U.S. patent application number 10/787138 was filed with the patent office on 2004-12-23 for pyk2 and inflammation.
This patent application is currently assigned to Sugen, Inc.. Invention is credited to Gishizky, Mikhail, Okigaki, Mitsuhiko, Schlessinger, Joseph.
Application Number | 20040259158 10/787138 |
Document ID | / |
Family ID | 33518687 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259158 |
Kind Code |
A1 |
Schlessinger, Joseph ; et
al. |
December 23, 2004 |
PYK2 and inflammation
Abstract
The present invention relates generally to the fields of
immunology and medicine, and more specifically to the field of
cellular signal transduction. The present invention relates, inter
alia, to methods for diagnosis, treatment, and identification of
therapeutics for particular inflammation-related diseases or
disorders characterized by an interaction between a PYK2
polypeptide and a natural binding partner.
Inventors: |
Schlessinger, Joseph;
(Woodbridge, CT) ; Okigaki, Mitsuhiko; (New York,
NY) ; Gishizky, Mikhail; (Menlo Park, CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Sugen, Inc.
New York University
|
Family ID: |
33518687 |
Appl. No.: |
10/787138 |
Filed: |
February 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10787138 |
Feb 27, 2004 |
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09476484 |
Dec 30, 1999 |
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60114465 |
Dec 30, 1998 |
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Current U.S.
Class: |
435/7.1 ;
514/266.1; 514/312; 514/418 |
Current CPC
Class: |
G01N 2500/00 20130101;
G01N 33/564 20130101; A61K 49/0004 20130101 |
Class at
Publication: |
435/007.1 ;
514/266.1; 514/312; 514/418 |
International
Class: |
G01N 033/53; A61K
031/517; A61K 031/47 |
Claims
1. A method for identifying compounds potentially useful to treat
or to prevent a disease or disorder, wherein said disease or
disorder is characterized by an inflammatory response involving an
abnormality in a signal transduction pathway that includes an
interaction between a PYK2 polypeptide and a natural binding
partner, comprising assaying one or more compounds for those able
to modulate said interaction as a means to identify said
potentially useful compounds.
2. The method of claim 1, wherein said disease or disorder
characterized by an inflammatory response is selected from the
group consisting of inflammatory bowel diseases and connective
tissue disease.
3. The method of claim 1, wherein said one or more compounds
modulate said interaction in vitro.
4. The method of claim 1, wherein said one or more compounds
modulate said interaction in vivo.
5. The method of claim 1, wherein said one or more compounds is
selected from the group consisting of tyrphostins, quinazolines,
quinoxolines, quinolines, and indolinones.
6. The method of claim 5, wherein one or more compounds is one or
more indolinones.
7. The method of claim 1, wherein said interaction is selected from
the group consisting of PYK2 phosphorylation, PYK2 natural binding
partner phosphorylation, PYK2 de-phosphorylation, PYK2 natural
binding partner de-phosphorylation, and complex formation between
PYK2 and a natural binding partner.
8. A method for diagnosis of a disease or disorder, wherein said
disease or disorder is characterized by an inflammatory response
involving an abnormality in a signal transduction pathway that
includes an interaction between a PYK2 polypeptide and a natural
binding partner, comprising detecting a change in said interaction
as an indication of said disease or disorder.
9. The method of claim 8, wherein said disease or disorder
characterized by an inflammatory response is selected from the
group consisting of inflammatory bowel diseases and connective
tissue diseases.
10. The method of claim 9, wherein said inflammatory bowel diseases
are selected from the group consisting of ulcerative colitis and
Crohn's Disease.
11. The method of claim 9, wherein said connective tissue diseases
are selected from the group consisting of rheumatoid arthritis,
systemic lupus erythematosus, progressive systemic sclerosis, mixed
connective tissue disease, and Sjogren's syndrome.
12. The method of claim 8, wherein said interaction is selected
from the group consisting of PYK2 phosphorylation, PYK2 natural
binding partner phosphorylation, PYK2 de-phosphorylation, PYK2
natural binding partner de-phosphorylation, and complex formation
between PYK2 and a natural binding partner.
13. The method of claim 8, wherein said change is an increase or
decrease in said interaction.
14-25. (Canceled).
Description
RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 60/114,465 by Schlessinger, Okigaki,
and Gishizky, entitled PYK2 and Inflammation, filed Dec. 30, 1998
(Lyon & Lyon Docket No. 236/075) which is hereby incorporated
by reference herein in its entirety, including any drawings,
tables, or figures. In addition, the present application is related
to U.S. application Ser. No. 08/357,642, by Lev and Schlessinger,
Lyon & Lyon Docket No. 209/070, entitled "PYK2 related Products
and Methods", filed Dec. 15, 1994; Ser. No. 08/460,626, by Lev and
Schlessinger, Lyon & Lyon Docket No. 211/121, entitled "PYK2
related Products and Methods", filed Jun. 2, 1995; and Ser. No.
08/987,689 by Lev and Schlessinger, Lyon & Lyon Docket No.
230/110, entitled "PYK2 related Products and Methods", filed Dec.
9, 1997, all of which are hereby incorporated by reference herein
in their entirety including any drawings, figures, or tables.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
immunology and medicine, and more specifically to the field of
cellular signal transduction.
BACKGROUND OF THE INVENTION
[0003] None of the following discussion of the background of the
invention, which is provided solely to aid the reader in
understanding the invention, is admitted to be or to describe prior
art to the invention.
[0004] Cellular signal transduction is a fundamental mechanism
whereby external stimuli that regulate diverse cellular processes
are relayed to the interior of cells. One of the key biochemical
mechanisms of signal transduction involves the reversible
phosphorylation of tyrosine residues on proteins. The
phosphorylation state of a protein is modified through the
reciprocal actions of tyrosine phosphatases (TPs) and tyrosine
kinases (TKs), including receptor tyrosine kinases and non-receptor
tyrosine kinases.
[0005] RTKs are composed of at least three domains: an
extracellular ligand binding domain, a transmembrane domain and a
cytoplasmic catalytic domain that can phosphorylate tyrosine
residues. The intracellular, cytoplasmic, non-receptor protein
tyrosine kinases do not contain a hydrophobic transmembrane domain
or an extracellular domain and instead contain non-catalytic
domains in addition to their catalytic kinase domains. Such
non-catalytic domains include the SH2 domains and SH3 domains. The
non-catalytic domains are thought to be important in the regulation
of protein-protein interactions during signal transduction.
[0006] FAK (focal adhesion kinase) and PYK2 (proline-rich tyrosine
kinase also known as RAFTK, CAK and CADTK (Lev, et al. (1995)
Nature 376:737-745; Avraham, et al. (1995) J. Biol. Chem.
270:27742-27751; Sasaki, et al. (1995) J. Biol. Chem.
270:21206-21219; Yu, et al. (1996) J. Biol. Chem. 271:29993-29998)
comprise one family of protein tyrosine kinases. FAK and PYK2
exhibit approximately 45% amino acid sequence identity to each
other and each contain an N terminus with similarity to band 4.1
homology domain, a centrally located protein tyrosine kinase domain
(Girault, et al. (1999) Trends Neurosci. 22:257-263), and two
proline rich regions at the C-terminus (Lev, et al. (1995) Nature
376:737-745). PYK2 and FAK bind to proteins that have been shown to
interact with the cytoskeleton such as paxillin (Salgia, et al.
(1996) J. Biol. Chem. 271:31222-31226), p130.sup.cas, the rhoGAP
protein Graf (Ohba, et al. (1998) Biochem. J. 330:1249-1254) and a
novel protein containing a LIM domain (Matsuya, et al. (1998) J.
Biol. Chem. 273:1003-1014; Lipsky, et al. (1998) J. Biol. Chem.
273:11709-11713).
[0007] PYK-2 is a non-receptor tyrosine kinase that is activated by
binding of ligand to G-coupled protein receptors such as bradykinin
and acetylcholine. PYK2 has a predicted molecular weight of 111 kD
and contains five domains: (1) a relatively long N-terminal domain;
(2) a kinase catalytic domain; (3) a proline rich domain; (4)
another proline rich domain; and (5) a C-terminal domain.
[0008] PYK2 is expressed in various tissues, including neural
tissues, hematopoietic cells, and in some tumor cell lines. PYK2 is
believed to regulate the activity of potassium channels in response
to neurotransmitter signaling. PYK2 may also regulate ion-channel
function by tyrosine phosphorylation.
[0009] PYK2 is activated by stimulation of G-protein coupled
receptors (Lev, et al. (1995) Nature 376:737-745), by stimulation
of antigen receptors on T cells (Qian, et al. (1997) J. Exp. Med.
185:1253-1259); B cells (Astier, et al. (1997) J. Biol. Chem.
272:228-232), and mast cells (Okazaki, et al. (1997) J. Biol. Chem.
272:32443-32447); as well as in response to inflammatory cytokines
(Tokiwa, et al. (1996) Science 273:792-794; Miyazaki, et al. (1998)
Genes Dev. 12:770-775; Takaoka, et al. (1999) EMBO J.
18:2480-2488), and stress signals (Tokiwa, et al. (1996) Science
273:792-794). In some cells, tyrosine phosphorylation and
activation of PYK2 was shown to be triggered by integrin-mediated
adhesion (Astier, et al. (1997) J. Biol. Chem. 272:228-232).
Furthermore, PYK2 can be activated by phorbol ester (PMA), or by a
variety of extracellular signals that elevate intracellular
Ca.sup.+2 concentration (Lev, et al. (1995) Nature
376:737-745).
[0010] It has been proposed that PYK2 acts in concert with Src to
link Gi or Gq coupled receptors with the MAP kinase signaling
pathway (Dikic, et al (1996) Nature 383:547-550).
Autophosphorylation of Y402 on PYK2 generates a binding site for
the SH2 domain of the docking protein Grb2, and subsequent
recruitment of the Grb2/Sos complex leads to activation of the
Ras/MAP kinase signal transduction cascade (Dikic, et al. (1996)
Nature 383:547-550). Activation of the JNK pathway can also be
mediated by PYK2, and this signaling pathway can be inhibited by
dominant-negative forms of rac and cdc42 (Tokiwa, et al. (1996)
Science 273:792-794). In addition, PYK2 activation leads to
suppression of outward potassium currents via tyrosine
phosphorylation of the delayed rectifier-type potassium channel
Kv1.2 (Lev, et al. (1995) Nature 376:737-745). PYK2 also interacts
with and phosphorylates a family of phosphatidylinositol transfer
proteins, designated Nirs (Lev, et al. (1999) Mol. Cell. Biol.
19:2278-2288), and with a novel ArfGAP, designated Pap (Andreev, et
al. (1999) Mol. Cell. Biol. 19:2338-2350), both in vitro and in
vivo. In addition, it was shown that activation of PYK2 leads to
tyrosine phosphorylation of other proteins that play a role in
signal transmission, including the adaptor proteins Shc (Lev, et
al. (1995) Nature 376:737-745) and Cas (Astier, et al. (1997) J.
Biol. Chem. 272:228-232).
[0011] PYK2 is activated by extracellular signals that lead to
calcium influx or calcium release from internal stores. PYK2 is
phosphorylated on tyrosine residues in response to a variety of
external stimuli. PYK2 may provide a link between G-protein coupled
receptors and calcium influx and the MAP kinase signaling pathway,
a pathway that relays signals from the cell surface to regulate
transcriptional events in the nucleus.
[0012] In the PCT Publication WO 98/07870 (Avraham, et al.), the
authors discuss PYK2 and state that
[0013] " . . . RAFTK therapeutics which modulate RAFTK activity in
B cells, T cells, and monocytes can be used to treat
immune-mediated disorders and mediate both cell mediated and
humoral immune responses.
[0014] Normal hematopoietic cells are dependent on growth factors
for growth and differentiation and the loss of this growth factor
dependence can lead to autonomous growth. The involvement of RAFTK
in several growth factor signaling pathways indicates that
misse[x]pression of RAFTK can lead to the development of cancers,
and the present invention contemplates modulating RAFTK expression
and/or activity to control aberrant cell growth. In a preferred
embodiment RAFTK is modulated to treat cancers of hematopoietic
cells. In another embodiment malignancy can be suppressed in
certain cells e.g., leukemic cells, by modulating RAFTK to induce
cellular differentiation . . . .
[0015] . . . In one embodiment the RAFTK proteins of the present
invention can modulate the differentiation or maturation of
hematopoietic cells; the subject RAFTK polypeptides are capable of
influencing both the differentiation and maturation of pluripotent
stem cells and the proliferation of differentiated cells."
SUMMARY OF THE INVENTION
[0016] The present invention relates, inter alia, to methods for
identification of compounds useful to treat or prevent
inflammation-related diseases or disorders characterized by an
interaction between a PYK2 polypeptide and a natural binding
partner. In addition, the present invention relates to methods for
diagnosis and for treatment of inflammation-related diseases or
disorders characterized by an interaction between a PYK2
polypeptide and a natural binding partner.
[0017] The current invention demonstrates for the first time in an
in vivo mouse model and a cellular model the link between PYK2 and
the inflammatory response. To demonstrate the role of PYK2 in vivo,
knockout mice lacking the pyk2 gene were created using molecular
genetic techniques. The inflammatory response of the knockout mice
was compared with the corresponding mice not containing a pyk2
deletion. Experiments are described in detail herein in the
Detailed Description of the Invention.
[0018] The data from these experiments confirm the role for PYK2 in
cytokine release and support the importance of PYK2 function in
inflammation. These experiments indicate that treatments that
inhibit the functioning of PYK2 will be useful to decrease
excessive inflammatory responses, whereas treatments to enhance the
functioning of PYK2 will be useful to augment inadequate immune
responses.
[0019] In a first aspect, the invention features a method for
identifying one or more potential compounds useful to treat or to
prevent a disease or disorder, wherein said disease or disorder is
characterized by a inflammatory response, wherein said inflammatory
response is characterized by an abnormality in a signal
transduction pathway, and wherein said signal transduction pathway
includes an interaction between a PYK2 polypeptide and a natural
binding partner, comprising assaying said one or more potential
compounds for those able to modulate said interaction as an
indication of a useful said compound.
[0020] By "identifying" is meant investigating for the presence or
absence of a property. The process may include measuring or
detecting various properties, including the level of signal
transduction and the level of interaction between a PYK2
polypeptide and a natural binding partner.
[0021] The term "compound" preferably refers to a non-peptide
organic molecule, and most preferably refers to a non-peptide
synthetic organic molecule. The term "non-peptide molecule" refers
to a compound that is not a polymer of amino acids. A non-peptide
molecule preferably does not contain chemical moieties that
hydrolyze in physiological conditions, e.g. a peptidomimetic.
Alternatively, a non-peptide molecule may be a peptoid, or modified
nucleic acid molecule. Examples of compounds are included in the
Description of the Invention, herein. Preferably, such molecules
have a molecular weight less than 3,000.
[0022] By "inflammatory response" is meant a protective response
elicited by injury or destruction of tissues, which serves to
destroy, dilute, or wall off (sequester) both the injurious agent
and the injured tissue. Histologically, it involves a complex
series of events, including dilatation of arterioles, capillaries,
and venules, with increased permeability and blood flow; exudation
of fluids, including plasma proteins; and leukocyte migration into
the inflammatory focus. A pathologic inflammatory response may be a
continuation of an acute form or a prolonged low-grade form, and
usually causes permanent tissue damage. Macrophage and T-cell
recruitment and functions, such as cytokine production, directly
contribute to inflammatory pathogenesis. There are many types of
diseases and disorders associated with inflammatory responses, all
of which are intended to be included under specific embodiments of
the present invention.
[0023] An "organism" can be single or multi-cellular. The term
includes mammals, and, most preferably, humans. Preferred organisms
include mice, as the ability to treat or diagnose mice is often
predictive of the ability to function in other organisms such as
humans.
[0024] By "disease or disorder" is meant a state in an organism
(e.g., a human) which is recognized as abnormal by members of the
medical community. The disease or disorder is characterized by an
abnormality in one or more signal transduction pathways in a cell,
where the components of the signal transduction pathway include a
PYK2 polypeptide and a natural binding partner.
