U.S. patent application number 14/592623 was filed with the patent office on 2015-07-23 for inhibition of gliadin peptides.
The applicant listed for this patent is Alba Therapeutics Corporation. Invention is credited to Sefik Alkan, Rosa A. Carrasco, Malarvizhi Durai, Kelly Marie Kitchens, Neil Poloso, Amir Tamiz.
Application Number | 20150203537 14/592623 |
Document ID | / |
Family ID | 41265370 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150203537 |
Kind Code |
A1 |
Alkan; Sefik ; et
al. |
July 23, 2015 |
INHIBITION OF GLIADIN PEPTIDES
Abstract
Novel compounds and methods for the inhibition of biological
barrier permeability and for the inhibition of peptide
translocation across biological barriers are identified. Assays for
determining modulators of biological barrier permeability and for
peptide translocation across biological barriers are provided.
Methods for treating diseases relating to aberrant biological
barrier permeability and peptide translocation across biological
barriers are provided. Such diseases include celiac disease,
necrotizing enterocolitis, diabetes, cancer, inflammatory bowel
diseases, asthma, COPD, excessive or undesirable immune response,
gluten sensitivity, gluten allergy, food allergy, rheumatoid
arthritis, multiple sclerosis, immune-mediated or type 1 diabetes
mellitus, systemic lupus erythematosus, psoriasis, scleroderma and
autoimmune thyroid diseases.
Inventors: |
Alkan; Sefik; (Baltimore,
MD) ; Tamiz; Amir; (Silver Spring, MD) ;
Kitchens; Kelly Marie; (Laurel, MD) ; Durai;
Malarvizhi; (Ellicott City, MD) ; Poloso; Neil;
(Rockville, MD) ; Carrasco; Rosa A.; (Baltimore,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alba Therapeutics Corporation |
Baltimore |
MD |
US |
|
|
Family ID: |
41265370 |
Appl. No.: |
14/592623 |
Filed: |
January 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12991658 |
Jun 15, 2011 |
8957032 |
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PCT/US2009/042973 |
May 6, 2009 |
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14592623 |
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61050915 |
May 6, 2008 |
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Current U.S.
Class: |
514/21.7 ;
435/375; 514/21.8; 514/21.9; 530/328; 530/329; 530/330;
530/331 |
Current CPC
Class: |
A61P 17/00 20180101;
C07K 5/101 20130101; C07K 7/06 20130101; C07K 5/0804 20130101; C07K
5/0808 20130101; A61K 38/07 20130101; A61K 38/06 20130101; C07K
5/1008 20130101; A61K 45/06 20130101; A61P 3/00 20180101; A61P 1/04
20180101; C07K 5/0815 20130101; C07K 5/0819 20130101; C07K 5/081
20130101; C07K 5/0817 20130101; C07K 5/0821 20130101; C07K 5/0812
20130101; C07K 5/0806 20130101; A61K 38/00 20130101; A61K 38/08
20130101; A61P 3/10 20180101 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 5/093 20060101 C07K005/093; A61K 45/06 20060101
A61K045/06; C07K 5/097 20060101 C07K005/097; C07K 5/09 20060101
C07K005/09; C07K 5/087 20060101 C07K005/087; C07K 5/103 20060101
C07K005/103; C07K 5/083 20060101 C07K005/083 |
Claims
1. A peptide permeability inhibitor consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1-162,
wherein said peptide permeability inhibitor inhibits translocation
of a gliadin-derived peptide across a biological barrier.
2. The peptide of claim 1, wherein the peptide does not consist of
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 15, 24 and 25.
3. The peptide of claim 1, wherein the peptide consists of an amino
acid sequence selected from the group consisting of SEQ ID NOs:
1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85,
87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147,
150, and 160-162.
4. A method of inhibiting gliadin-derived peptide translocation
across a biological barrier comprising contacting said barrier with
a peptide permeability inhibitor consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOs:
1-162.
5. The method of claim 4, wherein the peptide does not consist of
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 15, 24 and 25.
6. The method of claim 4, wherein the peptide consists of an amino
acid sequence selected from the group consisting of SEQ ID NOs:
1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85,
87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147,
150, and 160-162.
