This application is divided out of Indian application no. 205/KOLNP/2005.
Related Application
[001] This application relies on the benefit of priority of U.S. provisional
patent application Serial No. 60/400,044.
Field of the Invention
[002] The present invention relates to uses of proteins that bind MAPKAP
kinase 2 (MK2). More particularly, the invention relates to uses of proteins that bind
MK2 for treating conditions that are related to inflammation. The invention is useful
for treating conditions such as Crohn's disease, inflammatory bowel disease,
ulcerative colitis, rheumatoid arthritis, acute respiratory distress syndrome,
emphysema, delayed type hypersensitivity reaction, asthma, systemic lupus
erythematosus, and inflammation due to trauma, injury or stroke.
Background of the Invention
[003] A number of human and animal conditions are associated with
inflammation. To date, very few reliable or effective therapies exist for these
conditions. However, the terrible symptoms associated with these conditions may be
substantially reduced by employing therapies that decrease inflammation in patients
suffering from the condition. While not curing the conditions, such therapies would
significantly improve the quality of life for these patients and could ameliorate some
of the effects of these conditions. Thus, there is a need in the art to identify new
therapies that may contribute to an overall decrease in inflammation in patients
suffering from these conditions.
[004] Inflammatory conditions are often associated with inappropriate
regulation of cytokines (Han et al., Nature Cell Biol., E39-E40 (1999)). For this
1A

reason, the selective inhibitors of inflammatory cytokine expression are potential
agents for the treatment of conditions related to inflammation.
[005] MAPKAP kinase 2 (MK2) is thought to contribute to the regulation of
several cytokines and thus may be an essential component of the inflammatory
response. Mice with a null mutation for MK2 show an increased resistance to
lipopolysaccharide-induced endotoxic shock {Kotlyrov et al., Nature Cell BioL, 1:94-
97 (1999)). This stress resistance is thought to result from the decrease in the
biosynthesis of several inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-10,
and IFN-y. Because of the role of MK2 in the regulation of inflammatory cytokines,
proteins that bind and inhibit MK2 activity are potential agents for decreasing
inflammation.
[006] MK2 has been shown to associate with a number of proteins. MK2 is
phosphorylated by p38 MAP kinase in response to certain environmental stress or
inflammatory cytokines {Kotlyarov et al., Nature Cell Biology, 1:94-97 (1999)), as
shown in Figure 7. MK2 phosphorylates serum response factor (SRF) (Heidenreich
et al., J. Biol. Chem., 274:14434-14443 (1999)), CREB and ER81 {Janknecht, J.
Biol. Chem., 276:41856-41861 (2001)), small heat shock protein and leukocyte
specific protein 1 (reviewed in Neininger et al., EMBO Reports, 2:703-708 (2001)),
E47 (Neufeld et al., J. Biol. Chem., 275:20239-20242 (2000)), Akt (Rane et al., J.
Biol. Chem., 276:3517-3523 (2001)), tyrosine hydroxylase, and TTP {Mahtani et al.,
Mol. Cell Biol., 21:6461-6469 (2001)). In addition, MK2 interacts with 5-
lipoxygenase, which catalyzes important steps in the synthesis of leukotrienes, which
are a group of inflammatory mediators {Janknecht, J. Biol. Chem. 276:41856-41861
(2001)). One protein hnRNP A0, however, has been shown to be differentially
regulated in MK2 +/+ and -/- cells.
2

[007] Thus, due to MK2's involvement in inflammatory responses, it may be
a desirable target for therapeutic intervention. In particular, therapeutic agents that
inhibit the activity of MK2 may be used to treat human or animal conditions in which
a decrease in inflammation would be therapeutically beneficial.
Summary of the Invention
[008] Accordingly, the invention relates to proteins that interact with MK2.
Proteins that bind MK2, including splice variants, truncations, fragments,
substitutions, addition and deletion mutations, fusion protein, shuffling mutants and
motif sequences, and homologues of such proteins are potential novel anti-
inflammatory drug agents.
[009] The present invention further relates to protein complexes comprising
MK2 and an MK2 interacting protein such as, for example, STS, HPH2 and She and
variants thereof. Examples of additional MK2 interacting proteins include SRF,
CREB, ER81, tyrosine hydroxylase, TTP, small heat shock protein 1, E47, Akt and 5-
lipoxygenase. One or more of these proteins may also be present in a protein
complex including MK2. The invention also provides methods of making and using
such protein complexes for identifying potential compounds for treating conditions
and diseases where modulation of inflammation is desired.
[010] The present invention provides methods for modulating inflammatory
activity in cells that express MK2. Such methods comprise administering an
effective amount of a protein that binds MK2. The present invention also
encompasses methods for expressing a protein in a cell by administering a DNA
molecule encoding at least one protein that binds MK2.
[011] The present invention also includes drug screening methods to identify
anti-inflammatory drugs. In some embodiments, an anti-inflammatory drug is
3

identified by a method comprising, for example, providing a complex including MK2
and at least one MK2 interacting protein, adding an effective amount of a test
compound to the complex and determining whether the test compound inhibits
interaction of MK2 with an interacting protein. Anti-inflammatory drugs identified by a
method according to the invention include small molecules, chemical agents,
proteins, peptides and antibodies which inhibit an interaction between MK2 and an
MK2 interacting protein. Additionally, the present invention also provides methods of
identifying potential anti-inflammatory drugs which allow an interaction between MK2
and at least one MK2 interacting protein but block MK2 activity. Examples of anti-
inflammatory drugs include small molecules, chemical agents, proteins, peptides and
antibodies.
[012] According to the invention, compounds (such as proteins, peptides,
antibodies, chemical agents, and small molecules) that interact with at least one of
MK2 or an MK2 complex and modulate MK2 activity may be administered to a
patient, in a therapeutically effective dose, in order to treat or prevent medical
conditions in which a decrease in inflammation would be therapeutically beneficial.
Embodiments include treatment of conditions involving cells and tissue that are
associated with an increase in inflammation.
[013] Compounds that interact with at least one of MK2 or an MK2 complex
may be included in a pharmaceutical preparation. The pharmaceutical preparation
may contain other components, such as agents that aid in the binding of the
compound to MK2 or an MK2 complex.
[014] In addition, compounds that interact with at least one of MK2 or an
MK2 complex may be used as a diagnostic tool to quantitatively or qualitatively
detect MK2. For example, these compounds may be radioactively labeled, tissue
4

may be incubated with the labeled protein, and the excess, unbound protein may be
washed away. The tissue may then be assessed for the presence of radioactive
activity, which would indicate the presence of MK2. Compounds that interact with at
least one of MK2 or an MK2 complex may be used to detect the presence, absence,
or amount of MK2 in a cell, bodily fluid, tissue, or organism. The presence or
amount of MK2 detected may be correlated with one or more of the medical
conditions listed herein.
[015] The invention also includes compounds that promote interaction
between MK2 and an MK2 interacting protein, where the MK2 interacting protein
stimulates MK2 activity resulting in a inflammatory response. Such agents are
particularly useful in the treatment of conditions such as, for example, Listeria
monocytogenes infection, where stimulation of MK2 and a subsequent increase in
TNF-a production is desirable.
[016] Accordingly, the invention also encompasses a kit to be used for the
detection of the level of MK2 in a sample, comprising at least one compound that
interacts with MK2 or an MK2 complex, whether it is labeled or unlabeled, and at
least one agent that bind to this compound, such as a labeled antibody. The kit may
also include the appropriate biological standards and control samples to which one
could compare the results of the experimental detection. It may also include buffers
or washing solutions and instructions for using the kit. Structural components may
be included on which one may carry out the experiment, such as sticks, beads,
papers, columns, vials, or gels.
Brief Description of the Figures
[017] Figures 1A through 1B show the cDNA sequence encoding "similar to
smoothelin" (STS) protein, corresponding to SEQ ID NO:1.
5

[018] Figures 2A and 2B show the cDNA sequence encoding human
polyhomeotic 2 (HPH2) protein, corresponding to SEQ ID NO:2.
[019] Figures 3A and 3B show the cDNA sequence encoding a src homology
and collagen (She) protein, corresponding to SEQ ID NO:3.
[020] Figure 4 shows the amino acid sequence for "similar to smoothelin"
(STS) protein, corresponding to SEQ ID NO:4.
[021] Figure 5 shows the amino acid sequence for human polyhomeotic 2
(HPH2) protein, corresponding to SEQ ID NO:5.
[022] Figure 6 shows the amino acid sequence for src homology and
collagen (She) protein, corresponding to SEQ ID NO:6.
[023] Figure 7 shows a diagram of the p38/MK2 signalling pathway. P38
MAP kinase signaling pathways are activated in response to certain environmental
stresses or pro-inflammatory cytokines. P38 is directly phosphorylated by the
MKK3/6 MAP kinase kinases. Substrates of p38 include transcription factors as well
as a number of kinases which amplify and diversify p38 signaling. The MK2 kinase
is a p38 substrate which can phosphorylate a number of proteins including
transcription factors, cytoskeletal associated proteins, and an RNA binding protein.
MK2 also regulates TNF biosynthesis at a post-transcriptional level.
[024] Figure 8 shows the structural as well as MK2 interacting domains of
She A, HPH2, and STS. All three isoforms: 46, 52, and 66-kDa of She contain a src
homology 2 (SH2) domain, a phosphotyrosine binding (PTB) domain, and a collagen
homology domain 1, CH1. The 66 kDa isoform additionally contains a collagen
homology domain 2, CH2. Human polyhomeotic 2 has a sterile alpha motif (SAM)
protein interaction domain. STS contains actin binding domain (ABD) and a calponin
homology domain (CH).
6

[025] Figure 9A shows growth and color of yeast on selective media in
specificity assays for detecting interaction of various MK2 interacting proteins with
mutant MK2 in yeast. MK2 interacting proteins, She, HPH2 and STS bind
catalytically inactive MK2 K93R mutant. MK2 interacting proteins do not bind empty
BD vector (V) or lamin (L). Figure 9B summarizes the data obtained from the assays
of Figure 9A, where each of She, HPH2 and STS bind MK2 and MK2 K93R with
substantially the same affinity. Figure 9C shows domains in MK2 that interact with
She, HPH2, and STS. The MK2 N-terminal proline rich, catalytic, and C-terminal
localization domains are shown.
[026] Figure 10 depicts co-immunoprecipitation (IP) of proteins with MK2 in
293T cells as detected with Western blotting (WB) in presence or absence of
anisomycin. As shown in Figure 10A, Western blotting using antibodies against V5
or Myc shows that both V5-tagged She and Myc-tagged MK2 proteins are expressed
in 293T cells. Co-immunoprecipitation using the anti-V5 antibody and subsequent
immunoblotting with the anti-Myc antibody shows that MK2 co-immunoprecipitates
with She. Figure 10B shows that HA-HPH2, HA-p38, and Myc-MK2 were expressed,
as detected by Western blotting. Co-immunoprecipitation using the anti-HA antibody
and subsequent immunoblotting with the anti-Myc antibody shows that MK2 co-
immunoprecipitates with HPH2 and p38.
[027] Figure 11 depicts levels of TNF-a protein (pg/ml) in RAW264.7
macrophage cells. As shown, TNF-a levels are increased in anisomycin stimulated
RAW264.7 macrophage cells co-expressing either p38 and She or MK2 and She
compared to TNF-a levels in cells containing vector alone, p38 alone, MK2 alone or
She alone. Thus, She appears to be pro-inflammatory as it enhances MK2 activity.
7

[028] Figure 12A depicts a 2D autoradiography showing ^P labeled proteins
from MK2 +/+ and MK2 -/- mouse embryo fibroblasts resolved using two-dimensional
gel electrophoresis. A differentially phosphorylated protein is shown (arrow) which
has an isoelectric focusing point of 5.4. Figure 12B shows silver staining of the
same gel depicting the relative abundance of the resolved proteins.
[029] Figure 13A depicts a western blot for detecting phosphorylated Hsp 27
(pHsp 27) in the presence of MK2 or MK2 and She in both anisomycin stimulated
and unstimulated HeLa cells. As depicted, V5-p66 She A and MK2 are expressed in
HeLa cells and immunoblotting with an anti-pHsp 27 shows an increase in the levels
of basal pHsp 27 protein in cells expressing either MK2 or MK2 and She. Figure 13B
depicts levels of basal phosphorylated Hsp 27 protein in HeLa cells transfected with
either vector alone (V), MK2 alone, She alone or MK2 and She in both anisomycin
stimulated and unstimulated HeLa cells. The levels of phosphorylated Hsp 27
protein are normalized to levels in unstimulated cells transfected with vector alone.
Figure 13C depicts levels of basal phosphorylated Hsp 27 normalized to MK2 levels
in HeLa cells transfected with either MK2 and vector (V) or MK2 and She.
[030] Figure 14A depicts a western blot showing that She and MK2 are
expressed in RAW264.7 cells. An anti-actin antibody is used to show that equal
amounts of total protein were loaded in each lane. Figure 14B depicts the levels of
secreted TNF-a protein, as measured by ELISA, in both LPS-stimulated and
unstimulated RAW264.7 cells transfected with vector alone (V), MK2 alone, She
alone or MK2 and She.
[031] Figure 15A depicts a schematic representation of the p66Shc A protein
including a CH2 domain, a PTB domain, a CH1 domain and a SH2 domain. Also
shown is the MK2 interacting domain in the protein, the CAM kinase 2 consensus
8

sequence and GSK3 sequence. Figure 15B depicts phosphorylation of p66Shc A in
an in vitro kinase assay using recombinant MK2 and V5-Shc A immunoprecipitated
from transfected 293T cells. As depicted, phosphorylated She A is detected only in
immunoprecipitates from cells expressing She. Coomassie staining and western
blotting using an anti V5-antibody are used to confirm that She A was expressed in
She A transfected cells
[032] Figure 16 is a schematic representation of stress activated pathways
regulated by MK2 in response to cellular stress and phosphorylation of She in
response to MK2 activation by cellular stress.
[033] Figure 17 depicts western blots to show levels of phosphorylated AKT
and FKHR-L1 in response to hydrogen peroxide (H2O2) in MK2 -/- and MK2 +/+
mouse embryo fibroblasts (MEFs). As depicted, both phosphorylated AKT and
phosphorylated FKHR-L1 levels were reduced in MK2 -/- MEFs as compared to
levels in MK2 +/+ MEFs.
Brief Description of the Sequences
[034] The following table provides information on the sequences in this
application:

Sequence ID Figure Sequence Description
Number
SEQ ID NO:1 1A and 1B cDNA sequence encoding STS
SEQ ID NO:2 2A and 2B cDNA sequence encoding HPH2
SEQ ID NO:3 3A and 3B cDNA sequence encoding She
SEQ ID NO:4 4 Amino acid sequence of STS encoded by the
cDNA sequence of SEQ ID NO:1
SEQ ID NO:5 5 Amino acid sequence of HPH2 encoded by the
cDNA sequence of SEQ ID NO:2
SEQ ID NO:6 6 Amino acid sequence of She encoded by the cDNA
sequence of SEQ ID NO:3
9

Definitions
[035] The term "complex" refers to an association of two or more proteins.
Such an association may either be covalent or non-covalent including, for example,
ionic, hydrophilic and hydrophobic interactions between two proteins in a complex.
Typically, proteins that form a complex interact with each other such that
identification or detection of a first protein in the complex leads to identification or
detection of other protein or proteins that form a complex with the first protein. A
protein complex can either be identified in vivo, where two or more proteins naturally
associate with each other, for example, in a cell to form a complex. Alternatively, a
complex can be formed in vitro, where an interaction between two or more proteins
occurs when these proteins are added to a same reaction mixture. Methods that are
used for detection of proteins in a complex include, but are not limited to, co-
immunoprecipitation, yeast two-hybrid, fluorescence resonance energy transfer and
pull-down assays. An MK2 complex is a complex that contains MK2 and at least one
other protein.
[036] The term "co-immunoprecipitation" refers to a method for detecting an
interaction between two proteins. For example, interaction between an HA-tagged
MK2 interacting protein such as She, and MYC-tagged MK2 co-expressed in 293T
cells can be detected by immunoprecipitation. Cell lysates are prepared from cells
co-expressing both proteins that are subsequently immunoprecipitated with an anti-
HA antibody. Immunoprecipitates are resolved by SDS PAGE and immunoblotted
with an anti-MYC antibody to detect co-immunoprecipitated MK2.
10

[037] The term "HPH2" refers to human polyhomeotic homolog 2, which is
one of the polycomb group (PcG) of proteins and has a molecular weight of
approximately 51 kDa. HPH2 interacts with MK2 to form a complex, thereby
modulating MK2 activity. The term "HPH2" also includes variants of HPH2 including
splice variants, homologues, fusion proteins including HPH2, truncation and deletion
mutants, fragments, substitution mutants, addition mutants, shuffling mutants and
motif sequences of HPH2, which interact with MK2. Thus the term "HPH2" further
refers to functional variants of HPH2, including fragments of HPH2, which interact
with MK2. HPH2 is homologous to the Drosophila melanogaster PcG protein
'polyhomeotic1 as well as to the mouse Rae28/Mph1 protein {Gunster et ai, Molec.
Cell. Biol., 17: 2326-2335 (1997)). In Drosophila, the PcG genes are part of a
cellular memory system that is responsible for the stable inheritance of gene activity.
PcG proteins form a large multimeric, chromatin-associated protein complex and
contain a zinc finger motif and two regions designated homology domains I and II.
These complexes maintain transcriptional silencing/activation during development
and maintain transcriptional memory during the cell cycle, especially cell division.
Mutations in the PcG genes are associated with proliferation defects in
hematopoietic cells, implicating these proteins in regulation of hematopoiesis. The
cDNA sequence for HPH2 is provided in Figures 2A and 2B (corresponding to SEQ
ID NO:2) and the amino sequence of the protein is provided in Figure 5
(corresponding to SEQ ID NO:5). Figure 8B shows the structure of HPH2 and the
MK2 interacting domain in HPH2, and Figure 9C illustrates the HPH2 interacting
domain in MK2. HPH2 has a conserved C terminal sterile alpha motif (SAM) protein
interaction domain found in a number of signaling proteins including kinases,
11

scaffolding proteins, adaptor proteins and GTPAses as well as members of the ETS
family of transcription factors. It is believed that HPH2 is a transcriptional regulator.
[038] The term "inflammation" refers to a fundamental pathologic process
consisting of a dynamic complex of cytologic and histologic reactions that occur in
the affected blood vessels and surrounding tissues in response to an injury or
abnormal stimulation caused by a physical, chemical, or biological agent.
[039] The term "inflammatory condition" refers to conditions in which
inflammation is a symptom. Such conditions include, but are not limited to Crohn's
disease, inflammatory bowel disease, ulcerative colitis, rheumatoid arthritis, acute
respiratory distress syndrome, emphysema, delayed type hypersensitivity reaction,
asthma, systemic lupus erythematosus, and inflammation due to trauma injury or
stroke such as ischemia brain injury.
[040] The terms "MK2" and "MK2 polypeptide" refer to MAPKAP kinase 2, a
protein described in Stokoe etal. (Biochem. J. 296: 843-849 (1993)). This protein is
a Ser/Thr kinase originally identified as a hsp25/27 kinase. This protein kinase has
been shown to be active after phosphorylation by p38 mitogen-activated protein
kinase (p38 MAP kinase). MK2 is thought to regulate TNF post-transcriptionally, and
may be important in the regulation of other cytokines. Analysis of the cDNA
sequence for MK2 revealed the following features (in 5' to 3' order): a proline-rich
region containing 2 putative SH3-binding sites, a kinase catalytic domain, a
threonine residue phosphorylated by MAP kinase, and a nuclear localization signal.
MK2 -/- knockout mice are viable, however there is a 90% reduction in LPS-induced
TNF-a biosynthesis and these mice are resistant to LPS-induced shock. The term
"MK2" further includes variants of MK2 including splice variants, homologues, fusion
proteins including MK2, truncation and deletion mutants, fragments, substitution
12

mutants, addition mutants, shuffling mutants and motif sequences of MK2, where
these variants have MK2 activity. This term encompasses functional variants of
MK2, where a variant MK2 protein or a fragment thereof, has an MK2 activity, as
measured by one or more assays described herein and those that are known in the
art.
[041] The terms "protein that binds MK2" and "MK2 interacting protein" refer
to proteins that cohere or associate with MK2. The term encompasses proteins that
are found in a complex with MK2. The term also refers to any variants of such
proteins (including splice variants, truncations, fragments, substitutions, addition and
deletion mutations, fusion proteins, shuffling sequences and motif sequences, and
homologues) that have one or more of biological activities associated with native
proteins. These proteins further include amino acid sequences that have been
modified with conservative or non-conservative changes to the native proteins.
These proteins may be derived from any source, natural or synthetic. The protein
may be human or derived from animal sources, including bovine, chicken, murine,
rat, porcine, ovine, turkey, baboon, and fish. A protein that binds MK2 may stimulate
MK2, inhibit MK2, or have no effect on MK2 activity.
[042] The term "She" refers to sre homology and collagen (Migliaccio et al.,
Nature, 402:309-313 (1999)). The term "She" further includes variants of She
including splice variants, homologues, fusion proteins including She, truncation and
deletion mutants, fragments, substitution mutants, addition mutants, shuffling
mutants and motif sequences of She, which interact with MK2. This term
encompasses functional variants of She, where a variant She protein interacts with
MK2, thereby modulating MK2 activity. The cDNA sequence for a She protein (She
A) is provided in Figures 3A and 3B (corresponding to SEQ ID NO:3) and the amino
13

sequence of the protein is provided in Figure 6 (corresponding to SEQ ID NO:6).
Figures 8A and 16A show various domains in a She protein, including CH2, PTB,
CH1, SH2, and the MK2 interacting domain. Figure 9C shows the domain in MK2
which interacts with She. Three isoforms: 46, 52, and 66-kDa of the She A protein
are phosphorylated after engagement with cell surface receptors. The two smaller
isoforms are generated through different translation initiation while the 66 kDa
isoform, which has a unique N terminal CH domain (CH2), is generated through
alternative splicing. Src homology 2 (SH2) and phosphotyrosine binding (PTB)
domains bind phosphotyrosine on activated cell surface receptors, resulting in
tyrosine phosphorylation of the CH 1 domain, which promotes recruitment of Grb2
and SOS. Many activated cell surface receptors signal through the two smaller She
A Isoforms to activate the Ras MAP kinase pathway. In contrast, p66 binding to
these receptors has not been shown to activate this mitogenic pathway. The
collagen homology 2 (CH2) domain is unique to p66 and contains serine 36, which is
phosphorylated upon oxidative stress. The mammalian isoforms of She regulate
functions as diverse as growth (p52/p46Shc), apoptosis (p66Shc), and life-span
(p66Shc) {Luzietal., Curr. Opin. Genetics and Development, 10:668-674 (2000)).
[043] It is believed that She A is a signaling adapter phospho protein. The
p46 and p52 isoforms of the She A protein are ubiquitously expressed with the
exception of the brain and neurons, where they are developmentally regulated. p66
is expressed in specific cell types and tissues. Because p66 does not activate the
Ras MAPK pathway, its binding, which is proposed to compete with that of the two
smaller She A isoforms, is thought to modulate activation of the Ras MAPK pathway
through its differential expression. It has been shown in cells isolated from p66 -/-
mice that p66 acts downstream of p53 to mediate cellular responses to oxidative
14

stress including intracellular ROS and apoptosis. Phosphorylation of a serine
residue at position 36 in the p66 isoform (S36) is required for this activity. Two MAP
kinases: Erk and Jnk have been implicated in phosphorylating this serine.
[044] The term "similar to smoothelin" or "STS" refers to a specific protein
closely related to smoothelin. The term "STS" further includes variants of STS
including splice variants, homologues, fusion proteins including STS, truncation and
deletion mutants, fragments, substitution mutants, addition mutants, shuffling
mutants and motif sequences of STS, which interact with MK2. This term
encompasses functional variants of STS, where a variant STS protein interacts with
MK2. STS has not been fully characterized, however the predicted cDNA for STS is
documented in the National Center for Biotechnology Information (NCBI) data base,
National Institutes of Health, and its predicted molecular weight is 100 kDa. STS is
94% identical to smoothelin and both proteins have a calponin homology and an
actin binding domain based on sequence homology with known proteins in the NCBI
database. These proteins contain an actin binding domain (ABD) and a calponin
homology domain (CH). van derLoop et al., J. Cell Biol., 134: 401-411 (1996)
determined that smoothelin has significant homology to a sequence that flanks the
actin-binding domains of dystrophin, utrophin, beta-spectrin, and alpha-actinin. Cell
fractionation studies suggested to the authors that smoothelin is a part of the
cytoskeleton. Northern blot analysis revealed that the gene is expressed in several
tissues containing vascular smooth muscle, but not in brain, adipose tissue, cardiac
muscle, or skeletal muscle. The expression pattern of STS has yet to be
determined. The cDNA sequence encoding STS is provided in Figures 1A-1B
(corresponding to SEQ ID NO:1) and the amino sequence of the protein is provided
in Figure 4 (corresponding to SEQ ID NO:4). The structure of STS protein, including
15

the MK2 binding region, is shown in Figure 8C. It has a C terminal actin binding
domain (ABD) and a calponin homology domain (CH). Figure 9C shows the domain
in MK2 that interacts with STS. It is believed that STS is a cytoskeletal associated
protein.
[045] The term "therapeutic benefit" refers to an improvement in symptoms of
a condition, a slowing of the progression of a condition, or a cessation in the
progression of a condition. The therapeutic benefit is determined by comparing an
aspect of a condition, such as the amount of inflammation, before and after
administration of at least one protein that binds MK2. Therapeutic benefit can also
be determined by comparing an aspect of a condition, such as the amount of
inflammation, before and after administration of at least one agent that inhibits
interaction of MK2 with a protein, where the interaction stimulates MK2 activity.
Additionally, therapeutic benefit can also be determined by comparing an aspect of a
condition, before and after administration of at least one agent that promotes the
interaction between MK2 and a protein, where the interaction inhibits MK2 activity.
[046] In case of certain conditions, however, such as certain bacterial
infections, for example, Listeria monocytogenes infection, it is desirable to have an
enhanced MK2 activity. Accordingly, therapeutic benefit can also be determined by
an increased resistance to such an infection, before and after administration of an
agent that enhances MK2 activity or promotes the interaction between MK2 and a
protein, where the interaction enhances MK2 activity, resulting in, for example,
increased resistance to bacterial infection.
[047] The terms "treat", "treating" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative treatment. Those in need of treatment
may include individuals already having a particular medical condition as well as
16

those who may ultimately acquire the condition (i.e., those who are susceptible to the
condition and thus needing preventative measures). For example, these terms
encompass any treatment which leads to a reduction in severity of a disease or
condition, reduction in the duration of the disease course, amelioration of one or
more symptoms associated with a disease or condition, beneficial effects to the
patient with a disease or condition, without necessarily curing the disease or
condition and prophylaxis of one or more symptoms associated with a disease or
condition.
[048] The term "domain" as used herein means a region of a polypeptide
(including proteins) having some distinctive physical feature or role including, for
example, an independent structure or a function. Domains refer to a portion of a
polypeptide that may be either native or non-native to the polypeptide. A domain
may contain the amino acid sequence with a distinctive physical feature or it may
contain a fragment of the sequence. A domain may interact with other domains
within a polypeptide or protein. In some embodiments of the invention, an MK2
polypeptide and/or an MK2 interacting protein includes a domain chosen from affinity
tags, radionucleotides, enzymes and fluorophores. Such a domain can be used for
isolation or purification of a complex including MK2 and an interacting protein or for
isolation of a protein that includes the domain. Examples of domains include, but are
not limited to, polyhistidine, FLAG, Glu-Glu, glutathionine S transferase (GST),
thioredoxin, protein A, protein G, and an immunoglobulin heavy chain constant
region.
[049] The term "fusion protein" refers to a protein where a first amino acid
sequence derived from a first source is linked, covalently or non-covalently, to a
second amino acid sequence derived from a second source, wherein the first and
17

