No. 6,333,031.
Binding Assays
[0054] Agents capable of modulating surface receptor activity can be
identified by first screening
for the ability to bind an activation sequence of a receptor listed in Table
1. Some embodiments of
the various assays described herein utilize human cell surface receptors,
although other mammalian
receptors may also be used, including receptors from rodents (mice, rats,
hamsters, guinea pigs, etc.),
farm animals (cows, sheep, pigs, horses, etc.) and primates. Included within
the definition of cell
surface receptors are proteins having amino acid substitutions, insertions, or
deletions of the naturally
occurring sequence. Furthermore, included within the definition of cell
surface receptors are proteins
having portions of cell surface receptors; that is, either the full-length
receptor may be used, or
functional portions thereof. Thus, in one embodiment, binding to a candidate
agent to an oligopeptide
having a sequence identical to or substantially similar to SEQ ID NO:1 or 2,
or a receptor binding
fragment or variant thereof, is determined to identify compounds that bind the
activation domain and
potentially modulate receptor activity.
[0055] In one variation, the assay comprises combining an activation domain of
a TNF-RI (p55)
or IL-1RI (IL-1R-alpha) cell surface receptor, and a candidate bioactive
agent, and determining the
binding of the candidate agent to the activation domain. A wide variety of
assays may be used for this
purpose, including labeled in vitro protein-protein binding assays,
electrophoretic mobility shift
assays, immunoassays for protein binding, functional assays (e.g.,
phosphorylation assays), and the
like. In one variation, the candidate bioactive agent is labeled, and binding
determined directly. In
one approach, all or a portion of the cell-surface receptor is attached to a
solid support, a labeled
candidate agent (for example a fluorescent label) is added, excess and unbound
reagent is removed,
and the presence of the label is present on the solid support is determined.
Various blocking and
washing steps may be utilized as is known in the art. Alternatively, the
candidate agent can be
immobilized.
[0056] Another way to assess binding of an agent to an activation domain uses
competitive
binding assays to detecting competition between (i) the agent and (ii) a
competitor moiety that binds
13
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WO 2004/020608 PCT/US2003/027381
the receptor activation domain, for binding to a TNF-RI (p55) or IL-1RI (IL-1R-
alpha) activation
domain. Thus, in one variation, the method comprises combining a polypeptide
comprising a cell
surface receptor activation domain as listed in Table 1, a candidate bioactive
agent, and a competitor
moiety, and determining the binding of the candidate agent to the activation
domain. For example, in
one assay the ability of the agent to interfere with the binding of an
oligopeptide having a sequence
shown in Table 1 and its cognate receptor is determined. Exemplary competitor
moieties that bind a
receptor activation domain for use in competition assays include oligopeptides
having a sequence of
SEQ ID NO:1 or 2, or a receptor binding fragment or variant thereof (e.g., an
oligopeptide having a
sequence identical or substantially similar to one of SEQ ID NO:1 or 2). For
use in the assay,
examples of polypeptides that comprise a cell surface receptor activation
domain as listed in Table 1,
as described above, include an oligopeptide having a sequence identical or
substantially similar to one
of SEQ ID NO:1 or 2, a full-length receptor comprising an activation domain as
listed in Table 1
(either isolated or expressed by a cell), and a fragment of the receptor a
portion of the extracellular
portion of the receptor.
[0057] In one variation of the aforementioned assays, the candidate bioactive
agent is labeled.
Either the candidate bioactive agent, or the competitor moiety (e.g.,
oligopeptide) is added first to the
receptor for a time sufficient to allow binding. Incubations may be performed
at any temperature
which facilitates optimal activity, typically between 4°C-40°C.
Incubation periods are selected for
optimum activity, but may also be optimized to facilitate rapid high
throughput screening. Typically
between 0.1 and 2 hours will be sufficient. Excess reagent is generally
removed or washed away.
The second component is then added, and the presence or absence of the labeled
component is
followed, to indicate binding.
[0058] In another variation, the competitor moiety (e.g., oligopeptide) and
the candidate
bioactive agent are added together. Usually the candidate bioactive agent is
added first, and usually in
excess. Non-binding of the competitor moiety is an indication that the
candidate bioactive agent is
binding to the activation domain and thus is capable of modulating receptor
activity. Either
component can be labeled.
[0059] In an alternative variation, the candidate bioactive agent is added
first, with incubation
and washing, followed by the competitor moiety (e.g., oligopeptide). The
absence of binding by the
competitor moiety may indicate that the bioactive agent is bound to the
receptor with a higher affinity.
Thus, if the candidate bioactive agent is labeled, the presence of the label
on the support, coupled with
a lack of competitor moiety binding, may indicate that the candidate agent is
capable of binding to the
activation domain and modulating receptor activity.
[0060] In another variation, the methods comprise combining a cell surface
receptor and a
competitor moiety (e.g., oligopeptide) as described herein, to form a test
mixture. The candidate
bioactive agent is added to the test mixture, and the binding of the candidate
bioactive agent to the
activation domain of the receptor is determined. In this embodiment, either or
both of the competitor
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WO 2004/020608 PCT/US2003/027381
moiety or the candidate bioactive agent is labeled, with preferred variations
utilizing labeled
oligopeptides, such that displacement of the label indicates binding by the
candidate bioactive agent.
[0061] In another variation, the methods comprise differential screening to
identity bioactive
agents that bind a receptor activation domain. In this embodiment, the methods
comprise combining a
cell surface receptor and a competitor moiety (e.g., oligopeptide) that binds
the activation sequence in
a first sample. A second sample comprises a candidate bioactive agent, a cell
surface receptor and a
competitor moiety. The binding of the competitor moiety is determined for both
samples, and a
change, or difference in binding between the two samples indicates the
presence of an agent capable
of binding the activation domain. That is, if the binding of the competitor
moiety is different in the
second sample relative to the first sample, the agent is capable of binding
the activation domain.