[0025] By "abnormality" is meant a level which is statistically
different from the level observed in organisms not suffering from
such a disease or disorder and may be characterized as either an
excess amount, intensity or duration of signal or a deficient
amount, intensity or duration of signal. The abnormality in signal
transduction may be realized as an abnormality in cell function,
viability or differentiation state. Such abnormality in a pathway
can be alleviated by action at the PYK2:natural binding partner
interaction site in the pathway. An abnormal interaction level may
also either be greater or less than the normal level and may impair
the normal performance or function of the organism. Thus, it is
also possible to screen for agents that will be useful for treating
a disease or condition, characterized by an abnormality in the
signal transduction pathway, by testing compounds for their ability
to affect the interaction between a PYK2 polypeptide and a natural
binding partner, since the complex formed by such interaction is
part of the signal transduction pathway. However, in some
embodiments, the disease or condition may be characterized by an
abnormality in the signal transduction pathway even if the level of
interaction between the PYK2 polypeptide and natural binding is
normal. Further in some embodiments, the defect may result from an
inability of a natural binding partner to perform a function on
PYK2, or PYK2 to perform a function on a natural binding partner,
or both. Finally, in some preferred embodiments, the abnormality
does not fall within what is traditionally meant by the signal
transduction pathway, e.g. it may involve an activity of PYK2 that
does not directly relate to signal transduction.
[0026] The term "signal transduction pathway" refers to the
molecules that propagate an extracellular signal through the cell
membrane to become an intracellular signal. This signal can then
stimulate a cellular response. The polypeptide molecules involved
in signal transduction processes are typically (but not limited to)
receptor and non-receptor protein tyrosine kinases, receptor and
non-receptor protein phosphatases, SRC homology 2 and 3 domains,
PDZ domain containing proteins, phosphotyrosine binding proteins
(SRC homology 2 (SH2) and phosphotyrosine binding (PTB and PH)
domain containing proteins), PTB (phosphotyrosine binding) domain
that binds to phosphotyrosine as well as non-phosphorylated
peptides, PH (pleckstrin homology) domain that binds to
phosphoinositides, proline-rich binding proteins (SH3 domain
containing proteins), nucleotide exchange factors, and
transcription factors.
[0027] By "interact" is meant any physical association between
polypeptides, whether covalent or non-covalent. This linkage can
include many chemical mechanisms, for instance covalent binding,
affinity binding, intercalation, coordinate binding and
complexation. Examples of non-covalent bonds include electrostatic
bonds, hydrogen bonds, and Van der Waals bonds. Furthermore, the
interactions between polypeptides may either be direct or indirect.
Thus, the association between two given polypeptides may be
achieved with an intermediary agent, or several such agents, that
connects the two proteins of interest (e.g., a PYK2 polypeptide and
a natural binding partner). Another example of an indirect
interaction is the independent production, stimulation, or
inhibition of both a PYK2 polypeptide and natural binding partner
by a regulatory agent. Depending upon the type of interaction
present, various methods may be used to measure the level of
interaction. For example, the strengths of covalent bonds are often
measured in terms of the energy required to break a certain number
of bonds (i.e., kcal/mol). Non-covalent interactions are often
described as above, and also in terms of the distance between the
interacting molecules. Indirect interactions may be described in a
number of ways, including the number of intermediary agents
involved, or the degree of control exercised over the PYK2
polypeptide relative to the control exercised over the natural
binding partner.
[0028] By "a PYK2 polypeptide" is meant the PYK2 polypeptide
described in U.S. Pat. No. 5,837,815 to Lev et al. and WO
publication WO 96/18738 by Lev et al., both hereby incorporated by
reference herein in their entirety including tables, figures, and
drawings. The isolation and characterization of the PYK2
polypeptide is also fully described therein. The PYK2 polypeptide
can be encoded by a full-length nucleic acid sequence or any
portion of the full-length nucleic acid sequence, so long as a
functional activity of the polypeptide is retained. Preferred
functional activities include, but are not limited to, the ability
to phosphorylate and regulate RAK and/or other potassium channels.
A variety of methodologies known in the art can be utilized to
obtain PYK2 polypeptides for use in the methods of the
invention.
[0029] The term "natural binding partner" refers to polypeptides,
lipids, small molecules, or nucleic acids that bind to kinases in
cells. A change in the interaction between a kinase and a natural
binding partner can manifest itself as an increased or decreased
probability that the interaction forms, or an increased or
decreased concentration of kinase/natural binding partner complex.
Binding is understood to include interactions such as
phosphorylation or dephosphorylation, for example.
[0030] The term "modulates" refers to the ability of a compound to
alter the interaction of PYK2 and a natural binding partner. The
K.sub.m of a compound is preferably between 100 .mu.M and 1 .mu.M,
more preferably between 1 .mu.M and 100 nM, most preferably between
100 nM and 1 nM. A modulator preferably promotes or disrupts the
interaction of PYK2 and a natural binding partner. Alternatively,
the modulator may increase or decrease the cellular activity of the
kinase, including phosphorylation. Kinase activity is preferably
the phosphorylation of a natural binding partner on tyrosine,
serine, or threonine residues. Changes in the interaction with a
natural binding partner can also include increasing or decreasing
the probability that a complex forms between the kinase and a
natural binding partner. A modulator preferably increases the
probability that such a complex forms between the kinase and the
natural binding partner, and most preferably decreases the
probability that a complex forms between the kinase and the natural
binding partner. In some preferred embodiments the interaction
includes actions of the natural binding partner on PYK2.
[0031] The term "complex" refers to an assembly of at least two
molecules bound to one another. Signal transduction complexes often
contain at least two protein molecules bound to one another. For
instance, a protein tyrosine kinase receptor, GRB2, SOS, RAF, and
RAS assemble to form a signal transduction complex in response to a
mitogenic ligand.
[0032] By "disrupt" is meant that the interaction between the PYK2
polypeptide and a natural binding partner is reduced either by
preventing expression of the PYK2 polypeptide, or by preventing
expression of the natural binding partner, or by specifically
preventing interaction of the naturally synthesized proteins or by
interfering with the interaction of the proteins.
[0033] By "promote" is meant that the interaction between a PYK2
polypeptide and a natural binding partner is increased either by
increasing expression of a PYK2 polypeptide, or by increasing
expression of a natural binding partner, or by decreasing the
dephosphorylating activity of the corresponding regulatory TP (or
other phosphatase acting on other phosphorylated signaling
components), by promoting interaction of the PYK2 polypeptide and
natural binding partner or by prolonging the duration of the
interaction.
[0034] The term "activates" refers to increasing the cellular
activity of the kinase. The term "inhibit" refers to decreasing the
cellular activity of the kinase. Kinase activity is preferably the
phosphorylation of a natural binding partner on tyrosine,
threonine, or serine residues. Changes in the interaction with a
natural binding partner can also include increasing or decreasing
the probability that a complex forms between the kinase and a
natural binding partner. A modulator preferably increases the
probability that such a complex forms between the kinase and the
natural binding partner, and most preferably decreases the
probability that a complex forms between the kinase and the natural
binding partner.
[0035] In preferred embodiments of methods for screening for
compounds potentially useful for treating or preventing
inflammatory response-related diseases or disorders involving the
interaction of PYK2 and a natural binding partner, the inflammatory
response-related disease or disorder is selected from the group
consisting of inflammatory bowel diseases and connective tissue
diseases. Preferably, the inflammatory bowel diseases are selected
from the group consisting of ulcerative colitis and Crohn's Disease
and the connective tissue diseases are selected from the group
consisting of rheumatoid arthritis, systemic lupus erythematosus,
progressive systemic sclerosis, mixed connective tissue disease,
and Sjogren's syndrome.
[0036] Macrophage function and the production of cytokines by
macrophages and other cells associated with the inflammatory
response directly contribute to the pathophysiologic progression of
the diseases. The importance of PYK2 in these disease processes is
indicated by the decreased production of cytokines in cells from
pyk2-/- mice and in macrophage cell lines expressing kinase
inactive PYK2. Thus, inhibiting PYK2 function in inflammatory cells
should alleviate some of the pathologic consequences associated
with these diseases.
[0037] The term "inflammatory bowel disease" as used herein, refers
to inflammatory diseases of the bowel, many of which are of unknown
etiology, including Crohn's disease and ulcerative colitis.
[0038] The term "Crohn's Disease" as used herein, refers to a
chronic granulomatous inflammatory disease of unknown etiology,
involving any part of the gastrointestinal tract from mouth to
anus, but commonly involving the terminal ileum and/or colon with
scarring and thickening of the bowel wall. It frequently leads to
intestinal obstruction and fistula and abscess formation and has a
high rate of recurrence after treatment.
[0039] By "ulcerative colitis" is meant chronic, recurrent
ulceration in the colon, chiefly of the mucosa and submucosa, of
unknown cause. The rectum is almost always involved. It is
manifested clinically by cramping abdominal pain, rectal bleeding,
and loose discharges of blood, pus, and mucus with scanty fecal
particles. Complications include hemorroids, abscesses, fistulas,
perforation of the colon, pseudopolyps, and carcinoma.
[0040] The term "connective tissue diseases" as used herein refers
to heterogeneous disorders which share certain common features,
including inflammation of skin, joints, and other structures rich
in connective tissue, as well as altered patterns of
immunoregulation, including production of autoantibodies and
abnormalities of cell-mediated immunity. While certain distinct
clinical entities may be defined, manifestations may vary
considerably from one patient to the next and overlap of clinical
features between and among specific diseases is common.
[0041] The term "rheumatoid arthritis" as used herein refers to a
chronic systemic disease primarily of the joints, usually
polyarticular, marked by inflammatory changes in the synovial
membranes and articular structures and by muscle atrophy and
rarefaction of the bones. Persistent inflammatory synovitis usually
involves the peripheral joints in a symmetrical fashion, marked by
cartilaginous destruction, bony erosions, and joint deformation.
Infiltration of inflammatory cells is common. Forms of rheumatoid
arthritis include, but are not limited to, juvenile, chronic
villous, cricoarytenoid, deformans, degenerative, mutilans, and
proliferative.
[0042] The term "systemic lupus erythematosus" as used herein,
refers to a disease in which tissues and cells are damaged by
deposition of pathogenic antibodies and immune complexes. B-cell
hyperactivity, production of autoantibodies with specificity for
nuclear antigenic determinants, and abnormalities of T-cell
function occur. It may involve virtually any organ system and
follows a course of exacerabation followed by remission. A common
feature is the alar "butterfly" rash.
[0043] The term "progressive systemic sclerosis" as used herein
refers to a multisystem disorder characterized by inflammatory,
vascular, and fibrotic changes of skin and various internal organ
systems (chiefly GI tract, lungs, heart, and kidney). Primary event
may be endothelial cell injury with eventual intimal proliferation,
fibrosis, and vessel obliteration. Clinical manifestations include,
but are not limited to, Raynaud's phenomenon, scleroderma (fibrosis
of the skin), hypertension, and renal failure.
[0044] The term "mixed connective tissue disease" as used herein,
refers to syndrome characterized by a combination of clinical
features similar to those of systemic lupus erythematosus,
progressive systemic sclerosis, polymyositis, and rheumatoid
arthritis. Unusually high titers of circulating antibodies to a
nuclear ribonucleoprotein are found. Clinical manifestations
include Raynaud's phenomenon, polyarthritis and pulmonary fibrosis
among others.
[0045] The term "Sjogren's syndrome" as used herein refers to an
immunologic disorder characterized by progressive destruction of
exocrine glands leading to mucosal and conjunctival dryness (sicca
syndrome). Affected tissues show lymphocyte infiltration and
immune-complex deposition.
[0046] In preferred embodiments of methods of identifying
compounds, the one or more compounds modulate (inhibit or promote)
the interaction of PYK2 and a natural binding partner in vitro. An
example of an in vitro method involves growing cells (i.e., in a
dish) that either naturally or recombinantly express a G-coupled
protein receptor, PYK2, and RAK. The test compound preferably is
added at a concentration from 0.1 .mu.M to 100 .mu.M and the
mixture preferably is incubated from 5 minutes to 2 hours. The
ligand is added to the G-coupled protein receptor preferably for 5
to 30 minutes and the cells are lysed. RAK is isolated using
immunoprecipitation or ELISA by binding to a specific monoclonal
antibody. The amount of phosphorylation compared to cells that were
not exposed to a test compound is measured using an
anti-phosphotyrosine antibody (preferably polyclonal).
Alternatively, in other methods of identifying compounds, the one
or more compounds modulate (inhibit or promote) PYK2 and natural
binding partner interactions in vivo.
[0047] In other methods of identifying compounds, the interaction
is selected from the group consisting of PYK2 phosphorylation, PYK2
natural binding partner phosphorylation, PYK2 de-phosphorylation,
PYK2 natural binding partner de-phosphorylation, and complex
formation between PYK2 and a natural binding partner.
[0048] Examples of compounds that could be tested in such screening
methods include tyrphostins, quinazolines, quinoxolines,
quinolines, and indolinones. Publications describing representative
examples of these compounds and methods of making are given in the
Detailed Description of the Invention.
[0049] A second aspect of the invention features a method for
diagnosis of a disease or disorder, wherein said disease or
disorder is characterized by an inflammatory response involving an
abnormality in a signal transduction pathway that includes an
interaction between a PYK2 polypeptide and a natural binding
partner, comprising detecting the level of said interaction as an
indication of said disease or disorder.
[0050] By "diagnosis" is meant any method of identifying a symptom
normally associated with a given disease or condition. Thus, an
initial diagnosis may be conclusively established as correct by the
use of additional confirmatory evidence such as the presence of
other symptoms. Current classification of various diseases and
conditions is constantly changing as more is learned about the
mechanisms causing the diseases or conditions. Thus, the detection
of an important symptom, such as the detection of an abnormal level
of interaction between PYK2 polypeptides and natural binding
partners may form the basis to define and diagnose a newly named
disease or condition. For example, conventional cancers are
classified according to the presence of a particular set of
symptoms. However, a subset of these symptoms may both be
associated with an abnormality in a particular signaling pathway,
such as the ras.sup.21 pathway and in the future these diseases may
be reclassified as ras.sup.21 pathway diseases regardless of the
particular symptoms observed.
[0051] In preferred embodiments of methods for screening for
diagnosis of inflammatory response-related diseases or disorders
involving the interaction of PYK2 and a natural binding partner,
the inflammatory response-related disease or disorder is selected
from the group consisting of inflammatory bowel diseases and
connective tissue diseases. Preferably, the inflammatory bowel
diseases are selected from the group consisting of ulcerative
colitis and Crohn's Disease and the connective tissue diseases are
selected from the group consisting of rheumatoid arthritis,
systemic lupus erythematosus, progressive systemic sclerosis, mixed
connective tissue disease, and Sjogren's syndrome.
[0052] A third aspect of the invention features a method for
treating or preventing a disease or disorder, wherein said disease
or disorder is characterized by an inflammatory response involving
an abnormality in a signal transduction pathway that includes an
interaction between a PYK2 polypeptide and a natural binding
partner, comprising administering to a patient in need of such
treatment one or more compounds preferably in a pharmaceutically
acceptable composition, wherein said one or more compounds modulate
said interaction.
[0053] In preferred embodiments of methods for treating or
preventing inflammatory response-related diseases or disorders
involving the interaction of PYK2 and a natural binding partner,
the inflammatory response-related disease or disorder is selected
from the group consisting of inflammatory bowel diseases and
connective tissue diseases. Preferably, the inflammatory bowel
diseases are selected from the group consisting of ulcerative
colitis and Crohn's Disease and the connective tissue diseases are
selected from the group consisting of rheumatoid arthritis,
systemic lupus erythematosus, progressive systemic sclerosis, mixed
connective tissue disease, and Sjogren's syndrome.
[0054] In other preferred embodiments of the methods for treating
or preventing inflammatory response-related diseases or disorders
involving the interaction of PYK2 and a natural binding partner,
the one or more compounds modulate (inhibit or promote) the
interaction in vitro and/or in vivo. In some preferred embodiments,
the interaction is selected from the group consisting of PYK2
phosphorylation, PYK2 natural binding partner phosphorylation, PYK2
de-phosphorylation, PYK2 natural binding partner
de-phosphorylation, and complex formation between PYK2 and a
natural binding partner.
[0055] In yet other preferred embodiments of methods for treating
or preventing inflammation-related diseases or disorders involving
the interaction of PYK2 and a natural binding partner, the one or
more compounds is selected from the group consisting of
tyrphostins, quinazolines, quinoxolines, quinolines, and
indolinones.
[0056] In preferred embodiments the agent is therapeutically
effective and preferably has an EC.sub.50, or IC.sub.50 of less
than or equal to 100 .mu.M, even more preferably less than or equal
to 50 .mu.M, and most preferably less than or equal to 10 .mu.M.
Such lower EC.sub.50's or IC.sub.50's are advantageous since they
allow lower concentrations of molecules to be used in vivo or in
vitro for therapy or diagnosis. The discovery of molecules with
such low EC.sub.50's and IC.sub.50's enables the design and
synthesis of additional molecules having similar potency and
effectiveness. Generally, a therapeutically effective amount is
between about 1 nmol and 1 .mu.mol of the molecule, depending on
its EC.sub.50 or IC.sub.50, and on the age and size of the patient,
and the disease associated with the patient.