7. A composition for inhibiting gliadin-derived peptide
translocation across a biological barrier, wherein said composition
comprises a peptide permeability inhibitor consisting of an amino
acid sequence selected from the group consisting of SEQ ID NOs:
1-162.
8-9. (canceled)
10. A method for inhibiting gliadin-derived peptide translocation
across a biological barrier comprising administering to a subject
in need thereof the composition of claim 7 in an amount sufficient
to inhibit said gliadin-derived peptide translocation.
11-30. (canceled)
31. A method of treating a patient with celiac disease, comprising
administering to the patient a composition comprising a peptide
that inhibits translocation of a gliadin-derived peptide across a
biological barrier.
32. The method of claim 31, wherein said composition comprises
peptide consisting of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-162.
33. The method of claim 32, wherein the peptide does not consist of
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 15, 24 and 25.
34. The method of claim 32, wherein the peptide consists of an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77,
79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127,
128, 147, 150, and 160-162.
35. The method of claim 31, wherein said composition further
comprises an additional therapeutic agent selected from the group
consisting of aminosalicylates, corticosteroids, immunomodulators,
antibiotics, cytokines, chemokines and biologic therapeutics.
36-45. (canceled)
Description
PRIORITY
[0001] This application claims priority to U.S. Provisional
Application No. 61/050,915 filed May 6, 2008, which is hereby
incorporated by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of gastrointestinal
inflammation. In particular, it relates to compounds and methods
for the treatment of gastrointestinal inflammation.
BACKGROUND OF THE INVENTION
[0003] Environmental stimuli, such as microorganisms and gluten,
can lead to increased permeability of biological barriers and
initiate significant pathological events in the intestine, brain,
heart, and other organs. The pathological consequences of such
stimuli include the development of inflammatory diseases. Such
external stimuli are presumed to exert physiological effects on
biological barriers, possibly through interaction with specific
cell surface receptors. However, the mechanisms used remain
unclear, and specific cell surface receptors have yet to be
confirmed.
[0004] Many inflammatory diseases, including those that arc
understood to involve increased permeability of biological
barriers, are thought to be autoimmune. Such diseases include
celiac disease, rheumatoid arthritis, multiple sclerosis,
immune-mediated or type 1 diabetes mellitus, inflammatory bowel
diseases, systemic lupus erythematosus, psoriasis, scleroderma,
necrotizing enterocolitis and autoimmune thyroid diseases.
Prolonged inflammation is often associated with these diseases,
although the inflammation is thought to be a sequela rather than a
primary pathological insult.
Biological Barrier Dysfunction
[0005] Biological barrier function relies upon the structural and
functional integrity of tight junctions (TJ), which are one of the
hallmarks of absorptive and secretory epithelia. They act as a
boundary that physically separates apical and basolateral
compartments of epithelial cells, and they selectively regulate the
passage of materials through the epithelia by controlling access to
the space between the epithelial cells (the paracellular pathway).
To meet the many diverse physiological and pathological challenges
to which epithelia are subjected, the tight junctions must be
capable of rapid, physiologic, reversible, transient, energy
dependent, and coordinated responses that require the presence of a
complex regulatory system. Examples of epithelia containing tight
junctions include, but are not limited to, the intestines
(particularly the small intestine), and the blood brain
barrier.
[0006] In the absence of stimuli, tight junctions are closed
restricting access to the paracellular pathway. In the presence of
stimuli, the tight junctions are reversibly opened. Certain
bacteria have been shown to have toxins that stimulate the opening
of tight junctions. Vibrio cholerae infected with the filamentous
bacteriophage CTX.PHI., produces a toxin (zonula occludens toxin,
ZOT) that has been shown to cause opening of tight junctions. It
has been shown that 6 His-.DELTA.G, an N-terminal deletion of ZOT
in which the first 264 amino acids, have been deleted and replaced
with a six histidine purification tag, retains the ability to open
tight junctions.
[0007] Physiological changes in paracellular permeability, which
are due to TJ regulation, can be measured as variations in
transepithelial conductance. Such variations can usually be
attributed to changes in paracellular permeability since the
resistances of epithelial plasma membranes are relatively high. TJ
represent the major barrier in the paracellular pathway, and the
electrical resistance of epithelial tissues seems to depend on
their integrity.