(0062] A variety of assay formats will be apparent to those of skill. In one
variation of the
methods herein, the purified cell surface receptor or candidate agent is non-
diffusably bound to an
insoluble support having isolated sample receiving areas (e.g. a microtiter
plate, an array, etc.). The
insoluble supports may be made of any composition to which peptide or receptor
can be bound, is
readily separated from soluble material, and is otherwise compatible with the
overall method of
screening. The surface of such supports may be solid or porous and of any
convenient shape.
Examples of suitable insoluble supports include microtiter plates, arrays,
membranes and beads.
These are typically made of glass, plastic (e.g., polystyrene),
polysaccharides, nylon, or nitrocellulose,
TeflonTM, and the like. Microtiter plates and arrays are especially convenient
because a large number
of assays can be carried out simultaneously, using small amounts of reagents
and samples. The
particular manner of binding of the peptide or other protein is not crucial so
long as it is compatible
with the reagents and overall methods of the invention, maintains the activity
of the peptide and is
nondiffusable. Preferred methods of binding include the use of antibodies
(which do not sterically
block either the ligand binding site or activation sequence when the receptor
is bound to the support),
direct binding to "sticky" or ionic supports, chemical crosslinking, and the
synthesis of the receptor on
the support surface. Following binding of the peptide or receptor, excess
unbound material is
removed by washing. The sample receiving areas may then be blocked through
incubation with
bovine serum albumin (BSA), casein or other innocuous protein. In another
variation, cell lines that
overexpress the cell surface receptor are used to screen for candidate
bioactive agents.
(0063] In another variation a homogeneous LANCE assay format, or similar
solution phase
assay, is used to identify bioactive molecules. In the LANCE type assay, all
the reagents are in a
solution (in contrast to solid or semi-solid plate assay). Briefly, a receptor
of interest (e.g., TNF-RI
(p55) or IL-1RI (IL-1R-alpha)) is incubated with biotinylated peptide that
specifically binds the
modulation domain of the receptor under conditions in which a complex between
receptor and the
peptide is formed. A Europium-labeled anti-receptor antibody is allowed to
bind the complex and
streptavidin-APC conjugate is allowed to bind to biotinylated peptide. The
formation of the complex
is detected by signal emission which occurs as a result of close proximity
between Streptavidin-APC
CA 02496634 2005-02-24
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conjugate (that binds to biotinylated peptide) and Eu+-labeled anti- receptor
antibody. Thus, if the
complex between receptor and peptide is formed, strong signal emission occurs.
Lack of signal
indicates that anti-receptor antibody and streptavidin conjugate are not in a
close proximity, generally
because the receptor/peptide complex has not been formed. When used as a
screening assay, a
competition assay between the biotinylated peptide and candidate bioactive
molecules (e.g., from a
library of compounds) is usually carried out by adding the candidate bioactive
molecule'before the
formation of the peptide-receptor complex. An absence or diminution of signal
indicates competition
for the modulation site between the biotinyated peptide and the candidate
bioactive molecule.
[0064] "Labeled," as used herein in the context of binding and activity assays
refers to a
compound is either directly or indirectly labeled with a compound that
provides a detectable signal,
e.g., radioisotope, fluorescers, enzyme, antibodies, particles such as
magnetic particles,
chemiluminescers, or specific binding molecules. Specific binding molecules
include pairs, such as
biotin and streptavidin, digoxin and antidigoxin, and the like. For the
specific binding members, the
complementary member would normally be labeled with a molecule which provides
for detection, in
accordance with known procedures, as outlined above. The label can directly or
indirectly provide a
detectable signal. In some variations, only one of the components is labeled.
For example, the
oligopeptides may be labeled at tyrosine positions using 125 I, or with
fluorophores. Alternatively,
more than one component may be labeled with different labels; using'ZSI for
the oligopeptides, for
example, and a fluorophor for the candidate agents.
[0065] A variety of other reagents may be included in the screening assays.
These include
reagents like salts, neutral proteins, e.g., albumin and detergents, which may
be used to facilitate
optimal protein-protein binding and/or reduce non-specific or background
interactions. Also reagents
that otherwise improve the efficiency of the assay, such as protease
inhibitors, nuclease inhibitors, and
anti-microbial agents, may be used. The mixture of components may be added in
any order that
provides for the binding. An oligopeptide that binds a TNF-RI (p55) IL-1RI or
(IL-1R-alpha)
activation-domain can be referred to as an "activation-domain binding
oligopeptide: ' Examples of
such oligopeptides include oligopeptides having the sequence of SEQ ID NO:1 or
2 and others
identified using the binding assays described herein.
Receptor Activity Assays
[0066] Methods for detecting modulation of activity of a receptor listed in
Table 1 are known in
the art. As used herein, "receptor activity" has its usual meaning in the art
and refers to the biological
function associated with binding of natural ligand of the cell surface
receptor. Modulation of receptor
activity can refer to an increase in the activity (in the presence or absence
of the natural ligand) or a
decrease in natural-ligand induced activity (generally measured in the
presence of activating amounts
of natural ligand or ligand analogs that bind the ligand binding site, such as
homologs from other
species, variants, and the like).