second amino acid sequences are not the same. A first source and a second source
that are not the same can include two different biological entities, or two different
proteins from the same biological entity, or a biological entity and a non-biological
entity. A fusion protein can include for example, a protein derived from at least 2
different biological sources. A biological source can include any non-synthetically
produced nucleic acid or amino acid sequence {e.g. a genomic or cDNA sequence, a
plasmid or viral vector, a native virion or a mutant or analog, as further described
herein, of any of the above). A synthetic source can include a protein or nucleic acid
sequence produced chemically and not by a biological system {e.g. solid phase
synthesis of amino acid sequences). A fusion protein can also include a protein
derived from at least 2 different synthetic sources or a protein derived from at least
one biological source and at least one synthetic source.
[050] The term "isolated" in reference to a protein or a polypeptide refers to a
protein or polypeptide separated from its natural or native environment or source.
Thus, a protein or polypeptide isolated from a cell is prepared substantially free of
other polypeptides and components in the cell.
[051] The term "recombinant" as used herein refers to a polypeptide, which
by virtue of its origin or manipulation is not associated with all or portion of a
polypeptide with which it is naturally associated in nature or where such a
polypeptide does not naturally occur in nature.
[052] The term "Y2H" refers to the yeast two-hybrid system of detecting
interactions between two proteins. The two hybrid uses the yeast transcriptional
activator: GAL4, divided into two functionally distinct domains. The DNA binding
domain which when fused to a heterologous protein X retains its DNA binding
activity, and the activation domain which retains its transcriptional activation
18

properties when fused to a heterologous protein Y. The two fusion or hybrid proteins
are co-expressed in yeast. If there is an interaction between protein X and protein Y,
the association will bring the activation domain of the transcriptional activator into
close association with its binding domain, thereby reconstituting a functional
transcriptional activator. This reconstituted transcriptional activator can then drive
the expression of a number of reporter genes, integrated into the yeast genome,
which contain the binding site for the DNA binding domain. Reporter gene
expression is indicative of an interaction between protein X and protein Y.
Detailed Description of the Invention
A. MK2 Interacting Proteins
[053] The present invention relates to proteins that interact with MK2.
Examples of proteins known to interact with MK2 include, but are not limited to SRF,
CREB, ER81, small heat shock protein, leukocyte specific protein 1, E47, Akt, and 5-
lipoxygenase.
[054] HPH2 has previously been shown to bind MK2 by a Y2H system assay
(B. Neufield, Neue Interaktionspartner der MAPKAP-Kinasen 3pK und MK2: die
Polycomb-Proteine HPH2 und Bmi1 sowie der basische Helix-Loop-Helix-
Transkriptionsfaktor E47 (2000) (unpublished Ph.D. dissertation, University of
Wurzburg). This finding was confirmed in the present invention (Figures 2A and 2B,
Figure 9B and Example 5). In addition, STS (Figures 1A to 1B, Figure 9B and
Example 5) and She (Figures 3A and 3B, Figure 9B and Example 5) are shown to
bind MK2 using the Y2H system in the present invention.
[055] Proteins that bind MK2, may be isolated using a variety of methods.
For example, one may use co-immunoprecipitation, as exemplified in Example 7. A
V-5 or HA-tagged MK2 interacting protein, and MYC-tagged MK2 were co-expressed
19

in cells. Lysates of the cells were prepared and immunoprecipitated with an anti-HA
or anti-V5 antibody. Immunoprecipitates were resolved by SDS PAGE and
immunoblotted with an anti-MYC antibody to detect co-immunoprecipitated MK2.
[056] One could also use the yeast two-hybrid (Y2H) system, as exemplified
in Examples 1-5. This method was first formally described by Fields and Song.
(Nature, 340:245-246 (1989)). The two hybrid system uses a yeast transcriptional
activator, such as GAL4, divided into 2 functionally distinct domains. The GAL4 DNA
binding domain retains its DNA binding activity when fused to a heterologous protein
X. The GAL4 activation domain retains its transcriptional activation properties when
fused to a heterologous protein Y. The two fusion or hybrid proteins are co-
expressed in yeast. If there is an interaction between protein X and protein Y, the
association will bring the activation domain of GAL4 into close association with its
binding domain, thereby reconstituting a functional transcriptional activator. This
reconstituted transcriptional activator can then drive the expression of a number of
reporter genes, integrated into the yeast genome, which contain the binding site for
the DNA binding domain. Thus, reporter gene expression is indicative of an
interaction between protein X and protein Y.
[057] The Y2H system offers several advantages over more traditional
methods for studying protein-protein interactions (Luban etal., Curr. Opin. Biotech.,
6:59-64 (1995)). First, the detailed and laborious manipulation of the conditions
necessary for in vitro biochemical binding assays is not needed since the interaction
occurs in vivo. Second, the Y2H system is highly sensitive, and can detect
interactions not revealed by other methods {Fields etal., Trends in Genetics, 10:286-
291 (1994)). Finally, the Y2H system is particularly powerful when it is used to
screen a cDNA library for encoded proteins that interact with a protein of interest.
20

[058] In addition to the Y2H system, a mammalian 2-hybrid system can also
be used for studying the interaction between MK2 and another protein. For example,
293T cells can be transfected with: a plasmid containing a DNA sequence that binds
GAL4 upstream of a reporter gene such as luciferase or chloremphenicol acetyl
transferase (CAT); a plasmid containing cDNA encoding MK2 fused to the DNA-
binding domain of GAL4; and a plasmid containing cDNA encoding a protein that
interacts with MK2, as identified via Y2H, or a putative MK2 interacting protein, fused
to the VP16 activator. Transfected cells are lysed subsequent to co-expression of
MK2 and the interacting protein and the lysates are assayed for reporter gene
activity, which would be detected only when MK2 interacts with the protein. By this
assay, the interaction between MK2 and a protein can be confirmed in mammalian
cells. The mammalian two-hybrid system can also be used to validate the interaction
between MK2 and another protein, as identified via the Y2H system.
[059] In addition to using co-immunoprecipitation or two-hybrid systems, one
may use a low stringency screening of a cDNA library, or use degenerate PCR
techniques using a probe directed toward a sequence encoding a MK2 binding
domain of a protein that binds MK2. As more genomic data becomes available,
similarity searching using a number of sequence profiling and analysis programs,
such as MotifSearch (Genetics Computer Group, Madison, Wl), ProfileSearch
(GCG), and BLAST (NCBI) could be used to find novel proteins containing
sequences significant homology with MK2 binding domains of proteins that bind
MK2.
[060] One may also use a proteomics approach to identify MK2 interacting
proteins, as shown in Example 11. Wild type (+/+) or MK2 deficient (-/-) cells were
plated and labeled with 33P. MK2 was activated for 30 minutes following which
21

whole cell lysate were prepared and analyzed using two-dimensional gel
electrophoresis. Gels were compared to identify differentially phosphorylated
proteins. Figure 12 shows a differentially phosphorylated protein (arrow) using this
approach. Differentially phosphorylated proteins may also be identified using mass
spectrometry.
[061] A protein that binds MK2 may stimulate MK2, inhibit MK2, or have no
effect on MK2 activity. There are several ways to investigate whether a protein that
binds MK2 causes an inhibition or stimulation of MK2, thus having biological activity.
Proteins that bind MK2 that render a change in MK2 activity, particularly a decrease
in MK2 activity, are particularly good candidates for use as therapeutic agents and
as inhibitors of inflammation. In addition, a fragment or mutant of a protein that
naturally stimulates MK2 may be found to inhibit MK2, and would thus be a
candidate as an inhibitor of inflammation. For example, once a protein that binds
MK2 is identified and it is shown to stimulate MK2, mutations in the protein can be
made, such that the protein still interacts with MK2 but has an inhibitory effect on
MK2 activity. Mutant forms of an MK2 interacting protein can be tested for an effect
on MK2 activity in one or more of the assays provided herein. Proteins that bind
MK2 but have no effect on its activity can also be used as therapeutic agents. Such
proteins may, for example, compete with endogenous proteins that normally bind
MK2 to stimulate MK2 activity.
[062] To investigate whether a protein that binds MK2 affects its activity, one
could determine the effect of the binding proteins on MK2 activity such as, for
example, MK2 kinase activity. For example, an HA-tagged MK2 interacting protein
(for example, She), and Myc-tagged MK2 can be co-expressed in 293T cells. Cell
lysates are prepared and resolved by SDS PAGE. Subsequent immunoblotting with
22

an antibody to detect activated MK2 (for example, anti-phospho MK2 threonine 334),
will determine the activation state of MK2. Alternatively, the effect of the MK2
binding on MK2 kinase activity can be determined by quantitating the amount of
phosphorylated form of a known substrate for MK2.
[063] In addition, one could determine the effect of an MK2 interacting
protein on TNF-a biosynthesis, as exemplified in Example 9. An HA-tagged MK2
interacting protein (for example, She), and MYC-tagged MK2 can be co-transfected
into appropriate cells such as RAW, along with a TNF luciferase reporter gene. Cells
are either unstimulated or stimulated by anisomycin. Media is collected to assay for
TNF- production and cell lysates are prepared to determine luciferase activity. TNF
biosynthesis in the presence of an MK2 binding protein is compared to that in a
control sample.
[064] As exemplified in Example 10, one could also determine the effect of
MK2 on the phosphorylation state of an MK2 interacting protein. An HA-tagged MK2
interacting protein (for example, She) is expressed in 293T cells. Lysates are
prepared and immunoprecipitated with an anti-HA antibody. The immunoprecipitates
are used in an in vitro kinase assay with recombinant MK2 as the kinase. SDS
PAGE followed by phospho-imagery is used to detect phosphorylation of the MK2
interacting protein.
B. Nucleotide and Protein Sequences
[065] While not always necessary, if desired, one of ordinary skill in the art
may determine the amino acid or nucleic acid sequences of novel proteins that bind
MK2. For example, the present invention provides the cDNA sequences encoding
STS, HPH2, and She (Figures 1A and 1B; 2A and 2B; 3A and 3B; and SEQ ID NOS:
23

1-3). The present invention also provides the corresponding amino acid sequences
of these proteins (Figures 4-6; SEQ ID NOS: 4-6).
[066] The present invention also includes splice variants, truncations,
fragments, substitutions, additions or deletion mutations, fusion proteins, shuffling
mutants, motif sequences, and homologues of such nucleic and amino acid
sequences. For example, the nucleic or amino acid sequence may comprise a
sequence at least 70% to 79% identical to the nucleic acid or amino acid sequence
of the native protein, or at least 80% to 89% identical, or at least 90% to 95%
identical, or at least 96% to 100% identical. One of skill in the art will recognize that
the region that binds MK2 can tolerate less sequence variation than the other
portions of the protein not involved in binding. Thus, these non-binding regions of an
MK2 interacting protein may contain substantial variations without significantly
altering the binding of the protein to MK2. However, one of skill in the art will also
recognize that many changes can be made to specifically increase the affinity of the
protein for its target. Such affinity-increasing changes are typically determined
empirically by altering the amino acid sequence, for example, within the MK2-binding
region, and testing the ability of the protein to bind MK2 or by determining the
strength of such binding. All such alterations, whether within or outside a MK2
binding region within a protein, are included in the scope of the present invention.
[067] One of skill in the art will recognize that proteins that bind MK2 may
contain any number of conservative changes to their respective amino acid
sequences without altering their biological properties. Such conservative amino acid
modifications are based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the
like. Exemplary conservative substitutions which take such characteristics into
24

consideration are well known to those of skill in the art and include, for example,
arginine to lysine or lysine to arginine; glutamate to aspartate or aspartate to
glutamate; serine to threonine or threonine to serine; glutamine to asparagine or
asparagine to glutamine; valine to leucine or isoleucine, leucine to valine or
isoleucine, and isoleucine to valine or leucine. Furthermore, proteins that bind MK2
may be used to generate functional fragments that bind and inhibit MK2 activity.
[068] Relative sequence similarity or identity may be determined using the
"Best Fit" or "Gap" programs of the Sequence Analysis Software Package™ (Version
10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center,
Madison, Wl). "Gap" utilizes the algorithm of Needleman and Wunsch (Needleman
and Wunsch, 1970) to find the alignment of two sequences that maximizes the
number of matches and minimizes the number of gaps. "BestFit" performs an
optimal alignment of the best segment of similarity between two sequences. Optimal
alignments are found by inserting gaps to maximize the number of matches using
the local homology algorithm of Smith and Waterman {Smith and Waterman, J Theor
Biol., 91(2):379-80(1981)).
[069] The Sequence Analysis Software Package described above contains a
number of other useful sequence analysis tools for identifying homologues of the
presently disclosed nucleotide and amino acid sequences. For example, the
"BLAST" program (Altschul et al., J Mol Biol., 215(3):403-10 (1990)) searches for
sequences similar to a query sequence (either peptide or nucleic acid) in a specified
database (e.g., sequence databases maintained at the NCBI; "FastA" (Lipman and
Pearson, Science, 227(4693): 1435-41 (1985); Pearson and Lipman, Proc. Natl.
Acad. Sci. USA, (8):2444-8 (1988)) performs a Pearson and Lipman search for
similarity between a query sequence and a group of sequences of the same type
25