[0008] Environmental stimuli, including for example, microorganisms
and gluten, can increase permeability of biological barriers as
measured by a decrease in trans-epithelial electrical resistance
(TEER) (ex vivo) or the Lactulose/mannitol test (in vivo). Such
increases in barrier permeability arc due primarily to TJ
rearrangements, and they are believed to underlie many diseases
including a large number of inflammatory conditions.
[0009] TJ dysfunction occurs in a variety of clinical conditions,
including food allergies, infections of the gastrointestinal tract,
autoimmune diseases, celiac disease and inflammatory bowel
diseases. Healthy, mature gut mucosa with its intact tight junction
serves as the main barrier to the passage of macromolecules. During
the healthy state, small quantities of immunologically active
antigens cross the gut host barrier. These antigens are absorbed
across the mucosa through at least two pathways. Up to 90% of the
absorbed proteins cross the intestinal barrier via the
transcellular pathway, followed by lysosomal degradation that
converts proteins into smaller, non-immunogenic peptides. These
residual peptides are transported as intact proteins through the
paracellular pathway, which mediates a subtle, but sophisticated,
regulation of intercellular tight junction that leads to antigen
tolerance.
[0010] In normal bowels, the immune reaction is regulated to
maintain homeostasis of the gut. When TJ integrity is compromised,
in premature infants or on exposure to environmental stimuli,
radiation, chemotherapy, or toxins, a deleterious immune response
to environmental antigens may develop. This response can result in
autoimmune diseases and food allergies that lead to
inflammation.
[0011] Inflammatory bowel disease (IBD) is a phrase used to
describe an inappropriate immune response that occurs in the bowels
of affected individuals. Two major types of IBD have been
described: Crohn's disease and ulcerative colitis (UC). Both forms
of IBD show abnormal profiles of T cell mediated immunity. In the
gut of Crohn's disease a strong Th1 reaction is induced; the Th2
response is upregulated in the colon of UC.
[0012] The barrier function of the intestines is impaired in IBD.
For example, Crohn's disease is associated with increased
permeability of the intestinal barrier even in quiescent patients.
A TNF-.alpha.-induced increase in intestinal epithelial tight
junction (TJ) permeability has been proposed to be an important
proinflammatory mechanism contributing to intestinal inflammation
in Crohn's disease and other inflammatory conditions. Increased
intestinal permeability during episodes of active disease
correlates with destruction or rearrangement of TJ protein
complexes.
[0013] Examples of inflammatory diseases and disorders that may be
treated using the instant invention include, for example, celiac
disease, necrotizing enterocolitis, rheumatoid arthritis, multiple
sclerosis, immune-mediated or type 1 diabetes mellitus,
inflammatory bowel diseases (Crohn's disease and ulcerative
colitis), systemic lupus erythematosus, psoriasis, scleroderma, and
autoimmune thyroid diseases. Prolonged inflammation is often
associated with these diseases, although the inflammation is
thought to be a sequela rather than a primary pathological
insult.
[0014] Other diseases and disorders associated with biological
barrier dysfunction and which may be treated using the instant
inventions include, for example, celiac disease, asthma, acute lung
injury, acute respiratory distress syndrome, chronic obstructive
pulmonary disease, inflammation (e.g., psoriasis and other
inflammatory dermatoses), asthma, allergy, cell proliferative
disorders (e.g., hyperproliferative skin disorders including skin
cancer), metastasis of cancer cells, ion transport disorders such
as magnesium transport defects in the kidney, and exposure to
Clostridium perfringens enterotoxin (CPE). autoimmune
encephalomyelitis, optic neuritis, progressive multifocal
leukoencephalopathy (PML), primary biliary cirrhosis, IgA
nephropathy, Wegener's granulomatosis, multiple sclerosis,
scleroderma, systemic sclerosis, Hashimoto's thyroiditis
(underactive thyroid), Graves' disease (overactive thyroid),
autoimmune hepatitis, autoimmune inner ear disease, bullous
pemphigoid, Devic's syndrome, Goodpasture's syndrome, Lambert-Eaton
myasthenic syndrome (LEMS), autoimmune lymphproliferative syndrome
(ALPS), paraneoplastic syndromes, polyglandular autoimmune
syndromes (PGA), alopecia areata, gastrointestinal inflammation
that gives rise to increased intestinal permeability, intestinal
conditions that cause protein losing enteropathy, C. difficile
infection, enterocolitis, shigellosis, viral gastroenteritis,
parasite infestation, bacterial overgrowth, Whipple's disease,
diseases with mucosal erosion or ulcerations, gastritis, gastric
cancer, collagenous colitis, and mucosal diseases without
ulceration, Menetrier's disease, eosinophilic gastroenteritis,
diseases marked by lymphatic obstruction, congenital intestinal
lymphangiectasia, sarcoidosis lymphoma, mesenteric tuberculosis,
after surgical correction of congenital heart disease, and food
allergies, primarily to milk.