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[0067] As used herein, the terms "natural ligand," "naturally-occurring
ligand," and "endogenous
ligand" are used interchangeably, and refer to ligand that is natively
produced by the organism
expressing the subject receptor, and which binds the ligand binding site of
the receptor. For example,
tumor necrosis factor alpha is the natural ligand for the TNF-RI (p55) and IL-
1 is the natural ligand
for the IL-1RI (IL-1R-alpha). Natural ligands are examples of "activating
ligands." Other activating
ligands are compounds that compete with the natural ligand for binding to the
receptor ligand binding
site, and which when bound by the receptor cause receptor activation.
[0068] As will be appreciated by those in the art, the specific biological
function will vary
depending on the identity of the receptor. Generally ligand binding results in
a conformational
change in the receptor. Thus, in some embodiments, activation of a receptor is
detectable as a
conformational change either within the receptor, or as a result of monomeric
receptors becoming
multimeric, which allows the receptor to facilitate signaling. In some cases,
the conformational
change results in receptor phosphorylation, receptor association with another
cell biomolecule (e.g.,
protein), and/or phosphorylation of another cell protein. Methods for
detecting changes in receptor
conformation, receptor-biomolecule association, receptor phosphorylation or
phosphorylation of other
biomolecules in the receptor signaling pathway are well known. Illustrative
assays are described in
the Examples, infra. Receptor activation can also be detected as a downstream
effect mediated by
ligand binding to the receptor such as changes in concentration of
intracellular biomolecules (e.g.,
cAMP, see Example 2, infra), metabolic changes (see, e.g., Example 1, infra),
cell shape changes,
stimulation or inhibition of cell proliferation, activation of substrates in
down-stream signaling
pathways, and a variety of other measures of receptor activation. Suitable
activation assays can be
carried out in vitro (e.g., in immortalized cells lines or primary cell
cultures) or in vivo (e.g., in non-
human animal models, or human subjects). Another assay for receptor activity
is measurement of
TNF-alpha or IL-1 induced production of IL-8 or IL-6 (see, Bocker et al.,
2000, J. Biol. Chem
275:12207-13. See also Suzuki et al., 2000, FEBS Letters 465:23-27). An
examplary protocol for
measuring receptor activity using this method is provided in Example 3, infra,
although many
variations will be evident to one of ordinary skill.
Applications
[0069] The exogenous bioactive compounds, e.g., oligopeptides, of the present
invention are
used in methods of modulating receptor activity. Such modulation finds use in
screening assays,
studies of the mechanism of action of receptor ligands, therapeutic uses, and
other uses that will be
apparent to one of ordinary skill in the art.
[0070] In an aspect the invention provides a method for modulating activity of
a cell surface
receptor by contacting a mammalian cell surface receptor listed in Table 1 and
a compound that binds
the activation sequence of the receptor (set forth in Table 1 for the human
homology. In one respect,
the exogenous compounds may act as receptor antagonists, as seen with, e.g.,
TNF-al peptide and IL-
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1RI peptide (examples of agents that bind the receptor activation sequences of
TNF-RI (p55) and IL-
1RI (IL-IR-alpha)). Thus, for example, in an aspect the invention provides a
method for
antagonizing activity a cell surface receptor by contacting a mammalian cell
surface receptor and an
exogenous compound where the receptor and the exogenous compound are (a) a TNF-
RI (p55) and an
oligopeptide comprising a sequence substantially similar to SEQ ID NO:1; (b) a
TNF-RI (p55) and a
compound that competes with an oligopeptide of SEQ ID NO:1 for binding the TNF-
RI ~(p55); (c) an
IL-1RI (IL-1R-alpha) and an oligopeptide comprising a sequence substantially
similar to SEQ ID
N0:2; (d) an IL-1RI (IL-1R-alpha) and a compound that competes with an
oligopeptide of SEQ ID
N0:2 for binding the IL-1Ri (IL-1R-alpha).
[0071] It will be recognized by those of ordinary skill that modulation of
each of the TNF-RI
(p55) and IL-1RI (IL-1R-alpha) has a variety of therapeutic benefits. In
general, any of a variety of
diseases, symptoms, and conditions mediated, at least in part, by activation
of the TNF-RI (p55) or
IL-1RI (IL-1R-alpha) can be treated by administering agents that modulate
receptor activity. In this
context, "treatment" is an approach for obtaining beneficial or desired
results, such as alleviation or
amelioration of one or more symptoms, diminishment of extent of disease,
stabilized (i.e., not
worsening) state of disease, preventing spread of disease, delay or slowing of
disease progression,
amelioration or palliation of the disease state, and remission (whether
partial or total). Tumor
Necrosis Factor (TNF-alpha) and Interleukin-1 (IL-1) are important effector
cytokines for immune
responses and inflammation. These cytokines are produced.mainly by activated
macrophages, and
mediate pleiotropic inflammatory and immunoregulatory responses, as well as
cytotoxicity, antiviral
activity, and stimulation of cell growth.
[0072] In one aspect modulators of the IL-1 and/or TNF-alpha receptors are
used as anti-
inflammatory agents. Inflammation (e.g., peritonitis, carditis and arthritis)
is characterized by edema
and tissue injury due to the release of numerous chemotactic pro-inflammatory
cytokines including
IL-1 and TNF-alpha. Without intending to be bound by any particular mechanism,
inflammation
causes the induction of the Cox-2 enzyme, leading to the release of
prostanoids, which sensitize
peripheral nociceptor terminals and produce localized pain hypersensitivity.
Induction of Cox-2
results in elevated levels of PGE2 in the cerebrospinal fluid. Major inducers
of Cox-2 up-regulation
in the CNS are IL-1 and TNF-alpha. Thus, by preventing central prostanoid
production by inhibiting
the IL-1 or TNF-alpha mediated induction of Cox-2 in neurons or by inhibiting
central Cox-2 activity
centrally generated inflammatory pain hypersensitivity would be reduced.