(nucleic acid or protein); "TfastA" performs a Pearson and Lipman search for
similarity between a protein query sequence and any group of nucleotide sequences
(it translates the nucleotide sequences in all six reading frames before performing
the comparison); "FastX" performs a Pearson and Lipman search for similarity
between a nucleotide query sequence and a group of protein sequences, taking
frameshifts into account. "TfastX" performs a Pearson and Lipman search for
similarity between a protein query sequence and any group of nucleotide sequences,
taking frameshifts into account (it translates both strands of the nucleic sequence
before performing the comparison).
[070] The invention encompasses fragments of proteins that bind MK2.
Such fragments will likely include all or a part of an MK2 binding region in the
protein. Fragments may include all, a part, or none of the sequences between the
region that binds MK2 and the N-terminus of the protein and/or between the region
that binds MK2 and the C-terminus of the protein.
[071] It is understood by one of ordinary skill in the art that certain amino
acids may be substituted for other amino acids in a protein without adversely
affecting the activity of the protein, e.g., binding characteristics of a protein that binds
MK2. It is thus contemplated by the inventors that various changes may be made in
the amino acid sequences of proteins that bind MK2, or DNA sequences encoding
the proteins, without appreciable loss of their biological utility or activity. Such
changes may include splice variants, truncations, fragments, substitution, addition
and deletion mutations, shuffling mutations, motif sequences, fusion proteins,
homologues, and the like.
[072] In making such changes, the hydropathic index of amino acids may be
considered. The importance of the hydropathic amino acid index in conferring a
26

biological function of a protein is generally understood in the art (Kyte and Doolittle,
J. Mol. Biol., 157: 105-132 (1982)). It is accepted that the relative hydropathic
character of an amino acid contributes to the secondary structure of the resultant
protein, which in turn defines the interaction of the protein with other molecules, for
example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
[073] Each amino acid has been assigned a hydropathic index on the basis
of its hydrophobicity and charge characteristics; these are isoleucine (+4.5), valine
(±4.2), leucine (±3.8), phenylalanine (±2.8), cysteine/cystine (±2.5), methionine
(±1.9), alanine (±1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9),
tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5),
aspartate (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5). In making such
changes, the hydropathic indices of substituted amino acids may be within ±2, within
±1, and within ±0.5 of the amino acids that are replaced.
[074] It is also understood in the art that the substitution of like amino acids
can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101 states
that the greatest local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with a biological property of the
protein.
[075] As detailed in U.S. Patent 4,554,101, the following hydrophilicity values
have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate
(±3.0±1), glutamate (+3.0±1), serine (+0.3), asparagine (+0.2), glutamine (+0.2),
glycine (0), threonine (-0.4), proline (-0.5±1), alanine (-0.5), histidine (-0.5), cysteine
(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3),
phenylalanine (-2.5), and tryptophan (-3.4). In making such changes, the
27

hydropathic indices of substituted amino acids may be within ±2, within ±1, and
within ±0.5 of the amino acids that are replaced.
[076] The modifications may be conservative such that the structure or
biological function of the protein is not affected by the change. Such conservative
amino acid modifications are based on the relative similarity of the amino acid side-
chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and
the like. Exemplary conservative substitutions which take various of the foregoing
characteristics into consideration are well known to those of skill in the art and
include for example, arginine to lysine or lysine to arginine; glutamate to aspartate or
aspartate to glutamate; serine to threonine or threonine to serine; glutamine to
asparagine or asparagine to glutamine; valine to leucine or isoleucine, leucine to
valine or isoleucine, and isoleucine to valine or leucine. Amino acid sequences of
proteins that bind MK2 may be modified to have any number of conservative
changes, so long as the binding of the protein to MK2 is not adversely affected.
Such changes may be introduced within or outside of a portion of the protein that
binds the target. For example, changes introduced within the binding portion of the
protein may be designed to increase the affinity of the protein for its target.
C. Stabilizing Modification
[077] Stabilizing modifications are capable of stabilizing a protein, enhancing
the in vitro and/or in vivo half life of a protein, enhancing circulatory half life of a
protein and/or reducing proteolytic degradation of a protein. Such stabilizing
modifications include but are not limited to fusion proteins, modification of a
glycosylation site, and modification of carbohydrate moiety. As it is well known in the
art, fusion proteins are prepared such that a second protein is fused in frame with the
a first protein resulting in a translated protein comprising both the first and second
28

proteins. For example, in the present invention, a fusion protein may be prepared
such that a protein that binds MK2 is fused to a second protein (e.g. a stabilizer
protein portion). As will be recognized by one of ordinary skill in the art, such a
fusion protein may optionally comprise a linker peptide between the protein that
binds MK2 and the stabilizing protein portion. A stabilizer protein may be any protein
that enhances the overall stability of the protein that binds MK2.
[078] Proteins that bind MK2 can be glycosylated or be linked to albumin or a
nonproteinaceous polymer. For instance, proteins that bind MK2 may be linked to
one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol,
polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent
Numbers 4,640,835; 4,791,192; or 5,414,135. Proteins are chemically modified by
covalent conjugation to a polymer to increase their circulating half-life, for example.
Polymers, and methods to attach them to peptides, are also shown in U.S. Pat. Nos.
4,766,106 and 4,609,546.
[079] Proteins that bind MK2 can be stabilized by preparing a fusion protein
comprising a protein that binds MK2 and an immunoglobulin sequence, as
exemplified in U.S. Patent No. 5,864,020. The immunoglobulin sequence preferably,
but not necessarily, is an immunoglobulin constant domain. The immunoglobulin
moiety in the chimeras of the present invention may be obtained from lgG-1, lgG-2,
lgG-3 or lgG-4 subtypes, IgA, IgE, IgD or IgM, but preferably lgG-1 or lgG-3. In one
embodiment, the Fc fragment of IgG fused to the protein that binds MK2.
[080] Proteins that bind MK2 may be pegylated. Pegylation is a process
whereby polyethylene glycol (PEG) is attached to a protein in order to extend the
half-life of the protein in the body. Pegylation of proteins that bind MK2 may
decrease the dose or frequency of administration of the proteins needed for an
29

optimal decrease in inflammation. Reviews of the technique are provided in Bhadra
et al., Pharmazie, 57: 5-29 (2002), and in Harris et al., Clin. Pharmacokinet, 40: 539-
551 (2001).
[081] Proteins that bind MK2 may be modified to have an altered
glycosylation pattern (i.e., altered from the original or native glycosylation pattern).
As used herein, "altered" means having one or more carbohydrate moieties deleted,
and/or having at least one glycosylation site added to the original protein.
[082] Glycosylation of proteins is typically either N-linked or O-linked.
N-linked refers to the attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences, asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline, are the
recognition sequences for enzymatic attachment of the carbohydrate moiety to the
asparagine side chain. Thus, the presence of either of these tripeptide sequences in
a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to
the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a
hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[083] Addition of glycosylation sites to proteins that bind MK2 is conveniently
accomplished by altering the amino acid sequence of the protein such that it
contains one or more of the above-described tripeptide sequences (for N-linked
glycosylation sites). The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues in the sequence of the
original protein (for O-linked glycosylation sites). The protein's amino acid sequence
may also be altered by introducing changes at the DNA level.
30

[084] Another means of increasing the number of carbohydrate moieties on
proteins is by chemical or enzymatic coupling of glycosides to the amino acid
residues of the protein. Depending on the coupling mode used, the sugars may be
attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl
groups such as those of cysteine, (d) free hydroxyl groups such as those of serine,
threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine,
tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are
described in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev. Biochem., 22:
259-306(1981).
[085] Removal of any carbohydrate moieties present on proteins that bind
MK2 may be accomplished chemically or enzymatically. Chemical deglycosylation
requires exposure of the protein to trifluoromethanesulfonic acid, or an equivalent
compound. This treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the amino
acid sequence intact.
[086] Chemical deglycosylation is described by Hakimuddin et al., Arch.
Biochem. Biophys., 259: 52 (1987); and Edge etal., Anal. Biochem., 118: 131
(1981). Enzymatic cleavage of carbohydrate moieties on proteins can be achieved
by the use of a variety of endo- and exo-glycosidases as described by Thotakura et
a/., Meth. Enzymol., 138: 350 (1987).
[087] Proteins that bind MK2 may be linked to the protein albumin or a
derivative of albumin. Methods for linking proteins and polypeptides to albumin or
albumin derivatives are well known in the art. See, for example, U.S. Patent No.
5,116,944.
31

D. Screening for Compounds that Modulate MK2 Activity
1. Use of the Yeast-2 Hybrid System for Drug Screening
[088] The present invention also provides methods of screening for drugs
that modulate MK2 activity, including but not limited to, antibodies, chemical agents,
small molecules, proteins and peptides. In some embodiments, once a protein-
protein interaction has been detected between MK2 and another protein by Y2H, the
protein-protein interaction in Y2H can be used for high-throughput drug screening.
For example, once a protein-protein interaction is detected, the positive yeast
colonies (identified by color and growth, as described) harboring the two proteins
such as MK2 and an interacting protein, can be treated with a drug including
antibodies, chemical agents, peptides, proteins or small molecules. A change in
color or growth of the positive yeast colonies in the presence of the drug would be
indicative of an effect on the interaction between the two proteins.
[089] In some embodiments, an MK2 interacting protein stimulates MK2
activity, which has a pro-inflammatory effect in a host including a cell, a tissue or a
whole organism. Therefore, it would be desirable to identify drugs that would disrupt
such an interaction. Accordingly, the Y2H system can be used to identify drugs that
would disrupt the interaction between MK2 and a protein that stimulates MK2
activity, thereby leading to use of such drugs in the treatment or prevention of
inflammation.
[090] In other embodiments, an MK2 interacting protein inhibits MK2 activity,
resulting in an anti-inflammatory effect in a host including a cell, a tissue or a whole
organism. In such a case, the Y2H system can be used for identifying drugs that
strengthen such an interaction, which may be monitored by changes in color and
32

growth of yeast cells harboring the two proteins, as described herein. Such a drug
can subsequently be used in the treatment or prevention of inflammation.
[091] As discussed above, in case of certain conditions such as certain
bacterial infections, it is desirable to have enhanced MK2 activity. Accordingly, the
Y2H system can also be used for screening for drugs that strengthen the interaction
between MK2 and an interacting protein, resulting in enhanced MK2 activity, and
subsequently enhanced resistance to bacterial infection. Such drugs can be used
for treatment or prevention of, for example, certain bacterial infections such as
Listeria monocytogenes infection.
2. Use of an In Vitro Reconstitution System for Drug Screening
[092] An in vitro reconstitution system can also be used for identification of
drugs including but not limited to, small molecules, antibodies, peptides and
chemical agents that modulate MK2 activity. For example, subsequent to the
identification of a protein-protein interaction by any of the assays provided herein,
the proteins can be treated in vitro with drugs that will inhibit interaction between the
two proteins. As discussed above, it is desirable to identify drugs that would inhibit
the interaction between MK2 and another protein, where such an interaction
stimulates MK2 activity. In addition, an in vitro reconstitution system can be used for
formation and isolation of protein complexes which include MK2 and at least one
MK2 interacting protein. These complexes can subsequently be used for
identification of compounds that inhibit or promote complex formation, thereby
modulating inflammation and/or inhibit or stimulate activity of Mk2 in the complex,
thereby modulating inflammation.
[093] MK2 and an interacting protein are synthesized and 35S-labeled in an in
vitro translation system such as the rabbit reticulocyte lysate-coupled transcription-
33

translation system supplied by Promega (Madison, Wl). Interaction between MK2
and the protein is confirmed by co-immunoprecipitation as described. Alternatively,
recombinant MK2 and an interacting protein can be produced using an expression
system such as E. coli or baculovirus insect cells. The interaction between these two
proteins can then be detected by pull-down assays similar to co-
immunoprecipitation, ELISA or fluorescence resonance energy transfer (FRET). A
test compound is added to the mixture of in vitro translated proteins and co-
immunoprecipitation is performed as described. A desirable compound is one that
inhibits the interaction between MK2 and the protein, as determined by the lack of
interaction or reduced interaction in an immunoprecipitation assay or a pull-down
assay in case of recombinantly produced proteins. Such a compound can
subsequently be used for the treatment or prevention of inflammation. Examples of
compounds that can be tested for their effect on inflammation in assays of the
invention include chemical agents, small molecules, peptides, proteins and
antibodies.
[094] An in vitro reconstitution system can also be used to identify
compounds that bind MK2 and inhibit MK2 activity. For example, in vitro translated
MK2 or purified MK2 can be incubated with a compound and subsequently tested for
its ability to either phosphorylate a known substrate such as Hsp 27 or increase
TNF-a biosynthesis, as described here. However, any assay can be used which
measures MK2 activity. Thus, those compounds that inhibit MK2 activity are
identified as drugs that can be used for inhibition or prevention of inflammation.
Whereas, the compounds that increase MK2 activity can be used for treatment of
certain conditions where an increase in MK2 activity is desired.
34