Inflammation
[0015] Inflammation plays a central role in the pathology of
disease conditions that adversely affect a considerable proportion
of the population in developed countries. This process is mediated
by cytokines, a system of polypeptides that enable one cell to
signal to initiate events in another cell that initiate
inflammatory sequelae. Normally, the system acts as part of a
defensive reaction against infectious agents, harmful environmental
agents, or malignantly transformed cells. But when inflammation
exceeds the requirements of its defensive role, it can initiate
adverse clinical effects, such as arthritis, septic shock,
inflammatory bowel disease, and a range of other human disease
conditions.
[0016] Immune cells such as monocytes and macrophages secrete
cytokines including tumor necrosis factor-.alpha. (TNF.alpha.) and
tumor necrosis factor-.beta. (TNF.beta.) in response to endotoxin
or other stimuli. Cells other than monocytes or macrophages also
make cytokines including TNF.alpha.. For example, human
non-monocytic tumor cell lines produce TNF. CD4.sup.+ and CD8.sup.+
peripheral blood T lymphocytes and some cultured T and B cell lines
also produce TNF.alpha.. A large body of evidence associates
cytokines such as TNF.alpha. with infections, immune disorders,
neoplastic pathologies, autoimmune pathologies and graft-versus
host pathologies.
[0017] Small-molecule antirheumatic drugs such as methotrexate and
sulfasalazine are insufficient to control inflammation in about
two-thirds of arthritis patients. New biological agents developed
in the last decade have proved to be effective for a majority of
patients unresponsive to traditional drugs. The target for such
agents is often one of the cytokine pathways--either capturing the
ligand conveying the signal from one cell to another, or blocking
the receptor at the surface of the effector cell, preventing
transduction of the cytokine signal, thereby forestalling the
inflammatory events.
[0018] A leading biological agent for treating inflammatory
conditions is Enbrel.TM. (Etanercept), marketed by Amgen Corp. It
is a chimeric molecule comprising the extracellular portion of the
human TNF receptor linked as a dimer to the IgG Fc region. The
compound interferes with the binding of TNF to cell-surface TNF
receptors--showing the importance of modulating the TNF pathway for
clinical therapy of inflammatory conditions.
[0019] Other TNF.alpha. modulating agents currently licensed in the
U.S. for treating inflammatory conditions include Cimzia.TM.
(certolizumab pegol), a pegylated antibody fragment that binds to
TNF.alpha.; Remicade.TM. (Infliximab), a chimeric antibody that
binds TNF.alpha.; and Humira.TM. (adalimumab), a humanized
anti-TNF.alpha. antibody.
Celiac Disease
[0020] Celiac disease (CD) is a chronic autoimmune disease that is
HLA-DQ2/DQ8 haplotype restricted. Glutens, the major protein
fraction of wheat, and related proteins in rye and barley are the
triggering agents of the disease. Ingested gluten or its derivative
fractions (gliadin and subunits) elicit a harmful T cell-mediated
immune response after crossing the small bowel epithelial barrier,
undergoing deamidation by tissue transglutaminase (tTG) and
engaging class II MHC molecules.
[0021] While the earliest events leading to CD involve innate
immune responses, evidence in the literature seems to suggest that
a dysfunctional cross talk between innate and adaptive immunity is
also an important pathogenic element in the autoimmune process of
the disease. Under physiological circumstances, the intestinal
epithelium, with its intact intercellular tight junctions (tj),
serves as a key barrier to the passage of macromolecules such as
gluten. When the integrity of the tj system is compromised, as in
CD, a paracellular leak ("leaky gut") and an inappropriate immune
response to environmental antigens (i.e., gluten) may develop.