[0057] The term "preventing" refers to decreasing the probability
that an organism contracts or develops an abnormal condition. Thus,
in these cases a patient would include someone who is thought to be
at risk for contracting an abnormal condition. Persons skilled in
the art would be able to identify persons who would be considered
at risk from contracting an abnormal condition.
[0058] The term "treating" refers to having a therapeutic effect
and at least partially alleviating or abrogating an abnormal
condition in the organism. In this case the patient is already been
identified as having an abnormal condition.
[0059] The term "therapeutic effect" refers to the inhibition or
activation of factors causing or contributing to the abnormal
condition. A therapeutic effect relieves to some extent one or more
of the symptoms of the abnormal condition. In reference to the
treatment of abnormal conditions, a therapeutic effect can refer to
one or more of the following: (a) an increase or decrease in the
infiltration of cells; (b) inhibition of (i.e., slowing or
stopping) or increase in cell movement; (c) relieving to some
extent one or more of the symptoms associated with the abnormal
condition; and (d) enhancing or inhibiting the function of the
affected population of cells. Compounds demonstrating efficacy
against abnormal conditions can be identified as described
herein.
[0060] The term "abnormal condition" refers to a function in the
cells or tissues of an organism that deviates from their normal
functions in that organism. An abnormal condition can relate to
cell proliferation, cell differentiation, cell function, or cell
survival.
[0061] Abnormal cell infiltration conditions include, but are not
limited to, rheumatoid arthritis and chronic inflammatory bowel
disease.
[0062] The term "aberration", in conjunction with the function of a
kinase in a signal transduction process, refers to a kinase that is
over- or under-expressed in an organism, mutated such that its
catalytic activity is lower or higher than wild-type protein kinase
activity, mutated such that it can no longer interact with a
natural binding partner, is no longer modified by another protein
kinase or protein phosphatase, or no longer interacts with a
natural binding partner.
[0063] The term "administering" relates to a method of
incorporating a compound into cells or tissues of an organism. The
abnormal condition can be prevented or treated when the cells or
tissues of the organism exist within the organism or outside of the
organism. Cells existing outside the organism can be maintained or
grown in cell culture dishes. For cells harbored within the
organism, many techniques exist in the art to administer compounds,
including (but not limited to) oral, parenteral (e.g. intra-venous,
intramuscular, sub-cutaneous, and intra-articular) and aerosol
applications. The compounds may also be administered in a depot or
sustained release formulation. For cells outside of the organism,
multiple techniques exist in the art to administer the compounds,
including (but not limited to) cell microinjection techniques,
simple diffusion, and carrier techniques.
[0064] The term "pharmaceutically acceptable" or "pharmaceutical"
as used herein refers to solutions or components of the
pharmaceutical composition that do not prevent the therapeutic
compound from exerting a therapeutic effect and do not cause
unacceptable adverse side effects. Examples of pharmaceutically
acceptable reagents are provided in The United States Pharmacopeia
The National Formulary, United States Pharmacopeial Convention,
Inc., Rockville, Md. 1990 and FDA Inactive. Ingredient Guide 1990,
(1996) issued by the Division of Drug Information Resources (both
are hereby incorporated by reference herein, including any
drawings). Unacceptable side effects vary for different diseases.
Generally, the more severe the disease the more toxic effects which
will be tolerated. Unacceptable side effects for different diseases
are known in the art.
[0065] The term "physiologically acceptable" defines a carrier or
diluent that does not cause significant irritation to an organism
and preferably does not abrogate the biological activity and
properties of the compound.
[0066] The term "carrier" defines a chemical compound that
facilitates the incorporation of a compound into cells or tissues.
For example dimethyl sulfoxide (DMSO) is a commonly utilized
carrier as it facilitates the uptake of many organic compounds into
the cells or tissues of an organism.
[0067] The term "diluent" defines chemical compounds diluted in
water (or another solvent) that will dissolve the compound of
interest as well as stabilize the biologically active form of the
compound. Many salts dissolved in buffered solutions are utilized
as diluents in the art. One commonly used buffered solution is
phosphate buffered saline because it mimics the salt conditions of
human blood. Because buffer salts can control the pH of a solution
at low concentrations, a diluent rarely modifies the biological
activity of a compound.
[0068] The term "solvent" as used herein refers to a chemical
compound that facilitates the solubilization of compounds of the
invention. Examples of solvents include, but are not limited to,
pharmaceutically acceptable alcohols, such as ethanol and benzyl
alcohol; polyoxyhydrocarbyl compounds, such as poly(ethylene
glycol); pharmaceutically acceptable surfactants such as
CREMOPHOR.RTM. EL; polyglycolized lipids, such as GELUCIRE.RTM. and
LABRASOL.RTM.; and pharmaceutically acceptable oils, such as
miglyol 812.
[0069] The term "pharmaceutically acceptable alcohol" as used
herein refers to alcohols that are liquids at about room
temperature (approximately 20.degree. C.). These include propylene
glycol, ethanol, 2-(2-ethoxyethoxy)ethanol (TRANSCUTOL.RTM.,
Gattefosse, Westwood, N.J. 07675), benzyl alcohol, and
glycerol.
[0070] The term "polyoxyhydrocarbyl compound" as used herein refers
to a water soluble carbohydrate such as glucose, sucrose,
maltotriose, and the like; water soluble carbohydrate derivatives
such as gluconic acid and mannitol, and oligosaccharides; and water
soluble polymers such as polyvinylpyrrolidone, poly(vinyl alcohol),
and in particular, polyethers such as other polyoxyalkylenes
including poly(ethylene glycol) or other water soluble mixed
oxyalkylene polymers and the polymeric form of ethylene glycol.
Although polyoxyhydrocarbyl compounds preferably contain more than
one carbon, oxygen, and hydrogen atom, some molecules such as
poly(ethylene imine) are also included.
[0071] A particularly preferred class of solubilizing
polyoxyhydrocarbyl moieties comprises poly(ethylene glycol) (PEG)
and PEG derivatives, such as PEG monomethyl ether. Other suitable
PEG derivatives include PEG-silicon derived ethers. Many of these
polymers are commercially available in a variety of molecular
weights. Others may be conveniently prepared from commercially
available materials, such as by coupling of amino-PEG moiety to a
haloalkyl silyl or silane moiety.
[0072] Suitable PEGs may vary in molecular weight from about 200
g/mol to about 20,000 g/mol or more, more preferably 200 g/mol to
5,000 g/mol, even more preferably 250 g/mol to 1,000 g/mol, and
most preferably 250 g/mol to 500 g/mol. The choice of a particular
molecular weight may depend on the particular compound chosen and
its molecular weight and degree of hydrophobicity, as well as the
particular application for which the formulation is to be used.
[0073] The term "pharmaceutically acceptable surfactant" as used
herein refers to a compound that can solubilize compounds of the
invention into aqueous solutions, if necessary. Preferably for
parenteral formulations, the surfactant is a non-ionic surfactant.
Examples of pharmaceutically acceptable surfactants include
POLYSORBATE 80 and other polyoxyethylene sorbitan fatty acid
esters, glyceryl monooleate, polyvinyl alcohol, ethylene oxide
copolymers such as PLURONIC.RTM. (a polyether) and TETRONIC.RTM.
(BASF), polyol moieties, and sorbitan esters. Preferably
ethoxylated castor oils, such as CREMOPHOR.RTM. EL, are used for
the formulation of some compounds.
[0074] The term "ethoxylated castor oil" as used herein refers to
castor oil that is modified with at least one oxygen containing
moiety. In particular the term refers to castor oil comprising at
least one ethoxyl moiety.
[0075] Further, the term "pharmaceutically acceptable surfactant"
as used herein in reference to oral formulations, includes
pharmaceutically acceptable non-ionic surfactants (for example
polyoxyethylene-polypropyle- ne glycol, such as POLOXAMER.RTM. 68
(BASF Corp.) or a mono fatty acid ester of polyoxyethylene (20)
sorbitan monooleate (TWEEN.RTM. 80), polyoxyethylene (20) sorbitan
monostearate (TWEEN.RTM. 60), polyoxyethylene (20) sorbitan
monopalmitate (TWEEN.RTM. 40), polyoxyethylene (20) sorbitan
monolaurate (TWEEN.RTM. 20) and the like); polyoxyethylene castor
oil derivatives (for example,
polyoxyethyleneglycerol-triricinoleate or polyoxyl 35 castor oil
(CREMOPHOR.RTM. EL, BASF Corp.), polyoxyethyleneglycerol
oxystearate (CREMOPHOR.RTM. RH 40 (polyethyleneglycol 40
hydrogenated castor oil) or CREMOPHOR.RTM. RH 60
(polyethyleneglycol 60 hydrogenated castor oil), BASF Corp.) and
the like); or a pharmaceutically acceptable anionic surfactant.
[0076] The term "polyglycolized lipids" as used herein refers to
mixtures of monoglycerides, diglycerides, or triglycerides and
polyethyleneglycol monoesters and diesters formed by the partial
alcoholysis of vegetable oil using PEG of 200 g/mol to 2,000 g/mol
or by the esterification of fatty acids using PEG 200 g/mol to
2,000 g/mol and glycerols. Preferably these include GELUCIRE.RTM.
35/10, GELUCIRE.RTM. 44/14, GELUCIRE.RTM. 46/07, GELUCIRE.RTM.
50/13, GELUCIRE.RTM. 53/10, and LABRASOL.RTM..
[0077] The term "pharmaceutically acceptable oils" as used herein
refers to oils such as mineral oil or vegetable oil (including
safflower oil, peanut oil, and olive oil), fractionated coconut
oil, propylene glycol monolaurate, mixed triglycerides with
caprylic acid and capric acid, and the like. Preferred embodiments
of the invention feature mineral oil, vegetable oil, fractionated
coconut oil, mixed triglycerides with caprylic acid, and capric
acid. A highly preferred embodiment of the invention features
Miglyol.RTM. 812 (available from Huls America, USA).
[0078] In preferred embodiments, the methods described herein
involve identifying a patient in need of treatment. Those skilled
in the art will recognize that various techniques may be used to
identify such patients.
[0079] A fourth aspect of the invention features a composition
comprising one or more compounds identified by any of the methods
of the invention described above or herein. Preferably, this
composition is useful for treating or preventing a disease or
disorder, where the disease or disorder is characterized by an
inflammatory response involving an abnormality in a signal
transduction pathway that includes an interaction between a PYK2
polypeptide and a natural binding partner.
[0080] In preferred embodiments, the inflammatory response-related
disease or disorder is selected from the group consisting of
inflammatory bowel diseases and connective tissue diseases.
Preferably, the inflammatory bowel diseases are selected from the
group consisting of ulcerative colitis and Crohn's Disease and the
connective tissue diseases are selected from the group consisting
of rheumatoid arthritis, systemic lupus erythematosus, progressive
systemic sclerosis, mixed connective tissue disease, and Sjogren's
syndrome.
[0081] A fifth aspect of the invention features methods of making
compounds potentially useful to treat or to prevent a disease or
disorder, where the disease or disorder is characterized by an
inflammatory response that is characterized by an abnormality in a
signal transduction pathway, and where the signal transduction
pathway includes an interaction between a PYK2 polypeptide and a
natural binding partner, comprising assaying one or more potential
compounds for those able to modulate the interaction as an
indication of a useful compound and synthesizing the identified
compounds. References describing methods of synthesizing the
identified compounds are indicated in the Detailed Description of
the Invention.
[0082] In preferred embodiments, the inflammatory response-related
disease or disorder is selected from the group consisting of
inflammatory bowel diseases and connective tissue diseases.
Preferably, the inflammatory bowel diseases are selected from the
group consisting of ulcerative colitis and Crohn's Disease and the
connective tissue diseases are selected from the group consisting
of rheumatoid arthritis, systemic lupus erythematosus, progressive
systemic sclerosis, mixed connective tissue disease, and Sjogren's
syndrome.
[0083] A sixth aspect of the invention features kits comprising a
composition comprising one or more compounds identified by any of
the methods of the invention described above or herein. Preferably,
this composition is useful for treating or preventing a disease or
disorder, where the disease or disorder is characterized by an
inflammatory response involving an abnormality in a signal
transduction pathway that includes an interaction between a PYK2
polypeptide and a natural binding partner. The kit preferably
further comprises instructions for use either on a label or using
other suitable means as discussed herein in the Detailed
Description of the Invention.
[0084] In other preferred embodiments, the kit comprises additional
container means containing one or more of the following: diluents,
carriers, and solvents. Such containers include small glass
containers, plastic containers, or strips of plastic or paper. Such
containers allow the efficient transfer of contents from one
container to another, such that the contents are not contaminated
and the contents can be added in a quantitative fashion from one
compartment to another. The kit may additionally comprise means for
administering the composition. One skilled in the art will readily
recognize that the compositions of the instant invention can be
readily incorporated into one of the established kit formats that
are well-known in the art.
[0085] In preferred embodiments, the composition contained in the
kit is useful for treating or preventing an inflammatory
response-related disease or disorder selected from the group
consisting of inflammatory bowel diseases and connective tissue
diseases. Preferably, the inflammatory bowel diseases are selected
from the group consisting of ulcerative colitis and Crohn's Disease
and the connective tissue diseases are selected from the group
consisting of rheumatoid arthritis, systemic lupus erythematosus,
progressive systemic sclerosis, mixed connective tissue disease,
and Sjogren's syndrome.
[0086] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0087] These figures are provided for illustration only, and are
not considered necessary to disclose the invention.
[0088] FIGS. 1a, 1b, and 1c show the targeted disruption of the
pyk2 gene in mice. Partial restriction maps of murine pyk2 locus,
the regions used as a targeting vector, and the expected mutant
allele are shown in FIG 1a. Solid boxes indicate exons. The probe
was used for Southern blot hybridization analysis of the genomic
DNA from the ES cells and tail biopsy analysis. The solid line
under the pyk2 locus marks the PYK2 kinase domain. Arrows mark the
expected Apal fragments of the wild-type and mutant alleles. FIG.
1b shows Southern blot hybridization analysis of the mouse tail DNA
digested with Apal. FIG. 1c shows immunoblot analysis with
anti-PYK2 antibodies of lysates from Thymus(T), Brain (B), and
Spleen (S) from wild-type, pyk2+/-, and pyk2-/- mice. FIG. 1d shows
an immunoblot analysis of tissue lysates of FAK from wild type or
PYK2-/- mice. B, Brain; T, Thymus; S, spleen; H, Heart; K, Kidney;
L, Lung.
[0089] FIG. 2 shows RT-PCR (reverse transcriptase-polymerase chain
reaction) analysis of cytokine productions from wild-type +/+ and
pyk2-/- thioglycollated-elicited peritoneal macrophages stimulated
by LPS. Thioglycollate-elicited peritoneal macrophages were
incubated with or without LPS (10 .mu.g/mL) for 3 h, 6 h, 12 h, 24
h, and 48 h. Total RNAs were isolated from cells at each time point
and after normalization of the amount of mRNA in the total RNAs by
RT-PCR of actin mRNA, total RNAs were subjected to RT-PCR with
specific probes for various cytokines.
[0090] FIG. 3 shows RT-PCR analysis of the cytokine productions
from wild-type +/+ and pyk2-/- splenocytes stimulated by
anti-murine CD3 antibodies. Splenocytes were incubated with or
without anti-CD3 (1 .mu.g/mL) for 12 h, 24 h, 48 h and 72 h. Total
RNAs were isolated from cells at each time point and after
normalization of the amount of mRNA in the total RNAs by RT-PCR of
the actin mRNA, total RNAs were subjected to RT-PCR with specific
probes for various cytokines.
[0091] FIGS. 4a, 4b, and 4c demonstrate Carageenen induced cellular
infiltration in murine air pouches. FIG. 4a shows tissues from wild
type +/+ and pyk2-/- air pouches that were treated with Carageenen
for 10 hours after injection. Samples were formalin-fixed and
paraffin-embedded. Sections were stained with Hematoxylin and
Eosin. FIG. 4b shows a number of cells infiltrating into wild type
+/+ and pyk2-/- air pouches 10 hours after injection with
Carageenen. FIG. 4c shows a fraction of cells infiltrating into
wild-type +/+ and pyk2-/- mice air pouches 10 hours after injection
with Carageenen.