E. Pharmaceutical Compositions
[095] The present invention provides compositions including proteins that
bind MK2. Such compositions may be suitable for pharmaceutical use and
administration to patients. The compositions typically contain one or more proteins
that bind MK2 and a pharmaceutically acceptable excipient. The compositions also
include protein complexes that contain MK2 and at least one MK2 interacting protein.
As used herein, the phrase "pharmaceutically acceptable excipient" includes any and
all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents, and the like, that are compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. The compositions may
also contain other active compounds providing supplemental, additional, or
enhanced therapeutic functions. The pharmaceutical compositions may also be
included in a container, pack, or dispenser together with instructions for
administration.
[096] Pharmaceutical compositions of the invention also include
compositions that comprise compounds including small molecules, antibodies,
chemical agents, proteins and peptides that are identified by one or more methods
described herein. Appropriate dosages for administration of these compounds for
treatment or prevention of inflammation can easily be determined by a physician.
Examples of dosages include, but are not limited to, 5 mg to 500 mg, 50 mg to 250
mg, 100 mg to 200 mg, 50 mg to 100 mg, 15 mg to 85 mg, 30 mg to 70 mg, and 40
mg to 60 mg.
[097] A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Methods to accomplish the
35

administration are known to those of ordinary skill in the art. The administration may,
for example, be intravenous, intramuscular, rectal, or subcutaneous.
[098] Solutions or suspensions used for subcutaneous application typically
include one or more of the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol
or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl
parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents
such as ethylenediaminetetra acetic acid; buffers such as acetates, citrates or
phosphates; and agents for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or
sodium hydroxide. Such preparations may be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[099] Pharmaceutical compositions suitable for injection include sterile
aqueous solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline, bacteriostatic water,
Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all
cases, the composition must be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of microorganisms
such as bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the case of dispersion
36

and by the use of surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, one
may include isotonic agents, for example, sugars, polyalcohols such as manitol,
sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and gelatin.
[0100] In some embodiments, proteins that bind MK2 are prepared with
carriers that will protect the protein against rapid elimination from the body, such as a
controlled release formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will be apparent to
those skilled in the art. The materials can also be obtained commercially from Alza
Corporation (Mountain View, CA) and Nova Pharmaceuticals. Liposomal
suspensions containing proteins that bind MK2 can also be used as
pharmaceutically acceptable carriers. These can be prepared according to methods
known to those skilled in the art, for example, as described in U.S. Patent No.
4,522,811.
[0101] Additional therapeutically useful agents beneficial for the condition
being treated may optionally be included in or administered simultaneously or
sequentially with proteins that bind MK2.
[0102] It is especially advantageous to formulate compositions in dosage unit
form for ease of administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary dosages for the subject to
37

be treated. Each unit typically contains a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in association with
the required pharmaceutical carrier. The specification for the dosage unit forms of
the invention are dictated by and directly dependent on the unique characteristics of
an active compound and the particular therapeutic effect to be achieved, and the
limitations inherent in the art of using such an active compound for the treatment of
individuals.
F. Treatment Indications
[0103] Compounds that interact with at least one of MK2 or an MK2 complex
are useful to prevent, diagnose, or treat various medical conditions in humans or
animals. Accordingly, the present invention provides a method for treating
conditions related to inflammation, by administering to a subject a composition
comprising at least one compound (such as a protein, peptide, antibody, chemical
agent, and small molecule) that interacts with at least one of MK2 or an MK2
complex in an amount sufficient to ameliorate the symptoms of the condition. Such
conditions include Crohn's disease, inflammatory bowel disease, ulcerative colitis,
rheumatoid arthritis, acute respiratory distress syndrome, emphysema, delayed type
hypersensitivity reaction, asthma, systemic lupus erythematosus, and inflammation
due to trauma or injury or stroke.
G. Methods of Treatment Using Compounds that Interact with MK2 or an
MK2 Complex
[0104] Compounds that interact with MK2 or an MK2 complex, and modulate
MK2 activity may be used to inhibit or reduce one or more symptoms associated with
inflammation. In an embodiment, inflammation is inhibited at least 50%, or at least
60, 62, 64, 66, 68, 70, 72, 72, 76, 78, 80, 82, 84, 86, or 88%, or at least 90, 91, 92,
38

93, or 94%, or at least 95% to 100%. Compounds may be used individually or in
combination.
[0105] Pharmaceutical preparations comprising compounds, as described
herein, are administered in therapeutically effective amounts. A compound that
modulates MK2 activity can be selected from a protein, a peptide, an antibody, a
chemical agent or a small molecule. As used herein, an "effective amount" of the
compound is a dosage that is sufficient to reduce inflammation to achieve a desired
biological outcome. Such improvements may be measured by a variety of methods
including those that measure symptoms such as pain, swelling, or redness. For
example, an American College of Rheumatology (ACR) score is used to measure
inflammation in rheumatoid arthritis. An ACR score is defined as £20%, 50%, or
70% improvement in tender joint and swollen joint count plus > 20%, 50%, or 70%
improvement in at least 3 of the following 5 criteria: patient pain assessment,
physician and patient global assessments, patient self-assessed disability, and acute
phase reactant (erythrocyte sedimentation rate and C-reactive protein level).
However, it is understood that a physician will be able to diagnose and measure
inflammation in any disorder and determine whether a decrease in inflammation is
achieved using an anti-inflammatory drug identified using methods of the invention.
[0106] Generally, a therapeutically effective amount may vary with the
subject's age, condition, and sex, as well as the severity of the medical condition in
the subject. The dosage may be determined by a physician and adjusted, as
necessary, to suit observed effects of the treatment. Appropriate dosages for
administering at least one compound that interacts with MK2 or an MK2 complex,
and modulates MK2 activity, may range from 5 mg to 500 mg, 50 mg to 250 mg, 100
mg to 200 mg, 50 mg to 100 mg, 15 mg to 85 mg, 30 mg to 70 mg, and 40 mg to 60
39

mg. These compounds can be administered in one dose, or at intervals such as
once daily, once weekly, and once monthly. Dosage schedules can be adjusted
depending on the ability of the protein to decrease inflammation, the half-life of the
protein, or the severity of the patient's condition. Generally, the compositions are
administered as a bolus dose, to maximize the circulating levels of these compounds
for the greatest length of time after the dose. Continuous infusion may also be used
after the bolus dose.
[0107] Toxicity and therapeutic efficacy of such proteins or compounds can be
determined by standard pharmaceutical procedures. Experiments could be
performed in cell culture to determine an effect of the proteins or compounds on
cytokine expression or activity. Experiments could also be performed in
experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects of a compound is
its therapeutic index, which can be expressed as LD50/ED50. Compounds that
interact with MK2 or an MK2 complex, and modulate MK2 activity, including but not
limited to, peptides, proteins, antibodies, chemical agents and small molecules, and
which exhibit large therapeutic indices may be used in methods of treatment of the
invention.
[0108] Data obtained from cell culture assays and animal studies can be used
in evaluating a range of dosage for use in humans. A dosage of such proteins and
compounds may lie within a range of circulating concentrations that include the ED50
value with little or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration utilized. For any
compound that interacts with MK2 or an MK2 complex, the therapeutically effective
40

dose can be estimated initially from cell culture assays as described. A dose may be
formulated in animal models to achieve a circulating plasma concentration range that
includes the IC50 value (i.e., the concentration of the test protein which achieves a
half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma
may be measured, for example, by high performance liquid chromatography. Effects
of any particular dosage can be monitored by a suitable bioassay.
H. Methods of Treatment Using Cells
[0109] Another way to administer proteins, peptides, or antibodies that interact
with MK2 or an MK2 complex, and modulate MK2 activity, to a host is to administer
cells that express these compounds. Various methods can be used to deliver cells
expressing proteins, peptides or antibodies to a site for use in modulating
inflammation. In one embodiment of the invention, cells expressing a protein,
peptide or antibody can be administered by targeted delivery, for example, direct
injection of a sample of such cells into a specific site in a tissue that has
inflammation. The cells can be delivered in a medium or matrix that partially
impedes their mobility so as to localize the cells to a site of interest. Such a medium
or matrix could be semi-solid, such as a paste or gel, including a gel-like polymer.
Alternatively, the medium or matrix could be in the form of a solid, a porous solid
which will allow the migration of cells into the solid matrix, and hold them there while
allowing proliferation of the cells. In some embodiments, a host cell includes a first
nucleic acid encoding a recombinant MK2 polypeptide and a second nucleic acid
encoding an MK2 interacting protein such as, for example, STS, HPH2 and She. An
MK2 complex containing MK2 and at least one other protein can subsequently be
isolated from the host cell.
41

I. Methods of Expressing DNA in a Cell
[0110] DNA encoding proteins, peptides, and antibodies that interact with MK2
or an MK2 complex, and modulate MK2 activity, can be introduced into a cell.
Proteins, peptides and antibodies encoded by the DNA can then be expressed in
such a cell. In some embodiments of the invention, a DNA molecule encoding a
protein, peptide, or antibody that interacts with MK2 or an MK2 complex, thereby
modulating MK2 activity, could be introduced into a cell in order to alter the
production or activity of cytokines in the cell. Specifically, a DNA molecule encoding
a protein, peptide or antibody could be introduced into a cell to reduce or inhibit the
production or activity of cytokines.
[0111] Delivery of polynucleotide sequences of proteins, peptides or
antibodies can be achieved using a recombinant expression vector such as a
chimeric virus or a colloidal dispersion system. Target liposomes may be used for
therapeutic delivery of the polynucleotide sequences. Various viral vectors that can
be utilized for introducing DNA into cells include adenovirus, herpes virus, vaccinia,
or an RNA virus such as a retrovirus. A retroviral vector may be a derivative of a
murine or avian retrovirus. Examples of retroviral vectors in which a single foreign
gene can be inserted include, but are not limited to: Moloney murine leukemia virus
(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus
(MuMTV), and Rous sarcoma virus (RSV). A number of additional retroviral vectors
can incorporate multiple genes, for example, vectors that can encode polycistronic
messages or those that include multiple promoters. All of these vectors can transfer
or incorporate a gene for a selectable marker so that transduced cells can be
identified and generated.
42

[0112] Since recombinant retroviruses are defective, they require helper cell
lines that contain plasmids encoding all of the structural genes of a retrovirus under
the control of regulatory sequences within the long terminal repeat sequences of
viruses. These plasmids are missing a nucleotide sequence that enables the
packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell
lines that have deletions of the packaging signal include, but are not limited to, for
example, PSI.2, PA317 and PA12. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into cells in which the
packaging signal is intact, but the structural genes are replaced by other genes of
interest, the vector can be packaged and vector virion produced.
[0113] Alternatively, a second type of cell in tissue culture can be directly
transfected with a plasmid encoding the retroviral structural genes gag, pol and env,
by conventional calcium phosphate transfection. These cells are then transfected
with a vector plasmid containing the genes of interest. The resulting cells release
the retroviral vector into the culture medium, and the vectors are subsequently
introduced into appropriate cells.
[0114] Another targeted delivery system for a polynucleotide encoding a
protein, peptide or antibody is a colloidal dispersion system. Colloidal dispersion
systems include macromolecule complexes, nanocapsules, microspheres, beads,
and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes. Liposomes are artificial membrane vesicles that are useful as
delivery vehicles. RNA, DNA and intact virions can be encapsulated within the
aqueous interior and be delivered to cells in a biologically active form (see, for
example, Fraley et al., Trends Biochem. Sci., 6: 77 (1981)). Methods for efficient
gene transfer into a cell using a liposome vehicle are known in the art (see, for
43

example, Mannino et al., Biotechniques, 6: 682 (1988). The composition of liposome
usually includes a combination of phospholipids, typically in combination with
steroids, especially cholesterol. Other phospholipids or lipids may also be used. The
physical characteristics of liposomes depend on pH, ionic strength, and the presence
of divalent cations.
[0115] Examples of lipids useful in liposome production include phosphatidyl
compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
Illustrative phospholipids include egg phosphatidylcholine,
dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
J. Methods of Expressing DNA in a Tissue. Organ, or Organism
[0116] DNA encoding proteins that bind MK2, including antibodies, and DNA
encoding proteins, peptides and antibodies that modulate MK2 activity by binding an
MK2 complex can be introduced into a cell within a tissue, an organ, or an organism.
Proteins, peptides or antibodies encoded by the DNA can then be expressed in the
cell of the tissue, organ, or organism. In one embodiment of the invention, DNA
encoding proteins, peptides or antibodies could be introduced to a cell in order to
alter the production or activity of cytokines in the cells of the tissue, organ, or
organism. This method could be used to decrease inflammation in tissues, organ, or
organism. The invention is useful for treating conditions such as Crohn's disease,
inflammatory bowel disease, ulcerative colitis, rheumatoid arthritis, acute respiratory
distress syndrome, emphysema, delayed type hypersensitivity reaction, asthma,
systemic lupus erythematosus, and inflammation due to trauma or injury.
[0117] In one embodiment of the invention, DNA encoding proteins, peptides,
or antibodies that interact with MK2 or an MK2 complex, and modulate MK2 activity,
44

can be targeted to a specific cell type of interest. The cell type can be a component
of a tissue, organ, or organism. By inserting a sequence of interest into the viral
vector, along with another gene which encodes the ligand for a receptor on a specific
target cell, for example, the vector is now specific for a certain cell type, and thus
may be specific for a certain tissue, organ, or organism. Retroviral vectors can be
made target specific by attaching, for example, a sugar, a glycolipid, or a protein.
Targeting may be accomplished by using an antibody. Those of skill in the art will
recognize that specific polynucleotide sequences can be inserted into the retroviral
genome or attached to a viral envelope to allow target specific delivery of the
retroviral vector containing the polynucleotide of proteins, peptides or antibodies.
The targeting of liposomes is also possible based on cell specificity as known in the
art.
[0118] In another embodiment of the invention, cells can be removed from a
tissue, organ, or organism, DNA encoding a protein, peptide or antibody that
interacts with MK2 or an MK2 complex, and modulates MK2 activity, can be
introduced into the cells, and the cells can be reintroduced into the tissue, organ, or
organism. These cells would then produce the protein, peptide, or antibody when
delivered into a tissue, organ, or organism of interest. Cells can be reintroduced to
the tissue, organ, or organism by the methods described above.
K. Methods of Detection and Isolation of MK2
[0119] Compounds that interact with at least one of MK2 or an MK2 complex
may be used to detect the presence or amount of MK2, in vivo or in vitro. These
include proteins, peptides, antibodies, chemical agents, and small molecules. By
correlating the presence or level of MK2 interacting proteins with a medical condition,
45