[0022] In celiac intestinal tissues and in in vitro, ex vivo, and
in vivo animal experiments, gluten/gliadin causes a rapid increase
in permeability in normal and diseased states. Animal models
likewise have demonstrated the association of gluten, increased
paracellular permeability and other autoimmune diseases, including
type 1 diabetes (T1D).
[0023] AT-1001 is an orally administered octapeptide (Gly Gly Val
Leu Val Gln Pro Gly (SEQ ID NO:1), that appears to inhibit
gliadin-induced TJ disassembly and prevent the associated increase
in paracellular permeability. Experiments with ex vivo human tissue
and in mice demonstrate that AT-1001 blocks the peak of F-actin
increment induced by gliadin and inhibits gliadin induced reduction
in intestinal Rt (resistance).
[0024] There is a continuing need in the art for methods to treat
inflammatory and autoimmune diseases as well as diseases associated
with biological barrier dysfunction more effectively and to
discover or identify drugs which are suitable for treating
inflammatory and autoimmune diseases as well as diseases associated
with biological barrier dysfunction.
SUMMARY OF THE INVENTION
[0025] One object of the present invention is to inhibit increased
permeability of biological barriers in response to secreted
signals.
[0026] Another object of the present invention is to provide
compounds that inhibit secretion of signals that cause increased
permeability of biological barriers.
[0027] In particular embodiments the present invention provides
compounds that inhibit the secretion of signals that cause
increased permeability of biological barriers, wherein the signals
are secreted in response to exposure of lymphocytes to
lipopolysaccharide (LPS). In other particular embodiments the
present invention provides compounds that inhibit the secretion of
signals that cause increased permeability of biological barriers,
wherein the signals are secreted in response to exposure of
lymphocytes to pepsin/trypsin treated gliadin (PTG).
[0028] Another object of the present invention is to provide
pharmaceutical compositions that inhibit secretion of signals that
cause increased permeability of biological barriers.
[0029] In particular embodiments the present invention provides
pharmaceutical compositions that inhibit the secretion of signals
that cause increased permeability of biological barriers, wherein
the signals are secreted in response to exposure of lymphocytes to
lipopolysaccharide (LPS). In other particular embodiments the
present invention provides pharmaceutical compositions that inhibit
the secretion of signals that cause increased permeability of
biological barriers, wherein the signals are secreted in response
to exposure of lymphocytes to pepsin/trypsin treated gliadin
(PTG).
[0030] Another object of the present invention is to provide
methods of treating a patient showing an increased secretion of
signals that cause increased permeability of biological
barriers.
[0031] In particular embodiments the present invention provides
methods of treating a patient showing an increased secretion of
signals that cause increased permeability of biological barriers,
wherein the signals are secreted in response to exposure of
lymphocytes to lipopolysaccharide (LPS). In other particular
embodiments the present invention provides methods of treating a
patient showing an increased secretion of signals that cause
increased permeability of biological barriers, wherein the signals
are secreted in response to exposure of lymphocytes to
pepsin/trypsin treated gliadin (PTG).
[0032] In certain embodiments, the invention provides a method of
treating a patient with an autoimmune or inflammation-associated
disease. The disease is selected from the group consisting of
inflammatory bowel disease, including Crohn's disease and
ulcerative colitis, necrotizing enterocolitis, type 1 diabetes,
celiac disease, autoimmune hepatitis, multiple sclerosis, autism,
dermatitis herpetiformis, IgA nephropathy, primary biliary
chirrosis, rheumatoid arthritis, systemic lupus erythematosus,
Grave's disease, Hashimoto's disease, and depression. A compound
that inhibits the production, release and/or the biological effects
of TNF.alpha. is administered to the patient.