[0092] FIGS. 5a and 5b demonstrate influenza virus-induced
inflammation. FIG. 5a shows histologic sections that were made of
the lung from influenza virus-infected wild-type +/+ and pyk2-/-
mice 4 days after infection with the virus. Lungs were
formalin-fixed and paraffin-embedded. Sections were stained with
Hematoxylin and Eosin. FIG. 5b shows the time course of the
survival of mice at different doses of influenza virus.
[0093] FIGS. 6a and 6b show the tyrosine phosphorylation of PYK2 in
macrophage cell lines in response to LPS (lipopolysaccharide) and
tyrosine kinase inhibitors. Cells were either starved overnight in
0.5% serum and stimulated with LPS for variable time intervals
(FIG. 6a), or were pre-treated with either herbimycin A or
genestein for 4 hours and then stimulated with LPS for 30 minutes
(FIG. 6b). After stimulation, cells were washed, lysed, and
immunoprecipitated with anti-PYK2 antibody. Immunoprecipitates (IP)
were then analyzed by western blotting.
[0094] FIGS. 7a, 7b, 7c, and 7d demonstrate that expression of both
the PYK2 wild-type and the dominant negative kinase mutant are
inducible in P388D1 cells. The murine macrophage cell line (P388D1)
was serially transfected with doxycycline (DOX) inducible plasmids.
In the first transfection, a regulator plasmid was introduced, and
stable, drug-resistant clones were established. Two clones were
then selected and transfected with doxycycline-responsive plasmids
containing either HA-tagged (hemagglutinin), wild-type PYK2 or the
kinase dead, dominant-negative PYK2 mutant (also HA-tagged). After
drug selection, stable clones containing the wild-type (FIGS. 7b
and 7d) or the dominant-negative mutant (FIGS. 7a and 7c) were
screened for expression of the appropriate protein in the presence
(FIGS. 7a and 7b) and absence (FIGS. 7c and 7d) of doxycyclin by
western blotting.
[0095] FIGS. 8a, 8b, 8c, and 8d compare the secretion of
TNF-.alpha. by wild-type and kinase mutant P388D1 cells in response
to activators of macrophage function or PYK2 activity. FIGS. 8a and
8b show the effect of LPS in the presence and absence of DOX on
TNF-.alpha. secretion from kinase mutant (dominant negative; FIG.
8a) and wild-type (FIG. 8b) cells. FIGS. 8c and 8d show the effect
of PMA in the presence and absence of DOX on TNF-.alpha. secretion
from kinase mutant (FIG. 8c) and wild-type (FIG. 8d) cells.
Induction of P388D1 cells in the presence of doxicycline was
carried out for 48 hours followed by stimulation with LPS (1
.mu.g/mL) or PMA (1 .mu.g/mL) for 18 hrs. Secreted TNF-.alpha. was
measured by ELISA.
[0096] FIGS. 9a, and 9b show morphological abnormalities and
impaired cell migration in PYK2-/- macrophages. FIG. 9a shows
PYK2-/- (b, d, f) or wild type macrophages (a, c, e) were plated on
tissue culture dishes. Micrographs of unstimulated (a, b) or
SDF1a-stimulated macrophages (c, d, e, f). White arrows mark long
multidirectional processes and white broken arrows mark
multidirectional lamellipodia that are seen in PYK2-/- but not in
wild type macrophages. FIG. 9b shows micrographs of SDF-stimulated
or unstimulated PYK2-/- or wild type macrophages plated on tissue
culture dishes at different time points. White arrows mark the
original point of cell movement, large black arrows mark cell
contraction and small black arrows mark lamellipodia.
[0097] FIGS. 10a and 10b show measurement of contractile capacity
of lamellipodia in wild type or PYK2-/- macrophages by laser
tweezers. FIG. 10a shows plots of bead displacement from leading
edge as a function of time. Fibronectin coated beads were
positioned with tweezers on the lamellipodia of PYK2-/- or wild
type macrophages of unstimulated (top panels) or
MIP1.alpha.-stimulated (bottom panels) cells near the leading edge
at time 0. The trap remained on for approximately 60 sec. as
indicated by shaded area. Two upper plots present beads
displacement on non-stimulated macrophages and two lower plots
present displacement on MIP1.alpha.-stimulated macrophages. FIG.
10b shows a histogram of ratio (%) of beads displaying escape from
trapped field by laser tweezer. All the beads on the lamellipodia
of PYK2-/- or wild type macrophages before and after stimulation by
MIP1.alpha. were subjected to a restraining force after initial
bead-cell contact for 60 sec. Ratios of beads which escaped and
moved rearward were scored. Left column shows score of
non-stimulated macrophages and right column shows score of
MIP1.alpha.-stimulated macrophages.
[0098] FIGS. 11a, 11b, 11c, and 11 d show changes in cytoskeletal
organization in PYK2-/- macrophages. PYK2-/- and wild type
macrophage were plated on tissue culture dishes, fixed by 4%
paraformaldehyde and stained by fluorescently labeled phalloidin (A
and B) or anti-.alpha.-tubulin antibodies (D). In FIG. 11a white
arrows in
[0099] (b) and (d) indicate long multi-directional processes and
broken white arrows in (b) and (d) indicate membrane ruffles in
PYK2-/- macrophages. In FIG. 11b, top and side view of F-actin
distribution were visualized with a confocal microscope. Arrowheads
indicate reorganized F-actin in PYK2-/- macrophages. Black arrows
reveal the planes of the slices generated in top and side views. In
FIG. 11c, PYK2-/- and wild type macrophages were placed in
MIP1.alpha. gradient concentration for 60 min., fixed and then
stained with fluorescently labeled phalloidin. The top left part of
the field was exposed to the highest concentration of MIP1.alpha..
White arrows mark regions with strong phalloidin labeling. In FIG.
1d, nonstimulated (a, c) or MIP1.alpha.-stimulated (b, d)
macrophages. White arrowheads in (b) and (d) demonstrate
microtubules assembled at cell periphery in PYK2-/- macrophages.
White arrow in (d) demonstrates decreased intensity of MTOC in
PYK2-/- macrophages.
[0100] FIGS. 12a, 12b, 12c, and 12d show a comparison of cell
signaling in wild type and PYK2-/- deficient macrophages. For FIG.
12a, PYK2-/- and wild type macrophages were plated on fibronectin
coated dishes for 0.5, 1 and 2 hour, then lysed and incubated with
GST-RBD(rho-binding domain) bound to gluthatione beads as described
in material and methods. The amount of rhoA: GTP complex was
determined by immunoblotting with anti-rhoA antibodies. For FIG.
12b, the rho inhibitor C3 was microinjected together with
fluorescently labeled dextran into wild type macrophages. After
three hours incubation, the morphology of microinjected or
non-injected cells was compared by Nomarsky microscopy (right). The
injected macrophages were identified by their fluorescence (left).
Arrowheads mark microinjected macrophages, arrows mark lamellipodia
and broken arrows mark long process. FIG. 12c shows Ca.sup.+2
release in PYK2-/- or wild type macrophages. Macrophages loaded by
fura-2 were stimulated by MIP1.alpha. in Ringer's solution with 2
mM calcium. Changes in fluorescence intensity as a function of time
were traced in the fura-2 loaded cells following 1-0 stimulation
with MIP1.alpha. or ATP. FIG. 12d shows production of
Ins(1,4,5)P.sub.3 in wild type or PYK2-/- macrophages. Wild type or
PYK2-/- macrophages were labeled with myo-[.sup.3H]inositol for 24
hours. After MIP1.alpha. stimulation the lipid fraction was
extracted and analyzed by HPLC. Closed square indicate production
of inositol (1,4,5) triphosphate in wild type macrophages and open
circle indicate production of inositol (1,4,5) triphosphate in
PYK2-/- macrophages. The experiment was done in duplicates and
repeated three times.
DETAILED DESCRIPTION OF THE INVENTION
[0101] I. PYK2 and Signal Transduction
[0102] PYK-2 is a non-receptor tyrosine kinase that is activated by
binding of ligand to G-coupled protein receptors such as
bradykinin, acetylcholine, and CXCR4 or CCR5. PYK2 has a predicted
molecular weight of 111 kD and contains five domains: (1) a
relatively long N-terminal domain; (2) a kinase catalytic domain;
(3) a proline rich domain; (4) another proline rich domain; and (5)
a C-terminal domain.
[0103] The C-terminal domain of PYK2 has 62% similarity to the
C-terminal domain of another non-receptor tyrosine kinase, focal
adhesion kinase (FAK), which is also activated by G-coupled
proteins. The overall similarity between PYK2 and FAK is 52%.
However, the expression of PYK2 does not correspond with the
expression of FAK. PYK2 exhibits diffuse cytoplasmic localization
unlike the preferential localization of Fak in focal adhesion
areas. PYK2 is expressed in a variety of cells including neural
tissues, hematopoietic cells, some tumor cell lines, and
immune-related cells. PYK2 is highly expressed in the nervous
system and in the adult rat brain.
[0104] PYK2 enzymatic activity is positively regulated by
phosphorylation on tyrosine and results in response to binding of
bradykinin, TPA, calcium ionophore, carbachol, TPA+ forskolin, and
membrane depolarization. Activated PYK2 is known to phosphorylate
and thus suppress the activity of the delayed rectifier-type K+
channel, termed RAK (also called Kvi.2, RBK2, RCK5 and NGKI), that
is highly expressed in the brain and cardiac atria. In the same
system, FAK does not phosphorylate RAK. PYK2 activation may provide
a rapid and highly localized control mechanism for ion channel
function and kinase activation induced by stimuli that elevate
intracellular calcium.
[0105] PYK2 is activated by, and phosphorylated on tyrosine
residues in response to a variety of extracellular signals that
lead to calcium influx or calcium release from internal stores
resulting in elevation of intracellular Ca.sup.2+ concentration.
Calcium influx leads to the activation of PYK2, tyrosine
phosphorylation of Shc, recruitment of Grb2/Sos and activation of
the MAP kinase signaling pathway that relays signals to the cell
nucleus. Overexpression of PYK2 also leads to activation of MAP
kinase. PYK2 has also been shown to be activated by peptide
hormones that bind to G-protein coupled receptors that mediate
their intracellular responses via Gi and Gq type of G-proteins
(Lev, et al. (1995) Nature 376:737-745). Thus, PYK2 may provide a
link between G-protein coupled receptors and calcium influx and the
MAP kinase signaling pathway; a pathway that relays signals from
the cell surface to regulate transcriptional events in the
nucleus.
[0106] Additionally, in certain cells PYK2 is activated by integrin
mediated cell adhesion (Astier, et al. (1997) J. Biol. Chem.
272:228-232).
[0107] These results reveal a role for PYK2 in activation of the
MAP kinase signaling pathway by ion channels, calcium influx and
G-protein coupled receptors and provide a mechanism for signal
transduction induced by these stimuli. Furthermore, tyrosine
phosphorylation of Shc in response to membrane depolarization and
carbachol treatment was dependent on the presence of extracellular
calcium, indicating that calcium-influx plays a role in regulation
of Shc phosphorylation by these stimuli.
[0108] Similarly, PYK2 may modulate the action of ion channels that
mediate their responses via and are sensitive to intracellular
calcium concentration. PYK2 may therefore provide an autoregulatory
role for the very same channel responsible for PYK2 activation.
[0109] The expression pattern of PYK2 and the external stimuli that
activate this kinase together with its role in the control of MAP
kinase signaling pathway, suggests a potential role for PYK2 in the
control of a broad array of processes.
[0110] Since PYK2 activity is regulated by intracellular calcium
level, both the temporal and spatial pattern of PYK2 activation may
represent a carbon copy or a replica of the spatial and temporal
profile of intracellular calcium concentration. Calcium
concentration inside cells is highly localized because of a variety
of calcium binding proteins that provide a strong buffer. Moreover,
in excitable cells the level of calcium can be regulated by voltage
dependent calcium channels that induce large and transient increase
in intracellular calcium concentration leading to calcium
oscillations and calcium waves. PYK2 may provide a mechanism for
rapid and highly localized control of ion channel function, as well
as, localized activation of the MAP kinase signaling pathway.
[0111] II. Knockout Mice Lacking PYK2
[0112] The current invention demonstrates for the first time in an
in vivo mouse model and a cellular model the link between PYK2 and
the inflammatory response. To demonstrate the role of PYK2 in vivo,
knockout mice lacking the pyk2 gene were created using molecular
genetic techniques. FIG. 1 shows the altered size of a fragment of
the pyk2 gene following the knockout procedure along with the lack
of PYK2 expression in mice which are PYK2-/-. Additionally,
Northern blot hybridization analysis of total RNA isolated from
PYK2-/- brain, thymus and spleen tissues did not reveal any PYK2
expression.
[0113] PYK2-/- homozygous mice were fertile and did not show gross
anatomical abnormalities compared to wild-type litter-mates,
including tissues that normally express high levels of PYK2, such
as the brain, thymus, and spleen. Further analysis of lymphoid
subpopulations by flow cytometry did not reveal any obvious
difference in the distribution of T-cells, B-cells,
macrophage-monocytes, or NK cells in any lymphoid tissue, including
spleen, lymph node, thymus, bone marrow, and peritoneal cavity.
Serum levels of IgG1, IgG2a, IgG3, IgM and IgA were also similar in
PYK2-/- and wild-type littermates.
[0114] In the influenza model, the inflammatory response of the
knockout mice was compared with the corresponding mice not
containing a pyk2 deletion following infection with the influenza
virus which leads to an overwhelming pulmonary inflammatory
response and eventually death. It was found that the pyk2 knockout
mice survived significantly longer than control mice expressing the
pyk2 gene (48 hours). Histologic examination of the lungs of the
knockout and control mice showed that in the lungs of the pyk2
knockout mice there was a significantly lower infiltration of
inflammatory cells including PMNs (polymorphonuclear leukocytes),
macrophages, and T-cells. In vitro experiments showed that
macrophages and splenocytes from pyk2-/- mice produced decreased
amounts of cytokines and chemokines, including IL-1.alpha.,
IL-1.beta., IL-6, IL-10, TNF-.alpha., GMCSF, IFN-.gamma., and
MIP1-.alpha.. Lower production of cytokines and chemokines such as
TNF-.alpha. and MIP1-.alpha. (among others) results in attenuated
recruitment and decreased activation of macrophages, T-cells and
other hematopoietic and nonhematopoietic cells involved in the
inflammatory response. In addition, macrophages from knockout mice
had decreased motility in vitro. The attenuated cytokine response
and migration defect in vitro correlate with the enhanced survival
and decreased pulmonary cellular infiltrate in pyk2-/- mice
following influenza challenge.
[0115] In the subcutaneous carageenen air pouch model, a
subcutaneous air pouch is surgically induced on the hind flank of
the mouse. The air pouch is then filled with the immunogen
carageenen, a substance that induces an inflammatory response.
After 10 hours, the number and type of cells infiltrating the pouch
are enumerated. Histologic examination revealed a decrease in the
number of cells infiltrating the carageenen filled pouch of pyk2-/-
mice compared to controls. In addition there was a significant
decrease in the number of macrophages present at the site of
inflammation.
[0116] To evaluate the role of PYK2 in mature macrophage cells that
express wild-type PYK2, kinase inactive PYK2 protein was introduced
into a normal macrophage cell line. Expression of the kinase
inactive PYK2 should function as a "dominant-negative" inhibitor
and abrogate the function of the endogenous wild-type PYK2 protein
in these macrophages. Expression of the kinase inactive PYK2
protein decreased secretion of TNF-.alpha. in response to LPS, a
physiologically relevant inducer of the inflammatory response.
[0117] Defect in Cell Signaling in PYK2-/- Macrophages
[0118] The experiments presented below show that several
intracellular signals that are stimulated by chemokines are
impaired in PYK2-/- macrophages. We found that chemokine-induced
production of Ins(1,4,5)P.sub.3 as well as other inositol
phospholipids is impaired. In addition, chemokine-induced Ca.sup.+2
release and MAP kinase activation are also impaired. In certain
cell types intracellular release of Ca.sup.+2 leads to strong
activation of PYK2 (Lev, et al. (1995) Nature 376:737-745). Taken
together, these experiments suggest that PYK2 can function both as
a Ca.sup.+2 sensor as well as an element crucial for the control of
Ca.sup.+2 release.