[0073] In one aspect, modulators of the IL-1 and TNF-alpha receptors are used
as anti-
inflammatory agents for example in treatment of sepsis, cachexia, rheumatoid
arthritis, chronic
myelogenous leukemia, asthma, psoriasis, and inflammatory bowel disease (e.g.,
Crohn's disease,
e.g., for sustaining closure of draining fistulas in Crohn's disease
patients).
[0074] Depending on the type of disorder, administration of the pharmaceutical
composition can
serve to enhance the cellular response to endogenous or exogenous ligand,
e.g., in ligand resistant
1S
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states, to replace endogenous ligand, e.g., in ligand deficient states, or to
antagonize the effects of
ligand (e.g., in cases in which expression of endogenous ligand is detrimental
to the subject). Thus, in
one embodiment, the exogenous compound contacts the receptor in the presence
of the ligand which
normally activates the receptor. As above, there may be endogenous ligand
present, or exogenous
ligand added in addition to the exogenous compound.
Dosages and Fonnulations ofExogenous Compounds
[0075] An exogenous compound may be formulated as a pharmaceutical composition
by
combining the compound with a pharmaceutical carrier or diluent and optionally
other compounds
that enhance therapeutic utility and/or facilitate storage and administration.
Each carrier should be
both pharmaceutically and physiologically acceptable in the sense of being
compatible with the other
ingredients and not injurious to the patient. Thus, in one aspect the
invention provides pharmaceutical
compositions that include exogenous compounds of the invention along with
pharmaceutically
acceptable excipients, carrier or diluent and optionally other compounds that
enhance therapeutic
utility and/or facilitate storage and administration. Each carrier should be
both pharmaceutically and
physiologically acceptable in the sense of being compatible with the other
ingredients and not
injurious to the patient. Pharmaceutically acceptable excipients are well
known in the art and include
sterile water for pharmaceutical use, isotonic solutions such as saline and
phosphate buffered saline,
physiological saline, PBS, and dextrose solution. In addition, the
pharmaceutical composition or
formulation can include other carriers, adjuvants, or non-toxic,
nontherapeutic, nonimmunogenic
stabilizers, excipients and the like. The compositions can also include
additional substances to
approximate physiological conditions, such as pH adjusting and buffering
agents, toxicity adjusting
agents, wetting agents and detergents. Other excipients suitable for
administration to a human patient
known in the art. See, e.g., Remington: The Science and Practice of Pharmacy
(19th edition, 1995,
Gennavo, ed.). The pharmaceutical compositions are typically sterile (i.e.,
manufactured or
formulated as a sterile composition) and optionally can be prepared in
compliance with all Good
Manufacturing Practice (GMP) regulations of the U.S. Food and Drug
Administration.
[0076] The exogenous bioactive compounds of the invention may be administered
in a
physiologically acceptable Garner to a host. The agents may be administered in
a variety of ways,
e.g., orally, parenterally (e.g., by intravascular infusion or injection at an
epidermal, subcutaneous,
intramuscular, or intraperitoneal site), topically, transdermally, or by
transmucosal absorption.
Depending upon the manner of introduction, the agents may be formulated in a
variety of ways.
[0077] The formulation of bioactive agent will vary depending upon the purpose
of the
formulation, the particular mode employed for modulating the receptor
activity, the intended
treatment, and the like. The concentration of therapeutically active agents in
the formulation may
vary from about 0.1-100 wt. %. The formulation may involve patches, tablets,
capsules, liposomes,
time delayed coatings, injectables, or may be formulated in pumps for
continuous administration. For
19
CA 02496634 2005-02-24
WO 2004/020608 PCT/US2003/027381
example, formulations for injection may comprise a physiologically acceptable
medium, such as
water, saline, PBS, aqueous ethanol, aqueous ethylene glycols, or the like.
Water soluble
preservatives which may be employed include sodium bisulfate, sodium
thiosulfate, ascorbate,
benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate,
parabens, benzyl alcohol
and phenylethanol. These agents may be present in individual amounts of from
about 0.001 % to
about 5% by weight and preferably about 0.01% to about 2%. Suitable water
soluble buffering agents
that may be employed are alkali or alkaline earth carbonates, phosphates,
bicarbonates, citrates,
borates, acetates, succinates and the like, such as sodium phosphate, citrate,
borate, acetate,
bicarbonate and carbonate. Additives such as carboxymethylcellulose may be
used as a carrier in
amounts of from about 0.01% to about 5% by weight. The specific dosage may be
determined
empirically in accordance with known ways. See, for example, Harrison's
Principles of Internal
Medicine, 11th ed. Braunwald et al. ed, McGraw Hill Book Co., New York,1987.
[0078] Generally, a therapeutically effective dose of the exogenous bioactive
compound will be
administered. A therapeutically effective dose is an amount sufficient to
modulate receptor activity.
This amount is usually in the range of about 0.005-10, more usually from about
0.01-1 mg/kg of host
weight, and sometimes from about 0.1 to about 1 mg/kg. Administration may be
as often as daily;
sometimes not more than once or twice daily, or as infrequent as weekly. The
host may be any
mammal including domestic animals, pets, laboratory animals and primates,
particularly humans. The
amount will generally be adjusted depending upon the half life of the
compound, where dosages in the
lower portion of the range may be employed where the compound has an enhanced
half life or is
provided as a depot, such as a slow release composition comprising particles,
introduced in a matrix
which maintains the compound (e.g., peptide) over an extended period of time,
e.g., a collagen matrix,
use of a pump which continuously infuses the compound over an extended period
of time over a
substantially continuous rate, or the like. Heller (1987), Biodegradable
Polymers in Controlled Drug
Delivery, in: CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol.
l, CRC Press, Boca
Raton, Fla., pp 39-90, describes encapsulation for controlled drug delivery,
and Di Colo (1992)
Biomaterials 13:850-856 describes controlled drug release from hydrophobic
polymers.