[0033] Another object of the present invention is to provide
methods to inhibit paracellular passage of gluten derived peptides
across an epithelial barrier. Such methods comprise contacting the
epithelial barrier with one or more peptide permeability
inhibitors. Peptide permeability inhibitors for use in methods of
the invention may comprise a peptide of any length. Such peptide
permeability inhibitors may comprise a peptide from three to ten
amino acids in length. In some embodiments, a peptide permeability
inhibitor of the invention may comprise, consist essentially of, or
consist of a peptide that comprises, consists essentially of or
consists of an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-162. In some embodiments, a peptide
permeability inhibitor of the invention may comprise, consist
essentially of, or consist of a peptide that comprises, consists
essentially of or consists of an amino acid sequence selected from
the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34,
36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106,
110, 111, 113-125, 127, 128, 147, 150, and 160-162. In some
embodiments, the invention does not include SEQ ID NOs: 15, 24, and
25.
[0034] The present invention also provides novel methods to inhibit
increased paracellular permeability associated with exposure of a
biological barrier to gluten derived peptides. Such methods
comprise contacting the epithelial barrier with one or more peptide
permeability inhibitors. Peptide permeability inhibitors for use in
methods of the invention may comprise a peptide of any length. Such
peptide permeability inhibitors may comprise a peptide from three
to ten amino acids in length. In some embodiments, a peptide
permeability inhibitor of the invention may comprise, consist
essentially of, or consist of a peptide that comprises, consists
essentially of or consists of an amino acid sequence selected from
the group consisting of SEQ ID NOs: 1-162. In some embodiments, a
peptide permeability inhibitor of the invention may comprise,
consist essentially of, or consist of a peptide that comprises,
consists essentially of or consists of an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1-5, 10-17,
19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91,
92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and
160-162. In some embodiments, the invention does not include SEQ ID
NOs: 15, 24, and 25.
[0035] The present invention also provides compositions, e.g.,
pharmaceutical compositions, comprising one or more peptide
permeability inhibitors of the invention, useful to inhibit
paracellular passage of gluten derived peptides across an
epithelial barrier. Peptide permeability inhibitors for use in
compositions of the invention may comprise a peptide of any length.
In some embodiments, such peptide permeability inhibitors may
comprise a peptide of between three to ten amino acids in length.
Suitable peptide permeability inhibitors for use in the
compositions of the invention include, but are not limited to,
peptide permeability inhibitors that comprise, consist essentially
of, or consist of an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-162. In some embodiments, peptide
permeability inhibitors for use in the compositions of the
invention include, but are not limited to, peptide permeability
inhibitors comprising peptides that comprise, consist essentially
of, or consist of an amino acid sequence selected from the group
consisting of of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48,
49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110,
111, 113-125, 127, 128, 147, 150, and 160-162. In some embodiments,
the invention does not include SEQ ID NOs: 15, 24, and 25.
[0036] Compositions of the invention, for example, pharmaceutical
compositions, may be formulated for any type of delivery. For
example, compositions of the invention may be formulated for
intestinal delivery, e.g., may be delayed release compositions.
Compositions of the invention may also be formulated for pulmonary
delivery, oral delivery and/or transcutaneous delivery.
[0037] In one embodiment, the present invention provides a method
of treating a disease in a subject in need thereof. Methods of the
invention may comprise administering to the subject a
pharmaceutical composition comprising one or more peptide
permeability inhibitors of the invention. Methods of the invention
may comprise administering to the subject a pharmaceutical
composition comprising one or more peptide permeability inhibitors
and one or more additional therapeutic agents. In one embodiment,
the present invention provides a method of treating celiac disease
in a subject in need thereof. In another embodiment, the present
invention provides a method of treating necrotizing enterocolitis
in a subject in need thereof. In another embodiment, the present
invention provides a method of treating an excessive or undesirable
immune response in a subject in need thereof. In another
embodiment, the present invention provides a method of treating
inflammation in a subject in need thereof. In specific embodiments,
the present invention provides methods of treating inflammatory
bowel disease in a subject in need thereof. Inflammatory bowel
disease that can be treated using methods of the present invention
may be Crohn's disease or ulcerative colitis.
[0038] In further embodiments the invention provides methods of
treating an autoimmune or inflammation-associated disease in a
patient in need of such treatment. The disease is selected from the
group consisting of type 1 diabetes, celiac disease, autoimmune
hepatitis, multiple sclerosis, autism, dermatitis herpetiformis,
IgA nephropathy, primary biliary chirrosis, rheumatoid arthritis,
systemic lupus erythematosus, Grave's disease, Hashimoto's disease,
and depression.