[0119] Impairment in Macrophage Contractility and Locomotion Caused
by PYK2 Deficiency
[0120] The migration of cells in culture in response to
extracellular stimuli can be divided into a five-step cycle: (i)
extension of the leading edge towards to stimulus; (ii) adhesion of
the leading edge to the substrate, (iii) movement of the cytoplasm
towards the leading edge, (iv) release from contact sites at the
lagging edge and (V) recycling of membrane receptor from the
lagging edge to the leading edge of the cell (Sheetz, et al. (1999)
Biochem. Soc. Symp. 65:233-243). Each step of the cycle requires
orderly changes in cytoskeletal structures and focal contacts, a
process that is regulated by a variety of intracellular enzymes
including protein tyrosine kinases and protein tyrosine
phosphatases (Manes, et al. (1999) Mol. Cell. Biol.
19:3125-3135).
[0121] The experiments described below demonstrate that in
macrophages PYK2 plays an important role in the control of cell
migration. In fact, it appears that PYK2-/- macrophages display
alterations in most steps of the migration cycle. In PYK2-/-
macrophages, extension of the leading edge is delayed, and multiple
extensions are generated (steps i and ii). Furthermore, PYK2-/-
macrophages inefficiently detach the lagging edge from the matrix
to allow net movement (steps iv and v). It appears that the
migration of the cytoplasm is also impaired in the PYK2 deficient
macrophages. Alterations in the migration cycle are particularly
evident after the initial extension of lamellipodia, and altered
cell polarization, which could be observed as multi directional
lamellipodia and redistribution of F-actin in multiple sites.
[0122] The strength of the traction force in the lamellipodia of
wild type or PYK2-/- macrophages was analyzed by applying the laser
tweezer method (Choquet, et al. (1997) Cell 88:39-48). The ability
of plasma membrane bound beads to move rearward on the cell surface
in opposition to the restraining force imposed by the force field
of the laser provides a measurement for the ability of cells to
migrate over a fixed point on the matrix. Measurements of bead
movements on wild type or PYK2-/- lamellipodia revealed the
diminished capacity of integrin/cytoskeletal complex in PYK2-/-
macrophages to supply the contractile force necessary for cell
migration. This can be caused by decreased tightness of the linkage
between integrin and the cytoskeleton or decreased contractile
force of the cytoskeleton or decreases in both processes. It
appears that aberrant adhesion and diminished traction force in
PYK2-/- macrophages will stabilize lamellipodia that would normally
be retracted resulting in the disruption of cellular polarization
and migration.
[0123] It is thought that cells subjected to a chemotactic gradient
are "sampling" the environment by extending lamellipodia in several
directions. Stable attachments to the extracellular matrix are
formed in the direction of the highest concentration of the
stimulus, whereas other lamellipodia eventually retract into the
cell. The maintenance of an appropriate cellular polarization in a
gradient thus requires regulation of contact stability and
contractile capacity of the lamellipodia. In PYK2-/-macrophages
both process are impaired.
[0124] Aberrant regulation of the stability of contacts in PYK2-/-
macrophages leads to increased adherence of extended lamellipodia
to the substratum and impairment in subsequent retraction even when
the lamellipodia extends for the purpose of "sampling" the
environment into a direction which does not contain a high
concentration of chemokine. Increase in the stability of contacts
may result in change in cell polarization.
[0125] PYK2 Deficiency Impairs rho Activation and Causes
Cytoskeletal Changes
[0126] Cell morphological change such as contraction of
lamellipodia or formation of contact require vigorous changes in
cytoskeleton. Numerous studies have indicated that members of the
rho family of GTPases play an important role in modulating the
cytoskeleton in response to extracellular stimuli. Rho GTPases were
implicated in the control cytoskeletal organization, actomyosin
contraction, vesicle transport, phospholipid production (Exton, et
al. (1997) Eur. J. Biochem. 243:10-20) as well as in the control of
integrin clustering (Hotchin and Hall (1995) J. Cell. Biol.
131:1857-1865; Renshaw, et al. (1996) J. Biol. Chem.
271:21691-21694). In this report we show that activation of rho in
response to integrin-mediated cell adhesion on fibronectin is
impaired in PYK2-/- macrophages. Moreover, formation of long
processes and decreased cell contraction displayed by PYK2-/-
macrophages also occur in wild type macrophages that were treated
with a specific rho inhibitor. Since activation of rho has been
shown to be critical for contraction of lamellipodia (Kimura, et
al. (1996) Science 273:245-248), the decreased contractility in
PYK2-/- macrophages could be due to decreased activity of rho.
[0127] Degradation of molecules and disassembly of F-actin at the
rear end of migrating macrophages are required for cell detachment
from the substratum. Intracellular calcium plays an important role
in this process by regulating the activity of calpain and gelsolin
(Witke, et al. (1995) Cell 81:41-51; Huttenlocher, et al. (1997) J.
Biol. Chem. 272:32719-32722). The attenuated chemokine-induced
calcium release caused by PYK2 deficiency may lead to the
impairment of detachment of the rear end of migrating cell.
[0128] Phagocytotic cells such as macrophages or neutrophils as
well as lymphocytes migrate quickly into inflammatory regions in
response to chemokines and other cues. Recruited inflammatory cells
produce a battery of cytokines and a variety of inflammatory
mediators such as oxygen radicals, nitric oxide and lipid
mediators, which induce inflammatory reaction and systematic
effects. The onset of experimental allergic encephalitis (EAE)
induced by injection of a peptide corresponding to myelin specific
protein requires appropriate migration of macrophages into primary
inflammatory region that was triggered by infiltrated TH1 cells.
Experiments presented in this report demonstrate that the onset of
EAE was delayed by approximately two days in PYK2 deficient mice.
However, although EAE was delayed in PYK2 deficient mice the
outcome of the disease was more severe in these mice. It is also
demonstrated that the infiltration of macrophages into carageenen
induced inflammatory region is strongly inhibited in PYK2-/- mice.
Taken together, both the in vitro and in vivo experiments presented
in this study reveal an important regulatory role for PYK2 in
normal function of macrophages.
[0129] The above data confirm the role for PYK2 in cytokine release
and support the importance of PYK2 function in inflammation. These
experiments indicate that treatments that inhibit the functioning
of PYK2 will be useful to decrease excessive inflammatory
responses, whereas treatments to enhance the functioning of PYK2
will be useful to augment inadequate immune responses.
[0130] III. Identification of Compounds that Modulate PYK2
[0131] The present invention relates, inter alia, to methods of
detecting compounds that modulate the interaction of PYK2 with its
natural binding partners. The modulation can encompass either a
decrease, or an increase in the interaction between PYK2 and its
natural binding partners. The compounds thus identified would be
useful in the prevention or treatment of immune-related disorders
involving the signal transduction system and in particular
interactions among PYK2 and its natural binding partners. The
compounds may be present within a complex mixture, for example,
serum, body fluid, or cell extracts. Once the compounds are
identified, they can be isolated using techniques well known in the
art.
[0132] The present invention also encompasses a method of treating
or preventing immune-related diseases in a mammal with one or more
compounds, that modulate PYK2:natural binding partner interactions,
comprising administering the compounds to a mammal in an amount
sufficient to modulate PYK2:natural binding partner
interactions.
[0133] In an effort to discover novel treatments for diseases,
biomedical researchers and chemists have designed, synthesized, and
tested molecules that inhibit the function of protein kinases. Some
small organic molecules form a class of compounds that modulate the
function of protein kinases. Examples of molecules that have been
reported to inhibit the function of protein kinases include, but
are not limited to, bis monocyclic, bicyclic or heterocyclic aryl
compounds (PCT WO 92/20642, published Nov. 26, 1992 by Maguire, et
al.), vinylene-azaindole derivatives (PCT WO 94/14808, published
Jul. 7, 1994 by Ballinari, et al.),
1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992),
styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted
pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline
derivatives (EP Application No. 0 566 266 A1), selenoindoles and
selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny, et
al.), tricyclic polyhydroxylic compounds (PCT WO 92/21660,
published Dec. 10, 1992 by Dow), and benzylphosphonic acid
compounds (PCT WO 91/15495, published Oct. 17, 1991 by Dow, et
al.), all of which are hereby incorporated by reference herein
including any figures, drawings, or tables.
[0134] Compounds that can traverse cell membranes and are resistant
to acid hydrolysis are potentially advantageous as therapeutics as
they can become highly bioavailable after being administered orally
to patients. However, many of these protein kinase inhibitors only
weakly inhibit the function of protein kinases. In addition, many
inhibit a variety of protein kinases and will therefore cause
multiple side-effects as therapeutics for diseases.
[0135] Some indolinone compounds, however, form classes of acid
resistant and membrane permeable organic molecules. WO 96/22976
(published Aug. 1, 1996 by Ballinari, et al.; hereby incorporated
by reference herein including any figures, drawings, or tables)
describes hydrosoluble indolinone compounds that harbor tetralin,
naphthalene, quinoline, and indole substituents fused to the
oxindole ring. These bicyclic substituents are in turn substituted
with polar moieties including hydroxylated alkyl, phosphate, and
ether moieties. PCT Publication WO 98/07695, published March 26,
1998 by Tang, et al. (Lyon & Lyon Docket No. 221/187 PCT), PCT
Publication WO 96/40116, published Dec. 19, 1996 by Tang, et al.
(Lyon & Lyon Docket No. 223/298), and International Patent
Publication WO 96/22976, published Aug. 1, 1996 by Ballinari, et
al., all of which are incorporated herein by reference in their
entirety, including any drawings, figures, or tables describe
indolinone chemical libraries of indolinone compounds harboring
other bicyclic moieties as well as monocyclic moieties fused to the
oxindole ring. They also teach methods of indolinone synthesis,
methods of testing the biological activity of indolinone compounds
in cells, and inhibition patterns of indolinone derivatives.
[0136] Other examples of substances capable of modulating kinase
activity include, but are not limited to, tyrphostins,
quinazolines, quinoxolines, and quinolines. The quinazolines,
tyrphostins, quinolines, and quinoxolines referred to above include
well known compounds such as those described in the literature. For
example, representative publications describing quinazolines
include Barker, et al., EPO Publication No.0 520 722 A1; Jones, et
al., U.S. Pat. No. 4,447,608; Kabbe, et al., U.S. Pat. No.
4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5,316,553; Kreighbaum
and Corner, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO
Publication No. 0 562 734 A1; Barker, et al. (1991) Proc. of Am.
Assoc. for Cancer Research 32:327; Bertino, J. R. (1979) Cancer
Research 3:293-304; Bertino, J. R. (1979) Cancer Research 9(2 part
1)293-304; Curtin, et al. (1986) Br. J. Cancer 53:361-368;
Fernandes, et al.(1983) Cancer Research 43:1117-1123; Ferris, et
al. J. Org. Chem. 44(2), 173-178; Fry, et al. (1994) Science
265:1093-1095; Jackman, et al. (1981) Cancer Research 51:5579-5586;
Jones, et al. J. Med. Chem. 29(6), 1114-1118; Lee and Skibo (1987)
Biochemistry 26(23), 7355-7362; Lemus, et al.(1989) J. Org. Chem.
54:3511-3518; Ley and Seng, (1975) Synthesis 1975:415-522; Maxwell,
et al. (1991) Magnetic Resonance in Medicine 17:189-196; Mini, et
al. (1985) Cancer Research 45:325-330; Phillips and Castle (1980) J
Heterocyclic Chem. 17(19), 1489-1596; Reece, et al. (1977) Cancer
Research 47(11), 2996-2999; Sculier, et al. (1986) Cancer Immunol.
and Immunother. 23, A65; Sikora, et al. (1984) Cancer Letters
23:289-295; Sikora, et al. (1988) Analytical Biochem. 172:344-355;
all of which are incorporated herein by reference in their
entirety, including any drawings.
[0137] Quinoxaline is described in Kaul and Vougioukas, U.S. Pat.
No. 5,316,553, incorporated herein by reference in its entirety,
including any drawings.
[0138] Quinolines are described in Dolle, et al. (1994) J. Med.
Chem. 37:2627-2629; MaGuire (1994) J. Med. Chem. 37:2129-2131;
Burke, et al (1993) J. Med. Chem. 36:425-432; and Burke, et al.
(1992) BioOrganic Med. Chem. Letters 2:1771-1774, all of which are
incorporated by reference in their entirety, including any
drawings.
[0139] Tyrphostins are described in Allen, et al. (1993) Clin. Exp.
Immunol. 91:141-156; Anafi, et al. (1993) Blood 82, 12, 3524-3529;
Baker, et al. (1992) J. Cell Sci. 102:543-555; Bilder, et al.
(1991) Amer. Physiol. Soc. 6363-6143, C721-C730; Brunton, et al.
(1992) Proceedings of Amer. Assoc. Cancer Rsch. 33:558; Bryckaert,
et al (1992) Experimental Cell Research 199:255-261; Dong, et al.
(1993) J Leukocyte Biology 53:53-60; Dong, et al. (1993) J.
Immunol. 151(5), 2717-2724; Gazit, et al. (1989) J. Med. Chem.
32:2344-2352; Gazit, et al. (1993) J. Med. Chem. 36:3556-3564;
Kaur, et al. (1994) Anti-Cancer Drugs 5:13-222; King, et al. (1991)
Biochem. J. 275:413-418; Kuo, et al. (1993) Cancer Letters
74:197-202; Levitzki, A. (1992) The FASEB J 6:3275-3282; Lyall, et
al. (1989) J. Biol. Chem. 264:14503-14509; Peterson, et al. (1993)
The Prostate 22:335-345; Pillemer, et al. (1992) Int. J. Cancer
50:80-85; Posner, et al. (1993) Molecular Pharmacology 45:673-683;
Rendu, et al. (1992) Biol. Pharmacology 44(5), 881-888; Sauro and
Thomas, (1993) Life Sciences 53:371-376; Sauro and Thomas (1993) J.
Pharm. and Experimental Therapeutics 267(3), 119-1125; Wolbring, et
al. (1994) J. Biol. Chem. 269(36), 22470-22472; and Yoneda, et al.
(1991) Cancer Research 51:4430-4435; all of which are incorporated
herein by reference in their entirety, including any drawings.
[0140] Other compounds that could be used as modulators include
oxindolinones such as those described in U.S. patent application
Ser. No. 08/702,232 filed Aug. 23, 1996 and indolinones such as
those described in U.S. Pat. No. 5,792,783 issued Aug. 11, 1998,
entitled "3-Heteroaryl-2-Indolinone Compounds for the Treatment of
Disease" (Lyon & Lyon Docket No. 223/301, both of which are
hereby incorporated herein by reference in their entirety,
including any drawings, figures or tables.
[0141] IV. Pharmaceutical Formulations and Routes of
Administration
[0142] The compounds described herein can be administered to a
human patient per se, or in pharmaceutical compositions where they
are mixed with other active ingredients, as in combination therapy,
or suitable carriers or excipient(s). Techniques for formulation
and administration of the compounds of the instant application may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition. Preferred routes include oral,
transdermal, and parenteral delivery.
[0143] a) Routes Of Administration.
[0144] Suitable routes of administration may, for example, include
depot, oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intravenous, intramedullary injections, as well as intrathecal,
direct intraventricular, intraperitoneal, intranasal, or
intraocular injections.
[0145] Alternately, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a solid tumor, often in a depot or sustained
release formulation.
[0146] Furthermore, one may administer the drug in a targeted drug
delivery system, for example, in a liposome coated with
tumor-specific antibody. The liposomes will be targeted to and
taken up selectively by the tumor.
[0147] b) Composition/Formulation
[0148] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0149] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries that facilitate processing of the active
compounds into preparations that can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen.
[0150] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0151] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained by adding
a solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0152] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0153] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0154] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0155] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0156] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0157] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0158] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0159] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0160] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0161] A pharmaceutical carrier for the hydrophobic compounds of
the invention is a cosolvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. The cosolvent system may be the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD:D5W) consists of VPD diluted 1:1 with a 5% dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
may be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80; the fraction size of polyethylene glycol may be
varied; other biocompatible polymers may replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or
polysaccharides may substitute for dextrose.
[0162] Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds may be employed. Liposomes and emulsions
are well known examples of delivery vehicles or carriers for
hydrophobic drugs. Certain organic solvents such as
dimethylsulfoxide also may be employed, although usually at the
cost of greater toxicity. Additionally, the compounds may be
delivered using a sustained-release system, such as semipermeable
matrices of solid hydrophobic polymers containing the therapeutic
agent. Various types of sustained-release materials have been
established and are well known by those skilled in the art.
Sustained-release capsules may, depending on their chemical nature,
release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for protein
stabilization may be employed.
[0163] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0164] Many of the PTK modulating compounds of the invention may be
provided as salts with pharmaceutically compatible counterions.
Pharmaceutically compatible salts may be formed with many acids,
including but not limited to hydrochloric, sulfuric, acetic,
lactic, tartaric, malic, succinic, etc. Salts tend to be more
soluble in aqueous or other protonic solvents that are the
corresponding free base forms.