EXAMPLES
[0079] The following examples serve to more fully describe the manner of using
the above-
described invention. It is understood that these examples in no way serve to
limit the scope of this
invention, but rather are presented for illustrative purposes. All cells were
obtained from the
American Type Culture Collection.
CA 02496634 2005-02-24
WO 2004/020608 PCT/US2003/027381
EXAMPLE 1
BIOLOGICAL ACTIVITY OF TNF-RI (P55) DERIVED PEPTIDE (TNF-A1 PEPTIDE)
[0080] Example 1 illustrates that a TNF-RI (p55) derived peptide ("TNR-alp")
having the
sequence set forth as SEQ ID NO:1 in Table 1 is a TNF-a receptor antagonist.
The antagonistic effect
of TNR-alp binding to the TNF-a receptor is demonstrated by the inhibitory
effect on ligand-induced
phosphorylation of ERKl and p38.
[0081] The biological activity of TNF-al peptide was measured using HT-29 cell
and mouse
bone marrow macrophage assays. TNF-a activates members of the mitogen-
activated protein kinase
(MAPK) family whose downstream targets include both c-Jun and c-Fos. The MAPK
family
comprises three subfamilies: (i) the extracellular signal-regulated kinases of
ERK subfamily
represented by p42mapk/erk2 and p44mapk/erkl; (ii) the c-Jun NH2-terminal
kinases/stress-activated
protein kinases or JNK/SAPK subfamily represented by the p46 JNK/SAPK; and
(iii) the p38mapk
subfamily. These assays specifically addressed ERKl and p38 phosphorylation.
Assay Solutions
[0082] 2x Lysis Buffer is made by combining the following reagents: 50 ml 1M
HEPES, pH 7.6;
8.8 g NaCI; 10 ml Triton X-100; 10 ml O.SM EDTA, pH 8; HZO to 500 ml.
[0083] IOOx Protease Inhibitor Mix is made by combining the following
reagents: 20 mg
Aprotonin, 2 mg Pepstatin A, 2 mg Leupeptin; 2 mg Cymostatin, 467 mg AEBSF;
HZO to 20 ml.
[0084] Sanaple Buffer is 100 pl 50% Glycerol; 0.5% Bromphenyl Blue, 20 ~,1 ~3-
mercaptoethanol,
40 x.110% SDS, and 160 ~.l lx electrophoresis buffer.
[0085] IOx TST is made by combining the following reagents:12.1 g Tris base;
87.7 g NaCI; 7.5
ml Tween-20; 3 g NaN3; H20 to 1 liter, pH to 7.4.
(0086] Blotto is 100 ml l Ox TST, 5 g Dry Milk; Ha0 to 1 liter.
[0087] IOx TSM is made by combining the following reagents: 121.1 g Tris base;
58.4 g NaCl;
10.2 g MgCl2x 6Ha0, Hz0 to 1 liter, pH to 9Ø
[0088] BCIPlNBT Substrate: NBT stock: 50 mg/ml in 70% Dimethyl Formamide
stored at -
20°C; BCIP stock: SO mg/ml in 100% Dimethyl Formamide stored at -
20°C; Combine 10 ml lx TSM
with 66 pl 50 mg/ml NBT and 33 x,150 mg/ml BCIP just before using.
Cell signaling in HT-29 cells
HT ~9 cell growth and starvation
[0089] HT-29 (human colonic epithelial) cells were grown and maintained at
37°C and 5% COZ
in RPMI medium 1640 (ATCC, catalog no. 30-2001) containing 10% fetal bovine
serum (FBS,
ATCC, catalog no. 30-2020),100U penicillin/100~,~m1 streptomycin sulfate (P/S,
Applied Scientific,
catalog. no. 9366), and 2 ng/ml human granulocyte/macrophage colony-
stimulating factor (GM-CSF).
The cells were grown to a density no greater than 8 x 105 cells/ml.
21
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WO 2004/020608 PCT/US2003/027381
[0090] When ready to be used in the assay, the cells were starved overnight in
RPMI 1640
medium containing 3.5% FBS and PlS in T150 flasks (60 ml/flask) at a density
of 8 x 105 cells/ml at
environmental conditions of 37°C and S% C02. The cells were then
centrifuged at 200 x g for 5
minutes at room temperature. The supernatant was removed, and the cells pooled
into one SO ml
conical tube. The cells were washed twice with serum-free medium (RPMI
containing P/S) and
resuspended to a density of 1.5 x 10' cells/ml in serum-free medium. The cells
were then placed in 15
ml conical tubes in 1 ml aliquots, and incubated with the caps loosened for 1
hour at 37°C and 5%
COZ. The lower portion of each tube was tapped several times every 10 minutes
to prevent the cells
from settling.
TNF al peptide) modulation ofHT 29 cells and immunoprecipitation
[0091] TNF-al peptide stock was diluted with ice cold Hz0 (if necessary) to
obtain samples
having final peptide concentrations of 0.03 ~,M, 0.3 ~.M., 3 liM, and 30 ~M
after addition of 10 ~1 of
each dilution to the 1 ml of HT-29 cell suspension prepared above. The samples
were incubated at
37°C and 5% COZ for 30 minutes. Fourteen ml of ice cold PBS (Sigma,
catalog no. P-3813) was
added to each sample to stop the assays, and the samples centrifuged at 400 x
g for 5 minutes at 4°C.
The samples were then placed on ice, the supernatant aspirated, and the cells
lysed by adding 800 lCl
2x lysis buffer. The cells were incubated on ice for another 30 minutes.