[0039] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described herein, which form the subject of the claims of
the invention. It should be appreciated by those skilled in the art
that any conception and specific embodiment disclosed herein may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that any description, figure, example, etc. is
provided for the purpose of illustration and description only and
is by no means intended to define the limits the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a schematic representation of the events leading
to Celiac disease pathology. Gliadin fragments cross the intestinal
epithelium and activate immune cells to produce soluble factors
including cytokines that lead to increased permeability of the
intestinal epithelium.
[0041] FIG. 2 is a schematic representation of the blockade of the
gliadin fragment entry, the initial step leading to Celiac disease
pathology. Gliadin fragments cross the intestinal epithelium and
activate immune cells to produce soluble factors including
cytokines that lead to increased permeability of the intestinal
epithelium.
[0042] FIG. 3 shows the effect of a peptide permeability inhibitor
(SEQ ID NO:1) on permeability of a CaCO2 cell monolayer to a
gliadin fragment. Apical exposure of the monolayer to the gliadin
peptide PYPQPQLPY (SEQ ID NO:163) lead to an increase in
permeability to that peptide, which could be blocked by apical
treatment with a peptide permeability inhibitor (SEQ ID NO:1).
[0043] FIG. 4 shows the effect of a 13-mer gliadin peptide
(LGQQQPFPPQQPY; SEQ ID NO:164) on permeability of a CaCO2 cell
monolayer induced by a. Apical exposure of the monolayer to the
gliadin peptide FITC-C6-PYPQPQLPY lead to an increase in
permeability that could be blocked by treatment with a peptide
permeability inhibitor (SEQ ID NO:1).
[0044] FIG. 5A shows the effects on CaCO2 cell permeability of 72
hours treatment with peptide permeability inhibitor (SEQ ID NO:1)
in combination with culture supernatants prepared from donor PBMCs
(00022). After formation of tight junctions CaCO2 cells were
exposed basolaterally to control supernatant (control), untreated
PBMC supernatant (PBMC sup), LPS treated PBMC supernatant
(PBMC-LPS) and PTG treated PBMC supernatant (PBMC-PTG). Lucifer
yellow permeability was measured after 72 hours (day3).
Simultaneous apical addition of peptide permeability inhibitor (SEQ
ID NO:1) on day 0 abolished baseline permeability to Lucifer yellow
(control+AT-1001; and PBMC sup+AT1001) but had no significant
effect on permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
[0045] FIG. 5B shows the effects on CaCO2 cell permeability of 72
hours exposure to culture supernatants prepared from donor PBMCs
(00022) followed by addition of peptide permeability inhibitor (SEQ
ID NO:1) after 48 hours treatment. After formation of tight
junctions CaCO2 cells were exposed basolaterally to PBMC
supernatants as described above. Peptide permeability inhibitor
(SEQ ID NO:1) was added apically to the cultures after 48 hours
(day 2), and lucifer yellow permeability was measured after 72
hours (day3). Apical addition of peptide permeability inhibitor
(SEQ ID NO:1) on day 2 abolished baseline permeability to Lucifer
yellow (control+AT-1001; and PBMC sup+AT1001), and it significantly
reduced permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
[0046] FIG. 6A shows the effects on CaCO2 cell permeability of 72
hours treatment with peptide permeability inhibitor (SEQ ID NO:1)
in combination with culture supernatants prepared from donor PBMCs
(00023). After formation of tight junctions CaCO2 cells were
exposed basolaterally to control supernatant (control), untreated
PBMC supernatant (PBMC sup), LPS treated PBMC supernatant
(PBMC-LPS) and PTG treated PBMC supernatant (PBMC-PTG). Lucifer
yellow permeability was measured after 72 hours (day3).