[0165] c) Effective Dosage.
[0166] Pharmaceutical compositions suitable for use in the present
invention include compositions where the active ingredients are
contained in an amount effective to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount of compound effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being
treated. Determination of a therapeutically effective amount is
well within the capability of those skilled in the art, especially
in light of the detailed disclosure provided herein.
[0167] For any compound used in the methods of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating concentration range that includes
the IC.sub.50 as determined in cell culture (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of the PTK activity). Such information can be used to
more accurately determine useful doses in humans.
[0168] Toxicity and therapeutic efficacy of the compounds described
herein can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., for determining the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
between LD.sub.50 and ED.sub.50. Compounds which exhibit high
therapeutic indices are preferred. The data obtained from these
cell culture assays and animal studies can be used in formulating a
range of dosage for use in humans. The dosage of such compounds
lies preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon the dosage form employed and
the route of administration utilized. The exact formulation, route
of administration and dosage can be chosen by the individual
physician in view of the patient's condition. (See e.g., Fingl, et
al. (1975) The Pharmacological Basis of Therapeutics Ch. 1 p.
1).
[0169] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the kinase modulating effects, or minimal effective
concentration (MEC). The MEC will vary for each compound but can be
estimated from in vitro data; e.g., the concentration necessary to
achieve 50-90% inhibition of the kinase using the assays described
herein. Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration. However,
HPLC assays or bioassays can be used to determine plasma
concentrations.
[0170] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen that maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%.
[0171] In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
plasma concentration.
[0172] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0173] d) Packaging
[0174] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accompanied with
a notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use, or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compound for human or veterinary
administration. Such notice, for example, may be the labeling
approved by the U.S. Food and Drug Administration or other
government agency for prescription drugs, or the approved product
insert. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition. Suitable conditions indicated
on the label may include, for example, treatment of immune-related
diseases including inflammation, and the like.
[0175] V. Target Diseases to be Treated or Diagnosed by Methods of
the Invention
[0176] Target diseases to be treated or diagnosed by the methods of
the invention are generally those that have an aberrant
inflammatory response. A pathologic inflammatory response may be a
continuation of an acute inflammatory response or a prolonged
low-grade inflammatory response, and usually causes permanent
tissue damage. Macrophage and T-cell recruitment and functions,
such as cytokine production, directly contribute to inflammatory
pathogenesis. There are many types of diseases and disorders
associated with inflammatory responses, both acute and chronic, all
of which are intended to be included under specific embodiments of
the present invention.
[0177] Specific diseases of interest include, but are not limited
to the following:
[0178] Inflammatory bowel diseases include ulcerative colitis and
Crohn's disease. The majority of cases of ulcerative colitis are
mild, being limited to rectosigmoid involvement. However clinical
manifestations include bloody diarrhea, mucus, fever, abdominal
pain, tenesmus, and weight loss. Complications can include toxic
megacolon, colonic perforation, and cancer. The level of risk of
cancer is related to the extent and duration of colitis, and may be
preceded by dysplasia. Intractable disease (to drug treatment),
toxic megacolon, cancer and severe dysplasia may require a
colectomy.
[0179] Crohn's disease is generally more serious, occurring in any
part of the GI tract with transmural inflammation, bowel wall
thickening, linear ulcerations, granulomas, fissures and fistulas.
Clinical manifestations include fever, abdominal pain, diarrhea,
fatigue, weight loss, acute ilietis, anorectal fissures, fistulas,
and abscesses. Complications may include intestinal obstruction,
intestinal fistulas, and intestinal malignancy. Treatments include
parenteral nutrition as well as pharmaceuticals including
corticosteroids, immunosuppressive agents, and metronidazole.
Surgery may be required for fixed obstruction, abscesses,
persistent symptomatic fistulas, and intractability.
[0180] Connective tissue diseases involve inflammation of the
connective tissue as well as altered patterns of
immunoregulation.
[0181] Rheumatoid arthritis is a serious health care problem.
Progressive arthritic conditions in humans cause severe pain, loss
of joint mobility and disfigurement, and an overall reduction in
the quality of life. In rheumatoid arthritis, the synovium
hyperproliferates (aided by new blood vessels) and invades the
cartilage which is destroyed. Conventional treatment for rheumatoid
arthritis includes non-steroidal anti-inflammatory drugs (NSAIDs).
A need exists for an effective treatment for rheumatoid arthritis
that will disrupt disease progression in addition to suppression or
amelioration of symptoms.
[0182] Clinical manifestations of systemic lupus erythematosus
include fatigue, fever, malaise, weight loss, skin rashes (malar
"butterfly" rash), photosensitivity, arthritis, myositis, oral
ulcers, vasculitis, alopecia, anemia, neutropenia,
thrombocytopenia, lymphadenopathy, splenomegaly, organic brain
syndromes, seizures, psychosis, pleuritis, pericarditis,
myocarditis, pneumonitis, nephritis, venous or arterial thrombosis,
mesenteric vasculitis, and sicca syndrome. There is currently no
cure treatment is directed at controlling generalized inflammation.
Drugs include salicylates and NSAIDs. New, effective drugs are
desperately needed.
[0183] Clinical manifestations of progressive systemic sclerosis
include Raynaud's phenomenon, scleroderma, telangiectasis,
calcinosis, esophageal hypomotility, arthralgias and/or arthritis,
intestinal hypofunction, pulmonary fibrosis, hypertension, and
renal failure. Renal failure is the leading cause of death. There
is no definitive therapy.
[0184] Clinical manifestations of the mixed connective tissue
disease include Raynaud's phenomenon, polyarthritis, sclerodactyly,
esophageal dysfunction, pulmonary fibrosis, and inflammatory
myopathy. treatment is directed to controlling the inflammatory
process in general.
[0185] Clinical manifestations of Sjogren's syndrome include
xerostomia and keratoconjunctivitis sicca, nephritis, vasculitis,
polyneuropathy, interstitial pneumonitis, pseudolymphoma,
autoimmune thyroid disease, and congenital cardiac conduction
defects in women with SSA antibodies. Treatment includes
symptomatic relief of dryness as well as treatments associated with
autoimmune phenomena.
[0186] Although the primary target diseases to be treated or
diagnosed by the methods of the invention are those that have an
aberrant inflammatory response, other diseases that involve
alterations in macrophage or macrophage-like cell function (i.e.
osteoclast) are also intended to be included. Examples of diseases
that are not considered to be classic inflammatory
response-mediated diseases include, but are not limited to,
osteoarthritis, osteoperosis, osteopetrosis and
atherosclerosis.
[0187] VI. Other Embodiments
[0188] Methods for evaluation of disorders, methods for monitoring
changes in cells, methods of identifying compounds, methods of
isolating compounds which interact with a PTK, compositions of
compounds that interact with a PTK, and derivatives of complexes
are disclosed in detail with respect PYK-2 in PCT publication WO
96/18738 and U.S. Pat. No. 5,837,815. These publications are hereby
incorporated herein by reference in their entirety, including any
drawings, tables, or figures. Those skilled in the art will
appreciate that such descriptions are applicable to the present
invention and can be easily adapted to it. Those skilled in the art
will also appreciate that any modifications made to a complex can
be manifested in a modification to any of the molecules in that
complex. Thus, the invention includes any modifications to nucleic
acid molecules, polypeptides, antibodies, or compounds in a
complex.
[0189] In addition, the WO 96/18738 provides disclosure describing
the recombinant DNA techniques pertaining to the present invention,
nucleic acid vectors, the nucleic acid elements of these vectors,
the types of cells that can harbor these vectors, methods of
delivering these vectors to cells or tissues, methods of producing
and purifying antibodies, methods of constructing hybridomas that
produce these antibodies, methods of detecting signaling molecule
complexes, methods of detecting interactions with natural binding
partners, antibodies to complexes, disruption of PTK protein
complexes, purification and production of complexes, transgenic
animals containing nucleic acid vectors encoding a PTK, antisense
and ribozyme approaches, and gene therapy techniques. Those skilled
in the art will readily appreciate that such descriptions are
applicable to the present invention and can be easily adapted to
it.
[0190] Other methods associated with the invention are described in
the examples disclosed herein.
EXAMPLES
[0191] The examples below are non-limiting and are merely
representative of various aspects and features of the procedures
used to demonstrate the role of PYK-2 in signal transduction in
inflammation.
[0192] Materials and Methods
[0193] Chemicals
[0194] Bradykinin, pertusis toxin, cholera toxin, forskolin,
phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187,
carbachol, muscarine, atrophine, mecamylamine, and
1,1-dimethyl-4-phenyl piperazinium iodide (DMPP) were purchased
from Sigma.
[0195] Cells and Cell Culture
[0196] PC12-rat pheochromocytoma cells were cultured in Dulbecco's
modified Eagle's medium containing 10% horse serum, 5% fetal bovine
serum, 100 .mu.g/mL streptomycin and 100 units of penicillin/mL.
NIH3T3, 293, GP+E-86 and PA317 cells were grown in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum, 100
.mu.g/mL streptomycin and 100 units of penicillin/mL.
[0197] Antibodies
[0198] Antibodies against PYK2 were raised in rabbits immunized
(HTI) either with a GST fusion protein containing residues 362-647
of PYK2 or with a synthetic peptide corresponding the 15 amino
acids at the N-terminus of PYK2. Antisera were checked by
immunoprecipitation and immunoblot analysis. The specificity was
confirmed either by reactivity to the related protein Fak or by
competition with the antigenic or control peptides. The antibodies
were found to be specific to PYK-2; they do not cross react with
FAK.
[0199] Transfections and Infections
[0200] For stable expression in PC12 cells, PYK2 was subcloned into
the retroviral vector pLXSN (Miller and Rosman, Biotechniques
7:980, (1989)). The construct was used to transfect GP+E-86 cells
using lipofectamine reagent (GIBCO BRL). 48 hours after
transfection, virus containing supernatants were collected. Pure
retrovirus-containing cell-free supernatants were added to PC12
cells in the presence of polybrene (8 .mu.g/mL, Aldrich) for 4
hours (MCB 12 491, 1992). After 24 hours, infected PC12 cells were
split into growing medium containing 350 .mu.L/mg G418. G418
resistant colonies were isolated two to three weeks later and the
level of expression was determined by Western blot analysis.
[0201] Stable cell lines of NIH3T3 that overexpress PYK2 were
established by cotransfection of PYK2 subcloned into pLSV together
with pSV2neo utilizing lipofectamine reagent (GIBCO BRL). Following
transfection, the cells were grown in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum and 1 mg/mL G418.
Transient transfections into 293 cells were performed by using a
calcium phosphate technique, standard in the art.
[0202] Constructs
[0203] GST-PYK2 A DNA fragment of .lambda.900 bp corresponding to
residues 362-647 of PYK2 was amplified by PCR utilizing the
following oligonucleotide primers: 5'-
1 5'-CGGGATCCTCATCATCCATCCTAGGAAAGA-3' (sense) and
5'-CGGGAATTCGTCGTAGTCCCAGCAGCGGGT-3'. (antisense)
[0204] The PCR product was digested with BamHI and EcORI and
subcloned into pGEX3X (Pharmacia). Expression of the GST-PYK2
fusion protein was induced by 1 mM IPTG essentially as described by
Smith, et al. (1988) Gene 67:31. The fusion protein was isolated by
electroelution from SDS-PAGE.
[0205] PYK2
[0206] The full length cDNA sequence of PYK2 was subcloned into the
following mammalian expression vectors: pLSV; downstream from the
SV40 early promoter, pLXSN-retroviral vector; downstream from the
Mo-MuLV long terminal repeat; pRK5; downstream from the CMV
promoter.
[0207] PYK2-HA
[0208] The influenza virus hemagglutinin peptide (YPYDVPDYAS) was
fused to the C-terminus of PYK2 utilizing the following
oligonucleotide primers in the PCR reaction:
5'-CACAATGTCTTCAAACGCCAC-3' and 5'-GGCTCTAGATCACGATGCGT-
AGTCAGGGACATCGTATGGGRACTCTGCAGG TGGGTGGGCCAG-3'. The amplified
fragment was digested with RsrII and Xba1 and was substituted with
the corresponding fragment of PYK2. The nucleotide sequence of the
final construct was confirmed by DNA sequencing.
[0209] Kinase Negative Mutant/Dominant-Negative
[0210] In order to construct a kinase negative mutant, Lys (457)
was substituted to Ala by site directed mutagenesis utilizing the
`Transformer Site-Directed Mutagenesis Kit` (Clontech). The
oligonucleotide sequence was designed to create a new restriction
site of Nrul. The nucleotide sequence of the mutant was confirmed
by DNA sequencing. The oligonucleotide sequence that was used for
mutagenesis was: 5'-CAATGTAGCTGTCGCGACCTGCAAGAA-AGAC-3' (Nrul
site-, Lys-AAC substituted to Ala-GCG).
[0211] Immunoprecipitation and Immunoblot Analysis
[0212] Cells were lysed in lysis buffer containing 50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulferic acid (HEPES pH 7.5),
150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl.sub.2, 1 mM
ethyleneglycol-bis (.beta.-aminoethyl ether)-N,N,N'N'-tetraacetic
acid (EGTA), 10 .mu.g leupeptin per mL, 10 .mu.g aprotinin per mL,
1 mM phenylmethylsulfonyl fluoride (PMSF), 200 .mu.M sodium
orthovanadate and 100 mM sodium fluoride. Immunoprecipitations were
performed using protein A-sepharose (Pharmacia) coupled to specific
antibodies. Immunoprecipitates were washed either with HNTG'
solution (20 mM HEPES buffer at pH 7.5, 150 mM NaCl, 10% glycerol,
0.1% Triton X-100, 100 mM sodium fluoride, 200 .mu.M sodium
orthovanadate) or successively with H' solution (50 mM Tris-HCl pH
8, 500 mM NaCl, 0.1% SDS, 0.2% Triton X-100, 100 mM NaF, 200 .mu.M
sodium orthovanadate) and L' solution (10 mM Tris-HCl pH 8, 0.1%
Triton X-100, 100 mM NaF, 200 .mu.M sodium orthovanadate). The
washed immunoprecipitates were incubated for 5 min with gel sample
buffer at 100.degree. C. and analyzed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). In some experiments,
the gel-embedded proteins were electrophoretically transferred onto
nitrocellulose. The blot was then blocked with TBS (10 mM Tris pH
7.4, 150 mM NaCl) that contained 5% low fat milk and 1% ovalbumin.
Antisera or purified mAbs were then added in the same solution and
incubation was carried out for 1 h at 22.degree. C. For detection,
the filters were washed three times (5 min each wash) with
TBS/0.05% Tween-20 and reacted for 45 min at room temperature with
horseradish peroxidase-conjugated protein A. The enzyme was removed
by washing as described above, and the filters were reacted for 1
min with a chemiluminescence reagent (ECL, Amersham) and exposed to
an autoradiography film for 1-15 min.
[0213] In Vitro Kinase Assay
[0214] This was carried out on immunoprecipitates in 50 .mu.L HNTG
(20 mM Hepes pH 7.5, 150 mM NaCl, 20% glycerol, 0.1% Triton X-100)
containing 10 mM MnCl.sub.2 and 5 .mu.Ci or [mN-.sup.32P]ATP for 20
min at 22.degree. C. The samples were washed with H', M' and, L'
washing solutions, boiled for 5 min in sample buffer and separated
by SDS-PAGE.
[0215] Construction of Targeting Vector
[0216] A mouse genomic DNA clone corresponding to the N-terminal
domain and the kinase domain, was isolated from 129 strain PI phage
library (Genom Systems). 4.5 kb BamH1-Acc1 genomic DNA fragment
encoding the 5' terminal of the kinase domain was inserted a XhoI
site, flanking to a neo expression cassette in the pPNT vector
(Soriano, et al. (1991) Cell 64:693-702) and 2 kb AccI-SacI genomic
DNA fragment was inserted to BamHI site flanking to HSV thymidine
kinase expression cassette in the pPNT vector.
[0217] Electroporation of ES Cells and Generation of PYK2-/-
Mice
[0218] R1 ES cells were grown on mitomycin C-treated primary
embryonic fibroblast that are extracted from day 15 embryos at
37.degree. C. in Dulbecco's modified Eagles Medium (DMEM)
supplemented with 15% heat inactivated fetal bovine serum
(Hyclone), 0.1 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and
10.sup.3 U/ml leukemia inhibitory factor. (LIF) (GIBCO). Cells
(7.times.10.sup.6) were electroporated in 800 .mu.l of
phosphate-buffered saline(PBS) with 32 .mu.g of NotI linealized
targeting vector DNA at 240V, 500 mF using Gene Pulser (Bio-Rad)
and plated on gelatin coated plastic dishes. After 48 hours, the
cells were transferred to growth medium supplemented with G418 (150
mg/ml) (GIBCO) and Gancyclovir (2 mg/ml) (Syntex). G418- and
Gancyclovir resistant colonies were picked up 10-12 days after
electroporation. Homologous recombination was screened by Southern
blot hybridization. Four independently mutated ES cells clones were
used in embryo aggregation experiments for generation of mice.