Samples were then
transferred to 1.5 ml microcentrifuge tubes and spun for 10 minutes at 10,000
x g and 4°C. The
supernatant from each sample was then added to anti-phosphotyrosine-coated
GammaBind beads,
prepared as described below, and incubated overnight at 4°C with end-
over-end rotation.
[0092] Next, the samples were spun for 15 to 20 seconds and the supernatant
discarded. The
beads were washed twice with 800 ~,l lx lysis buffer and once with 800 ~,1 of
a 1:1 mix of lysis
buffer:125mM Tris, pH 6.8. To each sample was added 45 ltl lx sample buffer.
The samples were
then heated for 5 minutes at 95°C, centrifuged for approximately 20
seconds and examined by SDS-
PAGE.
(0093] The GammaBind beads were coated with antibody by washing them 3x with 1
ml ice cold
PBS, spinning them for 15 to 20 seconds, and then discarding the supernatant.
Ten microliters of
anti-phosphotyrosine antibody (PY99, Santa Cruz Biotech, catalog no. sc-7020)
was then added per
sample to the beads. Next, PBS was added so that the sample contained 50%
beads and SO% liquid.
The beads were then incubated for 2 hours at room temperature with end-over-
end rotation.
Western Blot Analysis
[0094] Samples were run on two 8% polyacrylamide gels, transferred to
Immobilon-P
membranes, blocked with blotto for 1 hour at room temperature, and one
membrane incubated with a
p38 antibody and the other with a ERKl antibody at 4°C overnight. The
membranes were then
washed 3 times with blotto at room temperature and incubated with a-Rabbit-AP
antibody diluted
22
CA 02496634 2005-02-24
WO 2004/020608 PCT/US2003/027381
1:2000, at room temperature for 2 hours. The membranes were then washed 3
times with blotto,
twice with lx TST, once with lx TSM, and developed with BCIP/NBT.
[0095] The Western Blot (digital image) in Figure 1 shows that phosphorylation
of both ERKl
and p38 is significantly diminished in the presence of TNR-alp. Furthermore,
TNR-alp at 0.3 ~.M
was shown to reduce the activation of kinases induced by 3, 10, or 30 ng/ml of
TNF-a. Thus, the
signaling assay using HT-29 cells demonstrated that TNR-al peptide is a TNF-a
receptor antagonist.
Cell Signaling in Mouse Bone Marrow Macrophages
[0096] Mouse bone marrow macrophages were isolated from femoral and tibial
bone marrow.
Cells were washed twice with PBS and resuspended to a density of 6.5 106
cell/ml in media. Three
hundred microliters (6.5 x 105) of cells were mixed with 1.5 ml of
methylcellulose media (Stem Cell
Technology, catalog no. M3234). At 5-7 days confluent monolayers of
macrophages were obtained
and stimulated with TNF-a in the presence or absence of TNR-alp. After cell
lysis, the activation of
ERK protein was measured. As shown in Figure 2, (digital image of Western blot
with
phosphospecific ERK antibody) TNR-al peptide inhibits TNF-a induced protein
phosphorylation.
The effect is observed at max TNF-a stimulation of 30 ng/ml. Kinetics indicate
peptide activity at 15
minutes, with a duration of activity as long as the hormone activity, which is
approximately 60
minutes.
[0097] Thus, the cell signaling assay in mouse bone marrow macrophages also
demonstrated that
TNR-al peptide is a TNF-a receptor antagonist.
EXAMPLE 2
ANTAGONIST EFFECT OF IL-1RI (IL-1R-ALPHAI DERIVED PEPTIDE (IL-1P1
[0098] This example demonstrates that IL-1RI (IL-1R-alpha) derived peptide
("IL-lp") having
the sequence set forth as SEQ ID N0:2 in Table 1 is an IL-1RI (IL-1R-alpha)
antagonist.
[0099] The in vitro biological activity of IL-lp was studied using a cell
signaling assay in HT-29
and HepG2 cells. TRAF6, IRAKl, and p38 are kinases in the downstream signaling
pathway of the
IL-1RI (IL-1R-alpha), and IL-1-induced activation of the IL-1RI (IL-1R-alpha)
results in
phosphorylation of TRAF6, IRAKl, and p38. The effect of IL-lp on IL-1 induced
phosphorylation
was determined using the techniques described in Example 1, with substitution
of appropriate
antibodies.
[0100] Figure 3 is a digital image of Western Blots probed with either anti-
TRAF6, anti-
IR.AKl, or anti-p38 showing dose-responsive inhibition of IL-1 induced
phosphorylation of
TRAF6, IRAKI, and p38 in HepG2 cells.
23
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WO 2004/020608 PCT/US2003/027381
EXAMPLE 3
MEASUREMENT OF TNF-a STIMULATED IL-8 PRODUCTION IN NHDF
[0101] This example provides exemplary protocols for measurement of receptor
activity.
This protocol can be easily modified to measure IL-1 stimulated IL-8
secretion. The assay
principle is that Normal Human Dermal Fibroblast (NHDF) cells respond to TNF-a
with the
secretion of soluble IL-8, which can be measured in a Sandwich ELISA.
[0102] Cells:
1. Grow NHDF cells in Clonetics Fibroblast Growth Medium (FGM-2) in a T-75
flask
until they are approximately 90% confluent.
2. Seed a 96-well tissue culture plate with 1 x 104 cells/well in FGM-2 medium
and ~.
incubate overnight.
3. Wash the cells lx, and starve overnight in FGM-2 containing 0.2% FBS.
[0103] Stimulation:
1. Wash the cells lx with FGM-2 containing 0.2% FBS. Aspirate the liquid from
the
wells.