one of skill in the art can diagnose the associated medical condition. The medical
conditions that may be diagnosed by these compounds are set forth herein.
[0120] Such detection methods are well known in the art and include ELISA,
radioimmunoassay, immunoblot, Western blot, immunofluorescence, immuno-
precipitation, and other comparable techniques. These compounds may further be
provided in a diagnostic kit that incorporates one or more of these techniques to
detect MK2. Such a kit may contain other components, packaging, instructions, or
other materials to aid detection of MK2 and uses of the kit.
[0121] Where compounds that interact with MK2 or an MK2 complex, and
modulate MK2 activity, are intended for diagnostic purposes, it may be desirable to
modify them, for example with a ligand group (such as biotin) or a detectable marker
group (such as a fluorescent group, a radioisotope or an enzyme). If desired, they
may be labeled using conventional techniques. Suitable labels include fluorophores,
chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands
having specific binding partners. Enzymes are typically detected by their activity.
For example, horseradish peroxidase is usually detected by its ability to convert
3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a
spectrophotometer. Other suitable binding partners include biotin and avidin or
streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in
the art. Other permutations and possibilities will be readily apparent to those of
ordinary skill in the art, and are considered as equivalents within the scope of the
instant invention.
[0122] Compounds that interact with at least one of MK2 or an MK2 complex
may also be useful for isolating MK2 in a purification process. In one type of
process, compounds may be immobilized, for example, through incorporation into a
46

column or resin. The compounds are used to bind MK2, and then subjected to
conditions which result in the release of the bound MK2. Such processes may be
used for the commercial production of MK2.
[0123] The following examples provide various embodiments of the invention.
One of ordinary skill in the art will recognize the numerous modifications and
variations that may be performed without altering the spirit or scope of the present
invention. Such modifications and variations are believed to be encompassed within
the scope of the invention. The examples do not in any way limit the invention. It is
understood that all of the numbers in the specification and claims are modified by the
term about, as small changes in dosages, for example, would be considered to be
within the scope of the invention.
EXAMPLES
Example 1: Preparation of the cDNA Library
[0124] Methods for performing the yeast two-hybrid screening were adapted
from the Pretransformed Matchmaker Libraries User Manual published by Clontech
(Palo Alto, California, USA; versions PT3183-1; PR1X299).
[0125] A pretransformed human bone marrow cDNA library was purchased
from Clontech (# HY4053AH). This cDNA library was pretransformed into
Saccharomyces cerevisiae host strain Y187 (Clontech). The yeast cells were kept
frozen before use.
[0126] DNA-bait construct in a yeast reporter strain AH109 (Clontech) served
as a mating partner for the Y187 yeast.
Example 2: Preparation of the MK2-Bait Construct
[0127] To prepare the MK2 construct comprising a binding domain (BD), an
MK2 gene fragment encoding the full length kinase in which amino acid lysine 93, in
47

the ATP binding pocket had been mutated to an arginine was generated by
directional cloning into the DNA-BD vector, pGBKT7 (Clontech) to generate MK
K93R-pGBKT7. The lysine at position 93 was converted to an arginine to produce a
catalytically inactive form of the kinase. Specifically, the pGBKT7 vector was
digested with Nde-Xho, and purified using agarose gel electrophoresis. The MK2
fragment was ligated into the pGBKT7 vector by using T4 DNA ligase. Plasmids
containing the inserts were identified by restriction analysis. Yeast were transformed
with the MK2 construct and MK2 expression was subsequently confirmed in the
transformed AH109 yeast strain.
[0128] Toxicity of the protein encoded by the MK2 construct on the host cell
was investigated by comparing the growth rate of cells transformed with the MK2
construct and cells transformed with the empty vector. The MK2 construct was
determined to be not toxic. Cells with the bait construct or with the empty vector
grew at substantially the same rate and cell number.
[0129] To analyze transcriptional activation, cells transformed with the MK2
construct were plated on SD/-Trp/X-a-Gal (Clontech), SD/-His/-Trp/X-a-Gal
(Clontech), and SD/-Ade/-Trp/X-a-Gal media (Clontech). Results showed that the
MK2 protein by itself does not activate transcription. The yeast containing the MK2
bait construct produced tryptophan, however, these yeast did not produce Adenine
or Histidine. Clones grew on the SD/-Trp/X-a-Gal medium, but did not appear blue.
Clones did not grow on SD/-His/-Trp/X-a-Gal or SD/-Ade/-Trp/X-a-Gal media,
showing that there was no general transcriptional activation of endogenous reporter
genes.
48

Example 3: Screening the Yeast Two-Hybrid Library
[0130] The MK2 used in the screening the library was full length and
catalytically inactive. This inactive mutant encodes an alanine substitution at the
conserved lysine in the MK2 ATP binding pocket rendering MK2 inactive (Kotlyarov
etal., Mol Cell Bio 22, 4827-4835 (2002)) form was used because inactive kinases
are known to bind interacting proteins more stably thereby allowing for stronger
transcriptional activation of 2 hybrid reporter genes.
[0131] A colony of yeast transformed with the MK2 construct was inoculated in
SD/-Trp (Clontech) medium at 30°C overnight with shaking at 250-270 rpm. The
next day, the OD600 was measured to be >0.8. The cells were spun down by
centrifugation at 1000 x g for 5 minutes. The supernatant was decanted and the cell
pellet was resuspended in residual liquid by vortexing.
[0132] One frozen aliquot (~1.0 ml) of the library culture was thawed in a room
temperature water bath. The cells were gently vortexed. The entire MK2 bait culture
and the 1 ml library culture were combined. 45 ml of 2X YPDA/Kan (a blend of yeast
extract, peptone, dextrose, adenine, and kanomycin; Clontech) was added to the
culture and swirled gently, and the volume was brought to 50 ml with 2X YPDA/Kan.
The cells were incubated overnight at 30°C with gentle swirling (30-50 rpm).
[0133] Cells were transferred to a sterile centrifuge bottle and spun down by
centrifugation at 1000 x g for 10 minutes. The pellet was resuspended in 50 ml 2X
YPDA/Kan, then the cells were spun down at 1000 x g for 10 minutes. This wash
step was repeated. Subsequently cells were resuspended in 10 ml of 0.5X
YPDA/Kan medium.
[0134] Two hundred /vl of the mating mixture was plated on approximately 50
large (150 mm) SD/-His/-Leu/-Trp plates (Clontech). The plates were incubated,
49

colony side down, at 30°C until colonies appeared on the plates. Clones were
scored for growth on SDALeu (Clontech), SD/-Trp, and SD/-Leu/-Trp (Clontech)
plates. Haploid and diploid cells grew on the SDALeu or SD/-Trp media. Only
diploid cells grew on the SD/-Leu/-Trp medium. Mating efficiency was determined to
be 5.4%.
[0135] Colonies growing on plates containing SD/-His/-Leu/-Trp X-a-Gal
medium were restreaked onto SD/-His/-Leu/-Trp X-a-Gal plates to verify the
phenotype. Next, the clones were screened on the more stringent SD/-Ade/-His/-
LeuATrp plates containing X-a-GAL (Clontech). Positive colonies were restreaked
onto SD/-Ade/-His/-Leu/-Trp in a grid fashion (Clontech) to generate master plates.
DNA preps and glycerol stocks of the yeast were made from these master plates.
Example 4: Analysis of Putative Positive Clones
[0136] The YEASTMAKER™ Yeast Plasmid Isolation Kit (Clontech; #K1611-
1) provided the reagents and tools for isolating plasmid from yeast. Yeast were
obtained from individual positives which had been streaked onto the master plates,
and were resuspended in 50 /v/Tris EDTA in a 96 well format. Ten JL/I lyticase was
added to each well. The plate was incubated at 37°C for 1 hour after which 20 /yl of
20% SDS was added. The DNA was purified using a Qiagen turbo prep (Qiagen,
Valencia, California, USA). Samples were PCR amplified using an Advantage 2
PCR enzyme (Clontech; # K1910-1). Inserts were identified by sequencing.
Example 5: Analysis of Positive Clones
[0137] Independent sequences were transformed into bacteria (DH5 a) from
which DNA was subsequently isolated. Next, the DNA was transformed into yeast
strain AH109. Several bates: MK2, catalytically inactive MK2 K93R, empty vector
pGBKT7, TPL2, p53, and Lamin (Clontech) were transformed into yeast strain Y187.
50

Each independent sequence strain transformed in AH109 was mated with Y187
yeast transformed with each of the three baits. Independent clones which interacted
with MK2 and MK2 K93R, but not with empty vector pGBKT7, TPL2, p53, or Lamin,
as assayed by their growth on SD/-Ade/-His/-I_eu/-Trp and blue color on SD /-Leu/-
Trp X-a-GAL, were identified as clones which included DNA inserts encoding specific
MK2 interacting proteins (Figure 9A). MK2 interacting proteins bound wild type MK2
and MK2 K93R with substantially the same affinity, illustrating that MK2 binding is
not an artifact of the kinase inactive mutant MK2 K93R. One protein encoded by an
independent DNA sequence characterized was "similar to smoothelin" (STS). The
cDNA sequence encoding the protein is provided in Figure 1 and SEQ ID NO: 1; the
amino acid sequence is provided in Figure 4 and SEQ ID NO: 4.
[0138] Another protein encoded by an independent clone was human
polyhomeotic2 (HPH2). The cDNA sequence encoding the protein is provided in
Figure 2 and SEQ ID NO: 2; the amino acid sequence is provided in Figure 5 and
SEQ ID NO: 5. HPH2 has a sterile alpha motif (SAM) protein interaction domain.
[0139] Yet another protein encoded by an independent DNA sequence
isolated was src homology and collagen (She). The cDNA sequence encoding the
protein is provided in Figure 3 and SEQ ID NO: 3; the amino acid sequence is
provided in Figure 6 and SEQ ID NO: 6. The longest isoform of She (p66 She A) has
an N terminal CH2 domain followed by a PTB, CH1, and an SH2 domain at the C
terminus of the protein.
Example 6: Delineation of MK2 Interaction Domains.
[0140] To delineate the MK2 domain required for interaction with She A,
HPH2, and STS several, MK2 deletion mutants were used. MK2 mutants tested
included MK2VN (MK2 amino acids 41-400), MK2VC (MK2 amino acids 1-370) as
51

well as MK2Cat (MK2 catalytic domain amino acids 41-338). MK2VN has the proline
rich N-terminus deleted, MK2VC has the MK2 nuclear localization signal (NLS) and
the p38 binding site deleted, and MK2Cat has the N terminal proline rich domain as
well as the C terminal auto-inhibitory domain, nuclear export signal (NES) and NLS
deleted. 2-hybrid analysis showed that She A interacts equally well with full length
MK2, MK2VC, and MK2Cat. Interaction with MK2VN, however, was barely
detectable. The interaction profile with She A indicates that minimally, She A
interacts with the MK2 catalytic domain and that deleting the MK2 N-terminus might
induce a conformational change such that She A no longer binds MK2 efficiently.
[0141] HPH2 binds full length MK2, MK2VN and MK2VC with substantially the
same affinity. Interaction with MK2Cat, however, was less pronounced. HPH2,
therefore, seems to require binding at the MK2 N- or C- terminus while the MK2
catalytic domain when expressed alone does not promote HPH2 binding with MK2 to
a comparable level.
[0142] Similar to smoothelin binds MK2 with higher affinity than either She A
or HPH2 as assayed by growth and color assays in yeast. Additionally, similar to
smoothelin binds each of MK2, MK2VN, MK2VC and MK2Cat with substantially the
same affinity indicating that multiple sites in MK2 interact with this protein.
Example 7: MK2 Co-lmmunoprecipitates with She A and HPH2 in Mammalian
Cells
[0143] V5-tagged She and MYC-tagged MK2 were co-expressed in 293T
cells, as shown in Figure 10A. Cells were unstimulated or stimulated with 10 μg/ml
anisomycin for 30 minutes. Western blotting of cell lysates using antibodies against
V5 or Myc show that both V5-tagged She and MYC-tagged MK2 proteins were
expressed. Cell lysates were immunoprecipitated with the anti-V5 antibody.
Immunoprecipitates were resolved by SDS PAGE and immunoblotted with anti-MYC
52

antibody. The anti-MYC antibody binds to the immunoblot, indicating that MYC-
tagged MK2 co-immunoprecipitated with V5-tagged She. This indicates an
interaction between MK2 and She.
[0144] In a similar experiment, co-immunoprecipitation using the anti-HA
antibody and subsequent immunoblotting with the anti-Myc antibody shows that MK2
co-immunoprecipitates with HPH2 and p38 (Figure 10B). HA-tagged p38, HA-
tagged HPH2, and Myc-tagged MK2 were expressed in 293T cells, as shown by
Western blotting.
[0145] Co-immunoprecipitation can be used to detect the binding of two
proteins or to confirm results of binding between two proteins as found in other
methods, such as the Y2H system.
Example 8: Effect of Binding Proteins on MK2 Activation
[0146] An HA-tagged MK2 interacting protein (for example, She), and MYC-
tagged MK2 are co-expressed in 293T cells. Cell lysates are prepared and resolved
by SDS PAGE. Subsequent immunoblotting with Myc and HA antibodies to detect
MYC-tagged MK2 and the HA-interacting protein will confirm that these proteins are
expressed. Since activation of MK2 comprises phosphorylation of MK2,
immunoblotting with an antibody to detect phosphorylated MK2 (for example, anti-
phospho MK2 threonine 334 (p334)), will determine the activation state of MK2. A
difference in the amount of MK2 344 when co-expressed with an MK2 activator,
compared to the amount of MK2 p334 when MK2 is expressed alone, will indicate
altered activity of MK2 in the presence of the MK2 interacting protein.
Example 9: Effect of MK2 Interacting Proteins on TNF Biosynthesis
[0147] Two empty vector constructs were co-expressed in RAW 264.7
macrophage cells to establish a basal level of TNF-a biosynthesis, as detected by
53