Chimeric mice were crossed to 129Sv/Ev females and germ line
transmission in heterozyous mice was identified in two independent
ES cell clone derived F1 mice by Southern blot analysis.
Heterozygote mice were intercrossed to produce homozygote mice.
[0219] Cell Culture
[0220] 2.0 ml of 3% Brewer thioglycollate (GIBCO) medium was
injected into peritoneal cavity 4 days prior to cell harvest. The
inflammatory cells, comprised of mixture of macrophages and
neutrophils, were harvested from euthanized animals as lavage by
PBS. Macrophages were isolated as an adherent cells after plating
harvested cells onto tissue cultured dishes.
[0221] Immunofluorescence Analysis
[0222] Peritoneal exudate cells were incubated on coverslips for 60
minutes. The non-adherent cells were washed off and the adherent
cells were treated for 1 hr with MIP1.alpha.: or SDF1.alpha.
(R&D systems) using by Zigmond glass slide chamber (Neuroprobe,
Cobin John, MD). The cells before and after treatment were fixed
with 4% paraformaldehyde, permabilized with 0.1% Triton X and
stained with TRITC-conjugated phalloidin (Sigma) to visualize
F-actin distribution. The slides were analyzed using the Leica TCS
confocal microscope and images collected using Leica TCS User
software. Images were further processed using NIH Image 1.61 and
Adobe Photoshop.
[0223] Migration Assays
[0224] Thioglycollate-induced peritoneal macrophages were plated on
tissue culture dishes and after addition of 100 ng/ml MIP1.alpha.,
image of migrating macrophages on the heated plate at 37.degree. C.
were captured every 10 minutes using CCD camera equipped with Nikon
Diaphot Inverted Microscope and processed using NIH image
software.
[0225] Experimental Inflammation in Air Pouch Model
[0226] The air pouch experiments were conducted essentially as
described previously (Wisniewski, et al. (1996) J. Immunol.
156:1609-1615). Briefly, air pouches were induced on the back of
wild type and PYK2-/- mice by three subcutaneous injections of air
every second day. To induce an acute inflammation, 1 ml of a 2%
(w/v) carrageenen solution in the saline was injected directly into
the air pouch. After 10 hour, the mice were sacrificed. 2 ml saline
was injected into each air pouch, and the exudate was aspirated.
Aliquots were diluted with saline and the cells were counted.
Further, air pouch was dissected, fixed and stained by hematoxyline
eosine. Three arbitrarily selected fields each containing
approximately 300 cells were distinguished morphologically and
counted per each mouse.
[0227] Microinjection
[0228] Cells were injected in Hepes-buffered culture medium at room
temperature using a Zeiss (Oberkochen, Germany) Axiopvert 35
microscope, an Eppendorf (Hamburg, Germany) micromanipulator 5170,
a microinjector 5242 and Sutter capillaries (Novato, Calif.). The
holding pressure was 450 hPa, the injection pressure was 500 hPa
and the duration of the injection was 200 ms. The concentration of
microinjected protein was 1-2 mg/ml in PBS. After microinjection,
cells were incubated in DMEM at 37.degree. C.
[0229] [.sup.3H]Inositol Phospholipids and Ca.sup.2+ Analyses
[0230] Cells grown in 6-well plates were labeled for 24 h in
medium-199 containing myo-[.sup.3H]inositol(20 .mu.Ci/ml). The
cells were washed once with PBS and pre-incubated for 10 min. in 2
ml of PBS containing 10 mM LiCl (PH 7.4) at 37.degree. C. prior to
the addition of chemokines. The cells were extracted with
methanol/1M HCl/chloroform 1:1:1 and analyzed by high-performance
liquid chromatography (HPLC) as described (Falasca, et al. (1998)
EMBO J. 17:414-422). [Ca.sup.2+ ]i measurements were performed in
single macrophages as described (Falasca, et al. 1995).
[0231] Laser Trapping Experiments.
[0232] For laser trap experiments, beads were prepared as described
(Choquet, et al. (1997) Cell 88:39-48; Felsenfeld, et al. (1999)
Nature Cell Biology 1:200-206). Briefly, carboxylated latex beads
(0.91 .mu.m; Polyscience) were derivitized with carbodimide and
coated with ovalbumin (Sigma). The ovalbumin was further
derivitized with covalently linked biotin (Sulpho NHS-LC biotin;
Pierce) to permit the binding of avidin (Neutravidin; Molecular
Probes) and finally, a recombinant fragment of fibronectin (FN type
3 domains 7-10; FNIII7-10; including the binding site for the
integrin .alpha.5.beta.1).
[0233] The optical gradient laser trap was formed using a
titanium-sapphire laser (800 nM; Coherent) which was excited with
an argon ion laser (5W; Coherent). The 800 nM laser beam was
focused through the bottom port of a Zeiss Axiovert100 TV to form a
trap that was parfocal with the image plane of the microscope. Bead
position was visualized using a newvicon camera (MTI Dage)
processed through a Hamamatsu Argus 10 image processor for
background subtraction and contrast stretching. Video data were
collected at 30 frames per second on an SVHS VCR for subsequent
analysis. Analysis of bead position was carried out using single
particle tracking routines (Gelles; 1987) implemented in the ISEE
image analysis software package (Innovision) running on an O.sub.2
workstation (Silicon Graphics Inc.).
[0234] Beads were placed at .about.0.5 mm from the edge of the
lamellipodia using a 30 mW (20 pN) trap. The laser trap remained
activated until the bead had moved >500 nm from the trap center.
Beads that moved <500 nm in 30 s.
[0235] Analysis of rho Activation
[0236] Measurement of amount of rho-GTP was performed as previously
described (Ren, et al. (1999) EMBO J. 18:578-585). Briefly,
macrophages plated on the fibronectin were washed with ice-cold PBS
and lysed in RIPA buffer (50 mM Tris, pH7.2, 1% Triton X-10, 0.5%
sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM MgCl.sub.2, 10
mM/ml each leupeptin and aprotinin, and 1 mM PMSF). Cell lysates
were centrifuged at 13,000 g at 4.degree. C. for 10 min and
incubated with GST-RBD (20 .mu.g) beads at 4.degree. C. for 45 min.
The beads were washed four times with washing buffer (Tris buffer
containing 1% Triton X-100, 150 mM NaCl, 10 nM MgCl.sub.2, 10
.mu.g/ml each leupeptin and aprotinin, and 0.1 mM PMSF). Bound Rho
proteins were detected by immunoblotting using a polyclonal
antibody against rho-A (Santa Cruz Biotechnology).
[0237] EAE Induction.
[0238] EAE was induced by subcutaneous injection at the base of the
tail of 100 .mu.g MOG 35-55 peptide emulsified in complete Freund's
Adjuvant and supplemented with 2 mg/ml heat-killed M. tuberculosis
H37RA (Difco). Pertussis toxin (200 ng/dose; Sigma) was injected
intravenously at the time of MOG injection and 40 hours later.
[0239] Clinical signs of EAE were monitored according to Baron, et
al. (1993): 1: limp tail; 2: hind leg weakness; 3: total hind leg
paralysis; 4: front leg weakness; 5: moribund. Lymphocyte cultures:
At various time-points, single-cell suspensions from draining
(periaortic) and mesenteric lymphnodes were prepared separately and
seeded at 10.sup.6 cells/ml in 96 well plates and analyzed for
proliferation to stimulation with anti CD3/28 (1 .mu.g/ml) and to 5
.mu.M MOG 35-55 peptide. After 48 hours in culture, plates were
pulsed with 1 .mu.Ci[.sup.3H] thymidine (NEN) for 6 hours,
harvested, and read in a betaplate (Wallac). Culture supernatants
were collected at 24, 48, 72 hours and IL-2, IL-4, IL-10 and
IFN-.gamma. accumulation was assessed by ELISA using antibody pairs
recommended by Phanningen. Data was collected from individual mice
(two mice per time-point).
[0240] CNS-Infiltrating leukocytes were prepared after total
bleeding of the mice, cerebellum and spinal cord dissection,
incubation with collagenase D (400 U/ml, Boehringer Mannheim) for
45 minutes at 37.degree. C. and centrifugation in 38% Percoll
(Pharmacia). The pellet was washed twice and stained with
anti-Mac1, anti-panCD45, anti I.A..sup.b, anti-granulocyte (Gr-1),
anti-CD3, and anti-B220, antibodies (Pharmingen).
Example I
PYK2 Knockout Experiments
[0241] The Pyk2 gene was knocked out of mice by targeted
disruption. FIG. 1a shows partial restriction maps of murine Pyk2
locus, the regions used as a targeting vector, and the expected
mutant allele. Solid boxes indicate exons. The probe was used for
Southern blot hybridization analysis of the genomic DNA from the ES
cells and tail biopsy analysis. The solid line under the Pyk2 locus
marks the Pyk2 kinase domain. Arrows mark the expected Apal
fragments of the wild-type and mutant alleles. Southern blot
hybridization analysis of the mouse tail DNA digested with Apal
indicates that the Pyk2 knockout was successful (FIG. 1b).
Immunoblot analysis (with anti-Pyk2 antibodies) of lysates from
Thymus (T), Brain (B), and Spleen (S) from wild-type, Pyk2+/-, and
Pyk2-/- mice also demonstrated the absence of Pyk2 polypeptide in
knockout mice (FIG. 1c).
EXAMPLE II
Analysis of Cytokine Production
[0242] RT-PCR (reverse transcriptase-polymerase chain reaction) was
used to analyze cytokine productions from wild-type +/+ and Pyk2-/-
thioglycollate-elicited peritoneal macrophages stimulated by LPS.
Thioglycollate-elicited peritoneal macrophages were incubated with
or without LPS (10 .mu.g/mL) for 3 h, 6 h, 12 h, 24 h, and 48 h.
Total RNAs were isolated from cells at each time point. After
normalization of the amount of mRNA in the total RNAs by RT-PCR of
actin r A, total RNAs were subjected to RT-PCR with specific probes
for various cytokines.
[0243] This experiment reveals a long delay of 24 to 48 hours in
the onset of production of cytokines in macrophages derived from
PYK2-/- mice (FIG. 2).
[0244] RT-PCR analysis was also used to determine the cytokine
productions from wild-type +/+ and Pyk2-/- splenocytes stimulated
by anti-murine CD3 antibodies. Splenocytes were incubated with or
without anti-CD3 (1 .mu.g/mL) for 12 h, 24 h, 48 h and 72 h. Total
RNAs were isolated from cells at each time point. After
normalization of the amount of mRNA in the total RNAs by RT-PCR of
the actin mRNA, total RNAs were subjected to RT-PCR with specific
probes for various cytokines.
[0245] This experiment reveals a delay of approximately 24 hours in
the production of cytokines in splenocytes derived from PYK2-/-
mice (FIG. 3).
[0246] Thus, these experiments showed that macrophages and
splenocytes from pyk2-/- mice produced decreased amounts of
cytokines and chemokines, including IL-1.alpha., IL-1.beta., IL-6,
IL-10, TNF-.alpha., GMCSF, IFN-.gamma., and MIP1-.alpha.. Lower
production of cytokines and chemokines such as TNF-.alpha. and
MIP1-.alpha. (among others) results in attenuated recruitment and
decreased activation of macrophages, T-cells and other
hematopoietic and nonhematopoietic cells involved in the
inflammatory response. In addition, macrophages from knockout mice
had decreased motility in vitro.
Example III
Carageenen Model of Inflammation
[0247] In the subcutaneous carageenen air pouch model, a
subcutaneous air pouch is surgically induced on the hind flank of
the mouse. The air pouch is then filled with the immunogen
carageenen, a substance that induces an inflammatory response.
[0248] Ten hours after carrageenan injection into wild type or
PYK2-/- mice, tissue sections of the injected lesion were examined
microscopically for the presence of infiltrating macrophages and
neutrophils (FIG. 4a). The average number of infiltrating cells in
wild type mice was 4.8.times.10.sup.6 per injected area, while in
the PYK2-/- mice there was an average of only 2.8.times.10.sup.6
cells per injected area (FIG. 4b). Morphological examination of the
infiltrating cells indicated that macrophages comprised
approximately 70% of the infiltrate in wild-type mice but only 20%
of the infiltrate in the PYK2-/- mice, the remaining cells were
primarily neutrophils (FIG. 4b). These data show that the failure
of PYK2-/- macrophages to migrate effectively in vitro is
correlated with a striking deficit in inflammatory infiltration in
vivo.
Example IV
Influenza Model of Inflammation
[0249] In the influenza model, infection with the influenza virus
leads to an overwhelming pulmonary inflammatory response and
eventually death.
[0250] Influenza virus-induced inflammation in mice lungs was
studied in lung sections from influenza virus-infected wild-type
+/+ and Pyk2-/- mice 4 days after infection with the virus. Lungs
were formalin-fixed and paraffin-embedded. Sections were stained
with Hematoxylin and Eosin (FIG. 5a). FIG. 5b shows a time course
of the survival of mice at different doses of influenza virus.
[0251] Histologic examination of the lungs of the knockout and
control mice showed that in the lungs of the pyk2 knockout mice
there was a significantly lower infiltration of inflammatory cells
including PMNs (polymorphonuclear leukocytes), macrophages, and
especially T-cells. Thus, on average the Pyk2 knockout mice
exhibited decreased pulmonary cellular infiltrate and lived about
48 hours longer than wild-type mice after challenge with the
influenza virus.
[0252] The attenuated cytokine response and migration defect in
vitro correlate with the enhanced survival and decreased pulmonary
cellular infiltrate in pyk2-/- mice following influenza
challenge.
Example V
Analysis of Cytokine Production in PYK2 Dominant-Negative
Mutants
[0253] To evaluate the role of PYK2 in mature macrophage cells that
express wild-type PYK2, kinase inactive PYK2 protein was introduced
into a normal macrophage cell line. Expression of the kinase
inactive PYK2 functions as a "dominant-negative" inhibitor and
abrogates the function of the endogenous wild-type PYK2 protein in
these macrophages.
[0254] By "dominant negative mutant protein" is meant a mutant
protein that interferes with the normal PYK2 signal transduction
pathway. The dominant negative mutant protein contains the domain
of interest (e.g., a PYK2 polypeptide or a NBP), but has a mutation
preventing proper signaling, for example by preventing binding of a
second domain from the same protein. One example of a dominant
negative protein is described in Millauer, et al Nature Feb. 10,
1994. Expression of the kinase inactive PYK2 protein decreased
secretion of TNF-.alpha. in response to LPS, a physiologically
relevant inducer of the inflammatory response.
[0255] Tyrosine phosphorylation of Pyk2 in response to LPS
stimulation was measured in P388D1 cells. Cells were starved
overnight in 0.5% serum and stimulated with LPS for variable time
intervals (FIG. 6a). After stimulation, cells were washed, lysed,
and immuno-precipitated with anti-Pyk2 antibody.
Immuno-precipitates were then analyzed by western blotting.
Tyrosine phosphorylated bands were detected with the
anti-phosphotyrosine antibody 4G10. Maximal phosphorylation was
observed after 30 minutes. Cells that had been pre-treated with
either herbimycin A or genestein for 4 hours were stimulated with
LPS for 30 minutes and analyzed as above (FIG. 6b). Tyrosine
phosphorylation was diminished by pre-treatment with non-specific
inhibitors of tyrosine phosphorylation.
[0256] The murine macrophage cell line (P388D1) was transfected
with doxycycline inducible plasmids in order to study the effect of
a dominant-negative Pyk2 protein on macrophage function. In the
first transfection, a regulator plasmid was introduced and stable,
drug-resistant clones were established. These were screened by
transient transfection with a doxycycline-responsive, luciferase
reporter plasmid. A range of luciferase responses was obtained from
different clones. Two clones were selected (based upon their
doxycycline response) and transfected with response plasmids
containing either HA-tagged, wild-type Pyk2 or the kinase dead Pyk2
mutant (also HA tagged). After drug selection, stable clones were
screened for expression of the appropriate protein by western
blotting (FIG. 7).