2. Dilute the exogenous agent (peptide or other compound) in..FGM-2 containing
0.2%
FBS to 2x the desired final concentration right before it is added to the
plate (peptide stock is 1mM in
H20, final concentration in the assay is 10~.M). Add 100,1 of each dilution to
the appropriate wells,
and incubate for 60 minutes at 37°C, 5% CO2.
3. Dilute hTNF-a to 2x its final concentration in FGM-2 containing 0.2% FBS
(i.e. for a
final conc. of l Ong/ml TNF- a, dilute to 20ng/ml; hTNF- a stock is 10~,g/ml
in PBS/0.1 % BSA).
Add 100p,1 of the dilution to each well, and incubate at 37°C, 5% COZ
for 4 hrs.
4. Spin plate at 200xg for 5 minutes. Remove 1501 of supernatant from each
well, and
place into the corresponding wells of an ice cold NUNC Minisorp plate. Samples
can be stored at -
80°C until they are needed for the IL-8 ELISA, or they can be used in
the assay immediately.
S. If the samples were frozen in Step 4, thaw them on ice (this can take a
while). Keep the
samples on ice until they are used in the ELISA. If the samples are too
concentrated, dilute them 1:4
in FGM-2 containing 0.2% FBS. Use 501 of each sample in an IL-8 ELISA.
[0104] Si~nalin~ in NHDF Cells in Response to TNF-a and Test Compound
Cell Growth and Starvation
1. Grow NHDF cells in Fibroblast Growth Medium (FGM-2, Cambrex) in a T-150
flask
until they are approximately 90% confluent. Grow the cells according to the
instructions provided by
Cambrex; do not use cells past passage 8.
2. Seed a 6-well tissue culture plate with 1.6 x 105 cells/well in FGM-2
medium and
incubate overnight.
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WO 2004/020608 PCT/US2003/027381
3. Wash the cells lx with Starvation Medium (FGM-2 containing growth factors
plus
0.2% FBS), and starve overnight in the same medium.
[0105] Cell Stimulation
1. Wash the cells lx with Starvation Medium. Aspirate the liquid from the
wells.
2. Add lml Starvation Medium to each well. Dilute the compounds in DMSO to
333.4x their
desired final concentrations. Add 3p.1 of each diluted compound to the
appropriate wells, and 3pl of
DMSO to each of the controls; incubate for 45 minutes at 37°C, 5%
COZ.
3. Dilute hTNF- a in Starvation Medium to 100x its final concentration (i.e.
for a final conc.of
6ng/ml TNF- a, dilute to 600ng/ml). Add 10.2p1 of the diluted TNF- a to each
well, and incubate at
37°C, 5% COa for l5minutes.
4. To stop the reactions, place the 6-well plate on ice, aspirate the medium,
and wash the cells
with ice cold PBS.
5. Remove as much of the PBS as possible, and lyse the cells with 0.3m12x
lysis buffer
containing 2x protease inhibitors, added just before use. Incubate on ice for
30 minutes.
6. Scrape the cells off of the bottom of each well, and place into clean
microcentrifuge tubes.
7. Spin the samples for 10 minutes. For each sample, place 20u14x Sample
Buffer, and 60u1
supernatant into a microcentrifuge tube. Heat the samples for 5 minutes at
95°C, and centrifuge
briefly to remove condensation from the top of the tubes. Vortex the samples
briefly and examine by
SDS-PAGE.
[0106] IL-8 ELISA:
1. On the day before the ELISA is to be performed, coat a NLJNC Maxisorp plate
with
SOpl/well SOOng/ml anti-hIL-8 (R&D, stock SOOpg/ml; diluted 1:1,000 in 25mM
NaHCO3, pH 9.6).
Incubate overnight at 4°C while shaking.
2. Block with 200pllwell Blocking Buffer for 1 hr at room temperature.
(Blocking
Buffer: TBS (20mM Tris, 137mM NaCI, pH 7.6), 0.05% Tween-20, 0.3% Safeway Dry
Milk).
3. Wash 3x with 250p.1/well Wash Buffer (TBS, 0.05% Tween-20). Do not let the
plates
sit after washing. Wash, then pipet plate by plate.
4. Prepare the hIL-8 standards in McCoy's medium containing 5% FBS (IL-8 stock
is
25~g/ml). Dilute samples to contain the following concentrations of IL-8: 0,
l2.Spg/ml, 25pg/ml,
SOpg~ml, 75pg/ml, 100pg/ml, 150pg/ml, and 200pg/ml.
S. Add SOpI of either the diluted IL-8 standards (from step #4 above) or the
diluted
sample (from step #3 in the previous section) to the appropriate wells of the
coated plates; each plate
should have a set of standards. Incubate for 3 hrs at room temperature.
6. Wash 3x with 250p.1/well Wash Buffer.
CA 02496634 2005-02-24
WO 2004/020608 PCT/US2003/027381
7. Dilute biotinylated anti-hIL-8 1:4,000 in Blocking Buffer (anti-hIL-8-
biotin stock is
100pg/ml), and add SOuI to each well. Incubate over night at 4°C while
shaking.
8. Wash 3x with 250~,1/well Wash Buffer.
9. Dilute HRP-Streptavidin conjugate 1:100 in Blocking Buffer. Add SOp,I to
each well.
Incubate for 20 minutes at 4°C.
10. Wash 3x with 250p1/well Wash Buffer.
11. Add 100p1 TMB substrate to each well. Incubate for 15 minutes at room
temperature.
12. Add SOp.l 2M HZS04 to each well. Read on the Wallac at 450nm.
13. Compare effect of added agent on IL-8 production. In the presence of
receptor
antagonists, IL-8 production is reduced.