ELISA (Figure 11). Cells were either unstimulated or stimulated with
lipopolysaccharide (LPS) to stimulate MK2 catalytic activity. The level TNF
expression-a was detected in cells in which MK2, p38, or She was co-expressed with
empty vector and levels were comparable to the basal level found with vector alone.
When cells co-expressed p38 and She, the level of TNF-a expression was greater
than that detected in controls. Similarly, co-expression of She and MK2 resulted in
an increase in TNF-a levels.
Example 10: Effect of MK2 on the Phosphorylation State of an MK2 Interacting
Protein.
[0148] An HA-tagged MK2 interacting protein is expressed in 293T cells. Cell
lysates are prepared and immunoprecipitated with an anti-HA antibody. The
immunoprecipitates are used in an in vitro kinase assay with recombinant MK2
added as kinase. SDS PAGE followed by phosphoimagery is used to detect
phosphorylation of the MK2 interacting protein. Phosphorylation of the MK2 binding
or interacting protein in the presence of MK2 and reduced or no phosphorylation of
the MK2 interacting protein in the absence of MK2 would indicate that MK2
phosphorylates the MK2 interacting protein. An MK2 interacting protein may or may
not be a substrate for MK2.
Example 11: Detection of MK2 Interacting Proteins Using a Proteomics
Approach
[0149] A proteomics approach can be used to identify MK2 interacting
proteins. Wild type (+/+) or MK2 deficient (-/-) cells were plated and labeled with 33P.
MK2 was activated for 30 minutes following which whole cell lysates were prepared
and analyzed using two dimensional gel electrophoresis. Gels were compared to
identify differentially phosphorylated proteins. Figure 12A shows a differentially
phosphorylated protein (arrow) with an isoelectric focusing point of 5.4. The
54

absence of phosphorylation in MK2 +/+ cells may be due to phosphorylation
dependent changes in protein migration. Figure 12B shows a silver stain of the
same region showing abundance of proteins resolved.
Example 12: MK2 is Activated when Co-Expressed with She A in HeLa Cells.
[0150] The activation state of MK2 when co-expressed with She A was
determined. Hsp 27 is a physiological substrate for MK2. Phosphorylation of
endogenous HSP 27 in HeLa cells is responsive to MK2 and therefore, can be used
to determine MK2 activity. HeLa cells were transfected either with vector alone, or
with vector and V5-Shc A or Myc-MK2 as controls. In parallel, cells were co-
transfected with both V5-Shc A and Myc-MK2. After allowing for expression, cells
were left either unstimulated or stimulated for 30 minutes with anisomycin to activate
MK2. Subsequent immunoblotting with an anti-phospho peptide specific antibody
against phosphorylated Hsp 27 (pHsp 27) shows an increase in pHsp 27 upon
stimulation. With expression of exogenous Myc-MK2 there was an increase in basal
pHsp 27 levels in unstimulated cells (Figure 13A). This increase in basal
phosphorylation is not observed in She A or vector control expressing cells.
Quantitation of these results using densitometry shows that the increase in basal
pHsp 27 seen with Myc-MK2 expression as compared with empty vector or V5-Shc
A is 1.5-2 fold. Basal pHsp 27 levels were further increased when Myc-MK2 was co-
expressed with V5-Shc A. Quantitation shows this increase to be 3 fold over levels
detected when Myc-MK2 was expressed alone (Figure 13B). As shown in 293T
cells, MK2 levels increased with co-expression of She A, further illustrating the
interaction between MK2 and She A. The observed increase in basal pHsp 27 is
likely to reflect increased MK2 activity as well as increased levels of MK2 protein.
Basal pHsp 27 was shown to increase 2 to 3 fold when normalized with MK2 levels
55

(Figure 13C). This increase is proposed to reflect the activation of MK2 with co-
expression of She A.
[0151] Increased MK2 activity is observed with co-expression of p66 She A.
Phosphorylation of endogenous Hsp 27 is responsive to MK2 activity in HeLa cells.
Increased pHsp 27 is observed with MK2-p66 She A co-expression indicating that
MK2 is activated with p66 She A binding. The observed increase in TNF-a levels
with MK2-p66 She A co-expression in RAW 264.7 cells confirms that MK2 is
activated with She A co-expression. MK2 activation with p66 She A may result in
MK2 localization and retention in the cytosol through She A binding. Cytosolic
localization may in turn increase MK2's access to cytoplasmic substrates such as
Hsp 27 and TNF-a mRNA. Alternatively, MK2 may bind cytosolic She promoting
MK2 cytoplasmic localization where it becomes further activated.
Example 13: MK2 is Activated when Co-Expressed with She A in RAW264.7
Cells
[0152] MK2 was activated when co-expressed with She A, as assayed by
levels of pHsp 27 in HeLa cells. To further confirm that MK2 is activated upon
association with She A, secreted TNF protein from RAW264.7 cells was assayed for
in presence of both MK2 and She A. Data from cells derived from mice deleted for
MK2 show a 90% decrease in TNF-a biosynthesis in response to LPS. Subsequent
experiments have shown that catalytically active MK2 is required to restore TNF
biosynthesis in these cells, thereby establishing MK2 enzymatic activity as
necessary for TNF-a biosynthesis. RAW264.7 cells were transfected with either
vector alone, or were co-transfected with vector and V5-Shc A or Myc-MK2 as
controls. In parallel, cells were co-transfected with both V5-Shc A and Myc-MK2.
After allowing for expression, cells were left either unstimulated or stimulated with
LPS for 30 minutes to activate MK2. Quantitative western blot analysis shows that
56

both Myc-MK2 and V5-Shc A are expressed in RAW264.7 cells. In contrast to co-
expression in 293T and HeLa cells, levels of both MK2 and She A decreased when
co-expressed in RAW264.7 cells. This decrease does not reflect an overall decrease
in protein levels because equal amounts of protein were loaded in each lane as
shown using an actin specific antibody (Figure 14A).
[0153] TNF ELISAs showed that TNF-a secretion increased 8 fold upon
stimulation by LPS and that expression of Myc-MK2 or V5-Shc A alone does not
potentiate TNF biosynthesis in these cells. In contrast, when MK2 and She A were
co-expressed TNF-a biosynthesis increases 1.5 fold after stimulation. (Figure 14B).
The increase in TNF biosynthesis is proposed to reflect an increase in MK2 activity
and not in MK2 expression since MK2 protein levels when co-expressed with She A,
are below those observed in cells expressing MK2 alone.
Example 14: MK2 Phosphorvlates She A In Vitro
[0154] P66 She is phosphorylated at serine 36 within the CH2 domain upon
oxidative stress. N-terminal to S36, a serine is located at position 17 (S17) within a
Cam kinase II consensus sequence: RXXS. Figure 15A depicts a schematic
representation of the She A protein, including various phosphorylation sites within
the protein. The RXXS motif has been shown to serve as a substrate for MK2
although it does not contain a hydrophobic amino acid often found -2 from the
conserved Arginine in the MK2 consensus motif. To determine if She A is a
substrate for MK2, V5-Shc A was expressed in 293T cells. After expression, She A
was immunoprecipitated using an anti-V5 antibody. Immunoprecipitates were
subsequently used in an in vitro kinase assay with exogenously added activated
recombinant MK2. Vector transfected control cells showed low levels of
phosphorylation at 66-kDa most likely resulting from the anti-V5 antibody
57

immunoprecipitating a protein endogenous to 293T cells which serves as a weak
substrate for MK2. Only She A transfected cells showed robust phosphorylation at
66-kDa demonstrating that She A is a substrate for MK2 in vitro. Coomassie staining
and immunoblotting using an anti-V5 antibody showed that She A was expressed in
She A transfected cells. (Figure 16B)
[0155] As depicted in Figure 15B, MK2 phosphorylates p66 She A in vitro.
MK2 may function in p66 She A regulated stress activated pathways. Although the
66 kDa isoform of She A does bind activated cell surface receptors, its binding does
not lead to activation of the Ras MAPK pathway. Since p66 She A is not ubiquitously
expressed, its cell type and tissue specific expression is proposed to selectively
regulate activation of the Ras MAPK pathway through its selective expression
pattern. Additionally, the p66 She A isoform acts downstream of p53 to regulate
cellular responses to oxidative stress. (Trinei et al., Oncogene 21 (24):3872-8
(2002)). The P38-MK2 pathway is activated by oxidative stress to phosphorylate
small heat shock proteins thereby modulating microfilament responses to stress.
{Huot et al., Circ Res. 80(3):383-92.. 1997)). Both p38 and MK2 have been
implicated in p53 phosphorylation upon oxidative stress. {She Q.B. et al., J Biol.
Chem. 7;275(27):20444-9 (2000); She Q.B. et al., Oncogene 21 (10): 1580-9 (2002)).
JNK and ERK have been implicated in serine phosphorylating p66. The data
presented in this application supports a role for MK2 in stress activated
phosphorylation of p66, as represented in Figure 16.
Example 15: Phospho Akt and Phospho FKHR-L1 Levels were Reduced in
MK2 -/- Cells
[0156] Data generated from animals that are knock-outs for p66Shc A has
shown that p66 regulates cellular responses to oxidative stress including generation
of intracellular ROS and apoptosis and that phosphorylation at S36 in the protein is
58

required for these responses. Cells derived from p66Shc A -/- animals have
decreased levels of intracellular free radicals and are resistant to stress induced
apoptosis compared with wild type littermates. In addition, p66Shc A -/- animals are
resistant to paraquot, an oxidant-generating compound and further show an
extended life span. Phosphorylation of both AKT and FKHR-L1 in response to
oxidative stress such as UV light or H202, is reduced in p66Shc -/- MEFs. Reduction
in FKHR-L1 phosphorylation correlates with an increase in FKHR-L1 activity, as this
transcription factor remains nuclear in its de-phosphorylated form. Cells with
activated FKHR-L1 are resistant to apoptosis consistent with the role of this
transcription factor in regulating the expression of several antioxidant enzymes
including superoxide dismutase and catalase.
[0157] A role for MK2 in phosphorylating and regulating p66Shc A in cellular
responses to stress suggests that cells deleted for MK2 should show reduced levels
of both phospho (p)-AKT and phospho (p)-FKHR-L1 in response to oxidative stress
as compared with wild type cells. In order to test this prediction, H202 induced p-AKT
and p-FKHR-L1 levels in MK2 -/- and +/+ MEFs were assayed. Quantitative western
blot analysis showed that both p-AKT and p-FKHR-L1 levels were reduced in MK2 -/-
MEFs as compared with +/+ MEFs suggesting that intracellular ROS levels are
reduced in MK2 -/- cells (Figure 17).
Example 16: Screening for Anti-Inflammatory Drugs
1. Yeast 2-hybrid System for Drug-Screening
[0158] Proteins that bind MK2 are used for identifying anti-inflammatory drugs
including small molecules, peptides, chemical agents and antibodies that are useful
for treatment or prevention of inflammation.
59

[0159] MK2-iinteracting proteins are identified using the yeast 2-hybrid system
described herein. MK2-interacting proteins are then assayed for their effect on MK2
activity by one or more of assays provided herein or those that are well known in the
art. For example, an MK2 interacting protein will either increase MK2 activity, for
example, as determined by MK2 kinase activity or TNF-a biosynthesis; inhibit MK2
activity; or have no effect on MK2 activity. MK2 interacting proteins that increase
MK2 activity are desirable to use as candidates for screening for anti-inflammatory
drugs.
[0160] A positive yeast clone showing an interaction between MK2 and an
MK2 interacting protein which increases MK2 activity, as described above, is
streaked on the appropriate selection plate as many times as the number of drug
candidates or test compounds to be tested. As expected, each streaked colony will
be positive for the interaction between MK2 and the interacting protein, as assayed
by color and growth assays. Each colony is subsequently contacted with a different
drug candidate to assay for an effect on the interaction between MK2 and the
interacting protein. A drug candidate that inhibits the interaction between MK2 and
the interacting protein, as assayed by a reduction in color and/or growth of the
colonies on the appropriate media, will be a identified as a potential candidate for the
treatment or prevention of inflammation.
[0161] The same assay can be used, for example, for identification of drug
candidates for identification of drug candidates which promote an interaction
between MK2 and an interacting protein, where the interacting protein inhibits MK2
activity. For example, a positive yeast clone showing an interaction between MK2
and an interaction protein which inhibits MK2 activity, at least partially, as assayed
by one or more assays provided herein, can be streaked on the appropriate media.
60