[0257] TNF secretion in response to stimulation with either LPS (1
.mu.g/mL) or PMA (1 .mu.g/mL) by the doxycycline inducible clones
is shown in FIG. 8. Induction in the presence of doxycycline was
carried out for 48 hours followed by stimulation with LPS or PMA
overnight (18 hours). Cells expressing the kinase-dead Pyk2 mutant
had a blunted response to either LPS or PMA compared with cells
expressing the wild-type Pyk2. Secreted TNF-.alpha. was measured by
ELISA. Both LPS and PMA caused secretion of TNF-.alpha.c. Secretion
of TNF-.alpha. was inhibited by herbimycin A and genestein, which
are non-specific inhibitors of tyrosine phosphorylation.
Example VI
PYK2-/- Macrophages Exhibit Abnormal Morphology, Enhanced Cell
Polarization and Impaired Migration
[0258] Macrophages normally express high levels of PYK2 and bearly
detectable levels of FAK (Lipsky, et al. (1998) J. Biol. Chem.
273:11709-11713). It is possible that these cells might be more
susceptible to the loss of PYK2. We have first compared the
morphology of peritoneal macrophages from wild type or PYK2-/- mice
30 minutes after plating these cells on coverslips or on
tissue-culture dishes. The micrograph depicted in FIG. 9 (panel a)
shows that after adhesion wild-type macrophages adopt a typical
round shape. Treatment of wild type macrophages with the chemokine
SDF1.alpha. resulted in rapid induction of lamellipodia and
enhancement of cell spreading (FIG. 9a, panel c and e). In
contrast, most PYK2-/- macrophages displayed a flattened morphology
with extensive membrane spreading even without chemokine treatment
(FIG. 9a, panel b). Addition of SDF1.alpha. further increased the
formation of pseudopodia as well as the appearance of long
processes (FIG. 9a, panel d and f).
[0259] In view of the enhanced spreading with multiple pseudopodia
and long processes as well as the enhanced substrate attachment
displayed by PYK2-/-macrophages, we considered that macrophage
migration in response to chemotactic stimulation could be impaired.
We treated wild type and PYK2-/- macrophages with SDF1.alpha. and
observed cell morphology and movement at ten minute intervals
following chemokine stimulation (FIG. 9b). This experiment shows
that within ten minutes wild-type macrophages become polarized,
developing lamellipodia, on one side of the cell. At later time
points (>20 minutes), the cell body moves in the direction
established by the leading edge, detaching from the substrate at
the trailing edge. In contrast, formation of new lamellipodia by
PYK2-/- macrophages in response to SDF1.alpha., was delayed as
compared to wild-type cells (FIG. 9b). Furthermore, the cell body
showed reduced ability to follow the leading edge and failed to
detach from the substratum. Over time, the PYK2-/- macrophages
extended lamellipodia in several directions with similar failure to
detach from the substrate. Eventually, most PYK2-/- macrophages
developed several pseudopodia-like processes with minimal net
migration. Overall, PYK2-/- macrophages are able to form a leading
edge in response to a chemotactic stimulus, albeit with slower
kinetics. However, these cells are unable to move the cell body
after the leading edge efficiently, and fail to detach the lagging
edge from the substratum.
Example VII
Impairment in Contractile Force in PYK2-/- Macrophages Revealed by
Optical Tweezer Analysis
[0260] Microscopic observation of migrating macrophages revealed
that PYK2-/- cells could extend lamellipodia but the cell body
failed to flow into the newly formed leading edge. We suggested
that the contractile activity of the cytoskelton in the
lamellipodia was impaired in PYK2-/- macrophages. The contractile
force was determined by measuring the rearward movement toward the
nucleus of beads coated with recombinant fragment of fibronectin
(FN type III domains 7-10) on lamellipodia in opposition to an
immobilizing force generated by optical tweezers (Choquet, et al.
(1997) Cell 88:39-48; Felsenfeld, et al. (1999) Nature Cell Biology
1:200-206). The velocity of rearward movement of the beads in
opposition to this force represents a function of (i) the strength
of association between the cytoskeleton and the integrins bound to
the fibronectin on the bead, and (ii) the strength of traction
force generated by the cytoskeleton itself (Choquet, et al. (1997)
Cell 88:39-48; Sheetz, et al. (1998) Trends Cell Biol. 8:51-54).
Immobilizing force by optical tweezers was applied to the beads on
the lamellipodia of the cells and the movement of the bound beads
was monitored. Representative plots of the distance of bead
displacement versus time in wild type or PYK2-/- macrophages is
presented in FIG. 10a.
[0261] The beads on the lamellipodia from wild type macrophages
exhibited rearward movement and escaped from the force field of the
laser trap. After chemokine stimulation, the velocity of bead
movement on the lamellipodia of wild type macrophages was increased
and the beads escaped more quickly. With the immobilizing force
exerted by the optical tweezers, more than 50% of the beads that
were attached to the lamellipodia of either stimulated or
unstimulated wild type macrophages were able to escape from the
force field of the optical trap. In contrast, beads bound to the
lamellipodia of PYK2-/- macrophages did not exhibit rearward
movement in presence or absence of chemokine stimulation (FIG.
10a). No beads that were attached to the lamellipodia of either
stimulated or unstimulated PYK2-/-macrophages were able to escape
from the optical trap (FIG. 10b). Overall, rearward movement, e.g.
the contractile force generated by the cytoskeleton, is impaired in
PYK2-/- macrophages in comparison to the contractile force
generated in wild type macrophages.
Example VIII
Altered Cytoskeletal Organization in PYK2-/- Macrophages
[0262] We next examined the status of the cytoskeleton in PYK2-/-
macrophages. Visualization of cells stained with fluorescent
phalloidin revealed an increase of F-actin in membrane ruffles in
PYK2-/- as compared to wild-type macrophages (FIG. 11a). Analysis
of F-actin distribution by confocal microscopy revealed an increase
in reorganized F-actin underneath the ruffles in PYK2-/-
macrophages (FIG. 11b). When wild-type macrophages were placed in a
chemotactic gradient, phalloidin staining revealed increase in the
relative amount of F-actin at the edge of the cell in the region
that is exposed to the greatest concentration of chemotactic
signal. The distribution of F-actin in stimulated PYK2-/-
macrophages was different, in these cells F-actin was distributed
at multiple sites along the cell periphery (FIG. 11c, left). In
migrating wild-type macrophages F-actin is continuously
redistributed towards the leading edge of the cell. This
redistribution of F-actin does not occur in PYK2-/- macrophages,
probably resulting in the failure of the cells to become properly
oriented in a chemotactic gradient.
[0263] We have also examined the distribution of microtubules in
wild type and PYK2-/- macrophages. It was proposed that
microtubules play an important role in driving actin polymerization
and leading-edge lamellipodia protrusion through specific
rho-GTPases during cell migration (Waterman-Storer, et al. (1999)
Nature Cell Biol. 1:45-50). The organization of microtubules in
wild type or PYK2-/- macrophages was visualized by staining
permaebilized cells with anti-tubulin antibodies. The experiment
depicted in FIG. 1d shows that the microtubules in PYK2-/-
macrophages are more assembled than the microtubules in wild type
macrophages. Upon chemokine stimulation, the microtubules of wild
type macrophages radiate from the microtubules organizing center
(MTOC) while PYK2-/- macrophages display long microtubules that are
assembled at the cell periphery into longitudinal directions with
decreased intensity towards the MTOC (FIG. 1d). The increased
assembly of microtubules in the periphery of PYK2-/- macrophages
could be linked to the enhancement in F-actin organization,
extensive lamelliopodia formation in this region leading to altered
cell polarization.
Example IX
Impairment in rho Activation in PYK2-/- Macrophages
[0264] The rho family of small G-protein has been implicated in the
control of cytoskelal organization leading to changes in cell
morphology and cell migration (Ridley, et al. (1999) Biochem. Soc.
Symp. 65:111-123). It was demonstrated that integrin-induced cell
adhesion leads to the activation of rho (Ren, et al. (1999) EMBO J.
18:578-585). The activated GTP bound form of rho binds to effector
proteins that are involved in the control of cytoskeletal
organization and contraction of lamellipodia (Allen, et al. (1997)
J Cell Sci. 110:707-720; Maekawa, et al. (1999) Science
285:895-898). We have analyzed activation of rho in macrophages by
applying a "pull-down" assay using a GST fusion protein containing
the binding site from rhotekin for the GTP bound form of rho (Ren,
et al. (1999) EMBO J. 18:578-585). The experiment presented in FIG.
12a shows fibronectininduced activation of rho as a function of
time. In contrast, a similar experiment performed with PYK2-/-
macrophages revealed reduced activation of rho in the mutant
macrophages in response to integrin-induced cell adhesion (FIG.
12a).
[0265] We have further examined the role played by rho in the
control of macrophage morphology by microinjecting into these cells
a specific inhibitor of rho designated C3 (Chardin, et al. (1989)
EMBO J. 8:1087-1092) together with fluorescently labeled dextran as
a specific marker (FIG. 12b. This experiment demonstrates that wild
type macrophages microinjected with C3, showed a rapid and
extensive cell spreading with strong ruffling and formation of long
processes similar to the morphological changes seen in PYK2-/-
macrophages. However, microinjection of C3 into PYK2-/-macrophages
did not cause further changes to those seen in untreated
PYK2--macrophages (FIG. 12b). Taken together, these experiments
show that rho is activated upon adhesion of macrophages and that
reduced activation of rho may be responsible for the enhanced
spreading, ruffling and formation of long processes in
PYK2-/-macrophages.
Example IX
Reduced Intracellular Calcium Release and Ins(1,4,5)P.sub.3
Production in PYK2Macrophages
[0266] Calcium plays an important role in the control of a variety
of intracellular events as well as in the control of cell shape,
and cell movement (Lawson and Maxfield, 1995). We therefore
measured cytoplasmic calcium release in single cells in response to
MIP1.alpha. stimulation by using quantitative fluorescence
microscopy of Fura-2 loaded cells. Treatment of wild type
macrophages attached to cover slips showed maximum increase in
cytoplasmic [Ca.sup.+2j concentration at approximately 300 nM of
MIP1.alpha.. By contrast, PYK2-/- adherent macrophages did not show
an obvious increase in [Ca.sup.+2] concentration (FIG. 12c). This
experiment shows that PYK2 plays an important role in the control
of MIP1.alpha.-induced Ca.sup.+2 release in adherent macrophages.
Defect in calcium release may contribute towards the failure of the
cells to detach at the rear end leading to impairment in cell
migration since proteins that regulate the degradation of focal
contact components and disassembly of F-actin require calcium for
their action (Witke, et al. (1995) Cell 81:41-51; Kulkarni, et al.
(1999) J. Biol. Chem. 274:21265-21275).
[0267] A significant proportion of intracellular Ca.sup.+2 released
in response to extracellular signals is mediated by inositol
(1,4,5) triphosphate [Ins(1,4,5)P.sub.3] production (Furuichi, et
al (1989) Nature 342:32-38). We therefore analyzed the production
of Ins(1,4,5)P.sub.3 in these cells. In this experiment wild type
or PYK2-/-macrophages were labeled with [.sup.3H] myo-inositol and
then stimulated with MIP1.alpha.. At various times after MI1.alpha.
stimulation the production of Ins(1,4,5)P.sub.3 was determined by
HPLC analysis (Falasca, et al. (1998) EMBO J. 17:414-422). Wild
type macrophages showed a biphasic production of Ins(1,4,5)P.sub.3
with peaks at 20 sec. and 2 min. after MIP1.alpha. (stimulation.
The experiment presented in FIG. 12d shows that Ins(1,4,5)P.sub.3
production was severely reduced in PYK2-/- macrophages; the peak at
20 sec post stimulation was reduced by approximately 50% as
compared to Ins(1,4,5,)P.sub.3 production in wild type macrophages
and no Ins(1,4,5)P.sub.3 was generated after 2 min. of MIP1.alpha.
stimulation (FIG. 12d). We have also detected impairment in the
production of glycerophosphoinositol,
phosphatidylinositol-3-phosphate and
phosphatidylinositol-4-phosphate in PYK2-/- macrophages. These
findings suggest that PYM deficiency may lead to a more general
impairment in phosphatidyl inositol metabolism. We have previously
shown that PYK2 forms a complex with a family of
phosphatidylinositol transfer proteins designated Nirs both in
vitro and in living cells (Lev, et al. (1999) Mol. Cell. Biol.
19:2278-2288). The interaction between PYK2 and the
phosphatidylinositol transfer proteins and its absence in PYK2-/-
macrophages may beresponsible for the impairment in
phosphatidylinositol metabolism described.
Example X
Delayed Onset of Experimental Autoimmune Encephalomyelitis (EAE)
and a More Severe Disease in PYK2-/- Mice.
[0268] Experimental Autoimmune Encephalomyelitis (EAE) is an
inflammatory demyelinating disease of the central nervous system
(CNS) which exhibits a predominantly mononuclear infiltrate, and is
widely used as an animal model of multiple sclerosis (Zamvil and
Steinman (1990) Annu. Rev. Immunol. 8:579-621): Since EAE is
largely dependent on the activity of macrophages, we have compared
the susceptibility of wild type or PYK2-/- mice to EAE by
immunizing the mice with Myelin Oligodendrocyte Glycoprotein (MOG)
(Johns, et al. (1995) J. Immunol. 154:5536-5541; Mendel, et al.
(1995) Eur. J. Immunol. 25:1951-1959) and monitoring EAE
progression. EAE was induced in PYK2-/- and wild type mice (129Sv)
and the clinical course of the disease was monitored daily. As
shown in FIG. 13a, both wild type and PYK2-/- mice are susceptible
to MOG-induced EAE. However, the onset of EAE was delayed by
approximately two days in PYK2 deficient mice as compared to the
onset of the disease in wild-type mice. In several experiments
PYK2-/- mice showed lower incidence of EAE; while not more than 75%
of the PYK2-/- mice came down with the disease, virtually all
wild-type mice became sick. However, the outcome of the disease was
more severe in PYK2-/- mice, as compared to wild-type mice. For
example, in the experiment depicted in FIG. 13, 50% of PYK2-/- mice
(4 of 8) died whereas only one of 8 wild-type mice succumbed, the
remaining mice showed partial or complete clinical recovery. EAE
recovery may not be due entirely to a reduction in the
proinflammatory stimulus. There is evidenece that cytokines and
regulatory cells are actively involved in the clinical improvement
(Welch, et al. (1980) J. Immunol. 125:186-189; Karpus, et al.
(1991) J. Immunol. 146:1163-1168; Kennedy, et al. (1992) J.
Immunol. 149:2496-2505); this process appears to be impaired in
PYK2-/- mice.
[0269] Next, draining lymphnode and central nervous system samples
were prepared from wild type or PYK2 deficient mice at different
times post induction. We have shown that T cells from wild type and
mutant mice proliferate equally well in response to anti-CD3
stimulation at all time-points. However, the experiment presented
in FIG. 13b shows that the proliferative response towards MOG was
delayed in T cells from draining lymphnodes from PYK2-/- mice as
compared to T cells from wild-type mice.
[0270] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The molecular complexes and the methods, procedures,
treatments, molecules, specific compounds described herein are
presently representative of preferred embodiments are exemplary and
are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention are
defined by the scope of the claims.
[0271] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0272] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains.
[0273] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed.
[0274] In particular, although some formulations described herein
have been identified by the excipients added to the formulations,
the invention is meant to also cover the final formulation formed
by the combination of these excipients. Specifically, the invention
includes formulations in which one to all of the added excipients
undergo a reaction during formulation and are no longer present in
the final formulation, or are present in modified forms.
[0275] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
For example, if X is described as selected from the group
consisting of bromine, chlorine, and iodine, claims for X being
bromine and claims for X being bromine and chlorine are fully
described.
[0276] Other embodiments are within the following claims.
Sequence CWU 1
1
6 1 30 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 cgggatcctc atcatccatc ctaggaaaga 30 2 30 DNA Artificial
Sequence Description of Artificial Sequence Primer 2 cgggaattcg
tcgtagtccc agcagcgggt 30 3 10 PRT Influenza virus 3 Tyr Pro Tyr Asp
Val Pro Asp Tyr Ala Ser 1 5 10 4 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 4 cacaatgtct tcaaacgcca c
21 5 63 DNA Artificial Sequence Description of Artificial Sequence
Primer 5 ggctctagat cacgatgcgt agtcagggac atcgtatggg ractctgcag
gtgggtgggc 60 cag 63 6 31 DNA Artificial Sequence Description of
Artificial Sequence Oligonucleotide 6 caatgtagct gtcgcgacct
gcaagaaaga c 31
* * * * *