EXAMPLE 4
ACTIVITY OF SMALL MOLECULE ANTAGONISTS AND
AGONISTS (SYNERGISTS) OF TNF-a
[0107] 48,000 compounds from a library of synthetic organic compounds with
molecular weight <S00 were screened in a peptide displacement assay for
binding to the
activation domain of the TNF-RI-receptor. Briefly, purified receptor was
incubated (in the
presence of library compounds) with biotinylated TNRal peptide and complex
formation was
assayed by (1) capture to a streptavidin coated plate and (2) detection using
an anti-TNF-RI
(p55) specific antibody in which colorimetrically detectable signal
corresponds to formation
of complex between TNRal peptide and TNF-RI-receptor. A reduction of signal is
indicative
of competition between the biotin-TNRal peptide and its binding site on the
TNF-RI-receptor
has occurred. Competition was detected using non-labeled peptide (control) or
certain
compounds from the library.
[0108] Molecules that competed with peptide binding were tested for their
biological
activity in two assays: (i) inhibition of TNF-a induced IL-8 production and
(ii)
phosphorylation of p38 protein (a substrate specific for the signal
transmitted through TNF-
a Receptor). Both small molecule antagonist and agonist to TNF-a activity were
identified.
Total of 67 compounds were identified as antagonists of TNF-a activity (TNF-a-
induced IL-
8 production and p38 phosphorylation), and 32 compounds were identified as
agonists of
TNF-a activity.
[0109] In summary, the primary screen (peptide displacement/binding assay) had
a hit
rate of 0.47% with 229 compounds showing >70% competition/inhibition.. A first
secondary screen (antagonists of TNF-a induced IL-8 production and p38
phosphorylation)
26
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WO 2004/020608 PCT/US2003/027381
had a hit rate of 0.14% (67 compounds). A second secondary screen (enhancers
of TNF-a
induced IL-8 production and p38 phosphorylation) had a hit rate of 0.07% (32
compounds).
[0110] Data representative of compounds with antagonist and agonist activity
is
presented in Figures 4 and 5.
[0111] Figure 4 shows the effect of two compounds (an antagonist and an
agonist) on
TNF-a-induced IL-8 production in Normal Human Dermal Fibroblasts (NHDF) cells.
Briefly, NHDF cells were seeded to a density of 104 cells/well in a 96-well
plate. After
starvation overnight in media without serum, cells were washed and compounds
of interest
were added. After 30 min of incubation, 300 pmols of TNF-a was added and
incubation
continued for 4 hours at 37°C, 5% COZ. Plates were spun down and
supernatants are
transferred to a fresh plate for IL-8 ELISA assay. Cell proliferation solution
was added to the
pelleted cells in order to determine number of viable cells in each individual
well. The final
reading of TNF-a induction was adjusted for the number of viable cells.
[0112] The amount of TNF-a used for IL-8 induction corresponds to its ECso
value, i.e.
300 pg/ml of TNF-a induces about 550 pg/ml of IL-8 (no TNF-a addition induces
about 20
pg/ml of IL-8, maximal induction corresponds to 1200 pg/ml of IL-8). The
presence of .°
small molecule agonist or antagonist (in the absence of TNF-a) does not induce
production
of IL-8. The presence of a small molecule antagonist in the presence of TNF-a
(300 pg/ml)
showed a dose-responsive decrease in IL-8 production; i.e. 5 p,M compound
reduced IL-8
production by ~ 70%. The presence of small molecule agonist in the presence of
TNF-a (300
pg/ml) showed a dose-responsive increase in IL-8 production; i.e. 5 ~M
compound reduced
IL-8 production by ~90%.
[0113] Figure 5 shows the effect of various compounds on TNF-RI downstream
substrate
phosphorylation (p38 protein) in Normal Human Dermal Fibroblasts (NHDF) cells.
Briefly,
NHDF cells were seeded to a density of 2x105 cells/well in a 6-well plate.
After starvation
overnight in media without serum, cells were washed and compounds of interest
were added.
After 30 min of incubation, TNF-a at 6 ng/ml was added and incubation
continued for 30
min at 37 C, 5% COa. Media was aspirated and cells were lysed on ice. Lysates
were run on
SDS-PAGE, transferred to PVDF membranes and incubated with anti-phospho-p38
antibody.
In Figure 5, the upper panel shows the antagonistic activity of different
small molecules.
Compounds B, D, E, F at a 3 pM concentration, and in the presence of 6 ng/ml
of TNF-a,
inhibited TNF-a-induced substrate phosphorylation. Compound A shows partial
antagonistic
activity, whereas compound C showed no antagonistic activity (control
compound).
27
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WO 2004/020608 PCT/US2003/027381
[0114] The lower panel of Figure 5 demonstrates compounds with agonistic
activity to
TNF-a. In the presence of 6 nglml of TNF-a, compounds H, I, J and K increased
TNF-a-
induced substrate (p38) phosphorylation. Control compound G showed no change
on TNF-a
induced substrate phosphorylation.
***
[0115] All publications, patents, patent applications, and accession numbers
(including
both polynucleotide and polypeptide sequences and corresponding annotations as
of the filing
andlor priority application ding dates) cited herein are hereby incorporated
by reference in
their entirety for all purposes to the same extent as if each individual
publication, patent or
patent application were specifically and individually indicated to be so
incorporated by
reference. Although the foregoing invention has been described in some detail
by way of
illustration and example for purposes of clarity of understanding, it will be
readily apparent to
those of ordinary skill in the art in light of the teachings of this invention
that certain changes
and modifications may be made thereto without departing from the spirit or
scope of the
appended claims.
28