Each colony of the positive clone is subsequently contacted with a potential drug
candidate or test compound. The drug candidates which lead to stronger growth as
well as color in yeast specificity assays, described herein, are potential candidates
for promoting an interaction between MK2 and an MK2 interacting protein, where the
interacting protein inhibits MK2 activity.
2. In Vitro Reconstitution Assays for Drug-Screening
[0162] In vitro reconstitution assays can be used both for identification of anti-
inflammatory drugs that block MK2 activity or block interaction between MK2 and an
interacting protein, where the protein increases MK2 activity.
[0163] Additionally, an in vitro reconstitution system can also be used for
formation of protein complexes including MK2 and at least one MK2 interacting
protein, where such complexes can be subsequently used for identification of test
compounds that modulate inflammation. The effect of a test compound on
inflammation can be assayed either by determining whether the test compound
inhibits or promotes complex formation or for its effect on MK2 activity. For example,
an amount of a protein complex including MK2 and an interacting protein can be
measured before and after contacting the protein complex with a test compound. A
test compound which leads to a decrease in the amount of protein complex relative
to the amount in absence of the test compound will be anti-inflammatory. Whereas,
a test compound which leads to an increase in the amount of protein complex
relative to the amount in the absence of the test compound will be pro-inflammatory.
[0164] For example, MK2 is synthesized using an in vitro transcription-
translation system, such as the rabbit reticulocyte system supplied by Promega. The
in vitro translated MK2 is first assayed for activity in one or more assays provided
herein. Various drug candidates are subsequently added to the translated MK2
61

under the appropriate conditions and for appropriate periods of time, and MK2
activity is assayed again subsequent to the contact with a potential drug candidate.
Those candidates which lead to inhibition or reduction in MK2 activity are identified
as potential anti-inflammatory drugs. These drugs can be further validated for their
effect on inflammation and on various pathways involved in inflammation in vivo in
cells and in animal models for inflammation.
[0165] Similarly, an in vitro reconstituted system can also be used for
identifying drugs which inhibit the interaction between MK2 and an interacting
protein, where the interacting protein stimulates MK2 activity. For example, a
composition including in vitro translated MK2 and an interacting protein, as identified
by the Y2H system or co-immunoprecipitation assays, is treated with potential drug
candidates. The candidates which inhibit the interaction between MK2 and the
interacting protein, are identified as potential anti-inflammatory drugs. These drugs
can subsequently be tested in vivo in cells and in animal models. In addition to in
vitro translated proteins, purified proteins may also be used in in vitro reconstitution
assays for identification of anti-inflammatory drugs.
Example 17: Treatment of Conditions Related to Inflammation
[0166] Compounds (such as proteins, peptides, antibodies, chemical agents,
and small molecules) that interact with at least one of MK2 or an MK2 complex may
be administered to patients suffering from a condition related to inflammation
according to Table 1. Patients take the composition one time or at intervals, such as
once daily, and the symptoms of their condition improve. For example, there will be
a decrease in inflammation. This shows that the composition of the invention is
predicted to be useful for the treatment of conditions related to inflammation.
62

Table 1: Administration of Compounds that Interact with MK2 or an MK2
Complex

[0167] The specification is most thoroughly understood in light of the
teachings of the references cited within the specification which are hereby
incorporated by reference. The embodiments within the specification provide an
illustration of embodiments of the invention and should not be construed to limit the
scope of the invention. The skilled artisan readily recognizes that many other
63

embodiments are encompassed by the invention. All publications, patent
applications and patents cited and sequences identified by accession or database
reference numbers in this disclosure are incorporated by reference in their entirety.
To the extent the material incorporated by reference contradicts or is inconsistent
with the present specification, the present specification will supercede any such
material. The citation of any references herein is not an admission that such
references are prior art to the present invention.
[0168] Many modifications and variations of this invention can be made
without departing from its spirit and scope, as will be apparent to those skilled in the
art. The specific embodiments described herein are offered by way of example only
and are not meant to be limiting in any way. Unless otherwise indicated, all numbers
expressing quantities of ingredients, cell culture, treatment conditions, and so forth
used in the specification, including claims, are to be understood as being modified in
all instances by the term "about." Accordingly, unless otherwise indicated to the
contrary, the numerical parameters are approximations and may very depending
upon the desired properties sought to be obtained by the present invention. Unless
otherwise indicated, the term "at least" preceding a series of elements is to be
understood to refer to every element in the series. Those skilled in the art will
recognize, or be able to ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following claims.
64

We Claim:
1. An isolated, purified, or recombinant protein complex comprising:
(i) an MK2 polypeptide; and
(ii) an MK2 interacting protein chosen from STS, HPH2 and She.
2. The complex of claim 1, comprising an MK2 polypeptide and at least
one MK2 interacting protein.
3. The complex of claim 2, wherein the MK2 interacting protein is chosen
from STS, HPH2 and She.
4. The complex of claim 1, comprising an MK2 polypeptide and at least
two MK2 interacting proteins.
5. The complex of claim 4, wherein the MK2 interacting proteins are
chosen from STS, HPH2 and She.
6. The complex of claim 1, wherein the MK2 polypeptide comprises a
fusion protein.
7. The complex of claim 6, wherein the fusion protein comprises a
domain for purifying, isolating or detecting the fusion protein.
65

8. The complex of claim 6, wherein the fusion protein comprises a
domain chosen from the group consisting of: affinity tags,
radionucleotides, enzymes, and fluorophores.
9. The complex of claim 7, wherein the domain is selected from the
group consisting of: polyhistidine, FLAG, Glu-Glu, glutathione S
transferase (GST), thioredoxin, protein A, protein G, and an
immunoglobulin heavy chain constant region.
10. A host cell comprising a first nucleic acid and a second nucleic acid,
wherein the first nucleic acid encodes a recombinant MK2 polypeptide
and the second nucleic acid encodes an MK2 interacting protein
chosen from STS, HPH2 and Shc.
11. The host cell of claim 10, further comprising a third nucleic acid
encoding a second MK2 interacting protein chosen from STS, HPH2
and Shc.
12. An assay for determining whether a test compound inhibits or
promotes formation of a protein complex comprising:

(a) forming a reaction mixture including an MK2 polypeptide, at
least one MK2 interacting protein and the test compound; and
(b) detecting the presence of the protein complex between MK2
and the MK2 interacting protein;
66

wherein a difference in the amount of complex in the presence of the
test compound, relative to the amount of complex in the absence of
the test compound indicates that the test compound inhibits or
promotes complex formation.
13. The assay of claim 12, wherein an increase in the amount of complex
in presence of the test compound indicates that the test compound
promotes complex formation.
14. The assay of claim 12, wherein a decrease in the amount of complex
in presence of the test compound indicates that the test compound
inhibits complex formation.
15. A method for determining whether a test compound affects MK2
activity comprising:

(a) forming a protein complex comprising an MK2 polypeptide and
an MK interacting protein;
(b) contacting the protein complex with the test compound, and
(c) determining the effect of the test compound on one or more
activities chosen from MK2 kinase activity, an amount of MK2 in the
complex, production of TNF, and amount of phosphorylated form of a
substrate of MK2.
16. A screening assay to identify compounds that inhibit or promote
formation of a protein complex, comprising
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(i) providing a two-hybrid assay system including a first fusion
protein comprising an MK2 polypeptide, and a second fusion
protein comprising a polypeptide chosen from one or more of
STS, HPH2 and Shc, under conditions wherein the two proteins
interact in the two hybrid assay system;
(ii) measuring a level of interaction between the fusion proteins in
the presence and in the absence of a test compound; and
(iii) comparing the level of interaction of the fusion proteins,
wherein a decrease in the level of interaction is indicative of a
compound that inhibits the interaction between the MK2 polypeptide
and a polypeptide chosen from one or more of STS, HPH2 and Shc.
17. An antibody that binds one or more proteins in a complex comprising
an MK2 polypeptide and an MK2 interacting protein chosen from STS,
HPH2 and Shc.
18. The antibody of claim 17, wherein the antibody inhibits interaction of
MK2 with the MK2 interacting protein.
19. A method for modulating formation of a protein complex in a cell
comprising at least a first protein and a second protein, wherein the
first protein is an MK2 polypeptide and the second protein is chosen
from one or more of STS, HPH2 and Shc, and wherein the method
comprises administering to the cell a compound capable of modulating
formation of the complex.
68

20. A method of producing a complex comprising:
transfecting a cell with one or more polynucleotides encoding an MK2
polypeptide and an MK2 interacting protein chosen from one or more
of STS, HPH2 and Shc, whereby the polypeptides form a complex.
21. A drug screening method for identifying anti-inflammatory drugs
comprising:
a) providing MK2 and at least one MK2-interacting protein;
b) allowing MK2 and the protein to interact to form a complex;
c) adding an effective amount of a potential drug to the complex;
and
d) determining whether the potential drug inhibits complex
formation.
22. The method of claim 21, wherein MK2 and the protein interact in vivo
in a yeast 2-hybrid system.
23. The method of claim 21, wherein MK2 and the protein interact in vivo
in a mammalian 2-hybrid system.
24. The method of claim 21, wherein MK2 and the protein interact in vitro.
25. The method of claim 21, wherein the protein is STS.
26. The method of claim 21, wherein the protein is She.
27. The method of claim 21, wherein the protein is HPH2.
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28. The method of claim 21, wherein the drug is a small molecule.
29. The method of claim 21, wherein the drug is a peptide or protein.
30. The method of claim 21, wherein the drug is an antibody.
31. The method of claim 21, wherein the drug is a chemical agent.
32. A method of modulating inflammation in a tissue comprising:

a) administering a nucleic acid to the tissue, wherein the nucleic
acid encodes an MK2 interacting protein; and
b) allowing the nucleic acid to express the MK2 interacting protein,
thereby to modulate inflammation in the tissue.

33. The method of claim 32, wherein the nucleic acid expresses a protein
chosen from STS, HPH2 and Shc.
34. A method of treating or preventing inflammation in a tissue comprising
administering to the tissue a therapeutically effective amount of at
least one agent, wherein the agent either

a) blocks the interaction between MK2 and an MK2 interacting
protein; or
b) allows the interaction, but blocks MK2 activity.
70

35. The method of claim 34, wherein the agent is an antibody.
36. The method of claim 35, wherein the antibody is a polyclonal antibody.
37. The method of claim 35, wherein the antibody is a monoclonal
antibody.
38. The method of claims 35-37, wherein the antibody binds MK2.
39. The method of claims 35-38, wherein the antibody binds the MK2-
interacting protein.
40. The method of claim 34, wherein the agent is a chemical agent.
41. The method of claim 34, wherein the agent is a peptide or protein.
42. The method of claim 34, wherein the agent is a small molecule.
43. A method of modulating inflammation in a tissue comprising:

a) contacting the tissue with at least one protein that binds MK2;
and
b) allowing the protein to modulate inflammation in the tissue.
44. A method of treating a patient suffering from at least one inflammatory
condition, comprising:
71

a) administering a therapeutically effective dose of at least one
compound chosen from a compound that interacts with at least
one of MK2 or an MK2 complex, wherein the compound is
chosen from an antibody, a chemical agent, a small molecule, a
protein and a peptide; and
b) allowing the compound to bind to At least one of MK2 or an
MK2 complex and modulate inflammation.

45. The method of claim 44, wherein the protein or peptide is a mutant
form of a wild-type protein or peptide which stimulates MK2 activity.
46. The method of claim 44, wherein the protein is chosen from STS,
HPH2 and Shc.
47. The method of claim 44, wherein the condition is chosen from Crohn's
disease, inflammatory bowel disease, ulcerative colitis, rheumatoid
arthritis, acute respiratory distress syndrome, emphysema, delayed
type hypersensitivity reaction, asthma, systemic lupus erythematosus,
and inflammation due to trauma or injury.
48. A method of expressing a nucleic acid in a cell to inhibit inflammation,
comprising
a) adding at least one nucleic acid encoding a compound chosen
from a compound that interacts with at least one of MK2 or an
MK2 complex, wherein the compound is chosen from an
72

antibody, a chemical agent, a small molecule, a protein and a
peptide; and
b) allowing the cell to express the compound and inhibit
inflammation.
49. The method of claim 48, wherein the nucleic acid encodes a protein
chosen from STS, HPH2 and Shc.
50. A method of detecting at least one of the absence, presence, and
amount of MK2 in a sample, comprising

a) administering at least one compound that interacts with at least
one of MK2 or an MK2 complex, wherein the compound is
chosen from an antibody, a chemical agent, a small molecule, a
protein and a peptide; and
b) correlating the absence, presence, or amount of bound protein
or compound with the absence, presence, or amount of MK2 in
the sample.

51. The method of claim 49, wherein the protein is chosen from STS,
HPH2 and Shc.
52. A kit, wherein the kit enables qualitative detection of MK2 comprising a
compound that interacts with at least one of MK2 or an MK2 complex,
wherein the compound is chosen from an antibody, a chemical agent,
73

a small molecule, a protein and a peptide; and at least one other kit
component chosen from:
a) at least one of buffer and solution;
b) at least one structural component.

53. The kit of claim 52, further comprising an agent that binds the protein
or compound.
54. The kit of claim 53, wherein the agent is an antibody.
55. The kit of claim 52, wherein the protein is chosen from STS, HPH2
and Shc.
56. A pharmaceutical composition comprising:

a) at least one protein that binds MK2, and
b) at least one pharmaceutically acceptable carrier.

57. The composition of claim 56, wherein the protein is chosen from STS,
HPH2 and Shc.
58. The protein complex of claim 1, wherein the MK2 interacting protein is
encoded by a cDNA molecule comprising a nucleotide sequence
chosen from SEQ ID NOs: 1, 2 and 3.
74

59. The protein complex of claim 1, wherein the MK2 interacting protein is
encoded by a cDNA molecule comprising a nucleotide sequence
chosen from fragments; splice variants; addition, deletion and
substitution mutants; and homologues of SEQ ID NOs:1, 2 and 3,
wherein the MK2 interacting protein binds MK2.
60. The protein complex of claim 1, wherein the MK2 interacting protein
comprises an amino acid sequence chosen from SEQ ID NOs: 4, 5,
and 6.
61. The protein complex of claim 1, wherein the MK2 interacting protein
comprises an amino acid sequence chosen from fragments; splice
variants; addition, deletion and substitution mutants; and homologues
of SEQ ID NOs: 4, 5, and 6, wherein the MK2 interacting protein binds
MK2.
75
62. An isolated, purified, or recombinant protein complex substantially as
herein described particularly with reference to the examples.

The present invention relates to uses of proteins that bind MK2 to modulate
inflammation. More particularly, the invention relates to uses of proteins that bind
MK2 for treating condition that are related to inflammation. The invention is useful
for treating inflammatory conditions, particularly those in which a decrease in
inflammation would be therapeutically beneficial.