Simultaneous apical addition of peptide permeability inhibitor (SEQ
ID NO:1) on day 0 abolished baseline permeability to Lucifer yellow
(control+AT-1001; and PBMC sup+AT1001) but had no significant
effect on permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
[0047] FIG. 6B shows the effects on CaCO2 cell permeability of 72
hours exposure to culture supernatants prepared from donor PBMCs
(00023) followed by addition of peptide permeability inhibitor (SEQ
ID NO:1) after 48 hours treatment. After formation of tight
junctions CaCO2 cells were exposed basolaterally to PBMC
supernatants as described above. Peptide permeability inhibitor
(SEQ ID NO:1) was added apically to the cultures after 48 hours
(day 2), and lucifer yellow permeability was measured after 72
hours (day3). Apical addition of peptide permeability inhibitor
(SEQ ID NO:1) on day 2 abolished baseline permeability to Lucifer
yellow (control+AT-1001; and PBMC sup+AT1001), and it significantly
reduced permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
[0048] FIG. 7A shows the effects on CaCO2 cell permeability of 72
hours treatment with peptide permeability inhibitor (SEQ ID NO:1)
in combination with culture supernatants prepared from donor PBMCs
(00064). After formation of tight junctions CaCO2 cells were
exposed basolaterally to control supernatant (control), untreated
PBMC supernatant (PBMC sup), LPS treated PBMC supernatant
(PBMC-LPS) and PTG treated PBMC supernatant (PBMC-PTG). Lucifer
yellow permeability was measured after 72 hours (day3).
Simultaneous apical addition of peptide permeability inhibitor (SEQ
ID NO:1) on day 0 abolished baseline permeability to Lucifer yellow
(control+AT-1001; and PBMC sup+AT1001) but had no significant
effect on permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
[0049] FIG. 7B shows the effects on CaCO2 cell permeability of 72
hours exposure to culture supernatants prepared from donor PBMCs
(00064) followed by addition of peptide permeability inhibitor (SEQ
ID NO:1) after 48 hours treatment. After formation of tight
junctions CaCO2 cells were exposed basolaterally to PBMC
supernatants as described above. Peptide permeability inhibitor
(SEQ ID NO:1) was added apically to the cultures after 48 hours
(day 2), and lucifer yellow permeability was measured after 72
hours (day3). Apical addition of peptide permeability inhibitor
(SEQ ID NO:1) on day 2 abolished baseline permeability to Lucifer
yellow (control+AT-1001; and PBMC sup+AT1001), and it significantly
reduced permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
[0050] FIG. 8A shows the effects on CaCO2 cell permeability of 72
hours treatment with peptide permeability inhibitor (SEQ ID NO:1)
in combination with culture supernatants prepared from donor PBMCs
(00065). After formation of tight junctions CaCO2 cells were
exposed basolaterally to control supernatant (control), untreated
PBMC supernatant (PBMC sup), LPS treated PBMC supernatant
(PBMC-LPS) and PTG treated PBMC supernatant (PBMC-PTG). Lucifer
yellow permeability was measured after 72 hours (day3).
Simultaneous apical addition of peptide permeability inhibitor (SEQ
ID NO:1) on day 0 abolished baseline permeability to Lucifer yellow
(control+AT-1001; and PBMC sup+AT1001) but had no significant
effect on permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
[0051] FIG. 8B shows the effects on CaCO2 cell permeability of 72
hours exposure to culture supernatants prepared from donor PBMCs
(00065) followed by addition of peptide permeability inhibitor (SEQ
ID NO:1) after 48 hours treatment. After formation of tight
junctions CaCO2 cells were exposed basolaterally to PBMC
supernatants as described above. Peptide permeability inhibitor
(SEQ ID NO:1) was added apically to the cultures after 48 hours
(day 2), and lucifer yellow permeability was measured after 72
hours (day3). Apical addition of peptide permeability inhibitor
(SEQ ID NO:1) on day 2 abolished baseline permeability to Lucifer
yellow (control+AT-1001; and PBMC sup+AT1001), and it significantly
reduced permeability changes induced by LPS (PBMC-LPS+AT1001) or
PTG treated PBMC supernatant (PBMC-PTG+AT1001).
DETAILED DESCRIPTION OF THE INVENTION
[0052] The inventors have discovered that peripheral blood
mononuclear cells (PBMCs) secrete signals that increase epithelial
monolayer permeability on response to stimulation with
lipopolysaccharide (PLPS) and pepsin/trypsin treated gliadin (PTG).
These secreted signals are present in PBMC culture supernatant, and
they increase permeability of CaCO2 cell monolayers to Lucifer
yellow when presented to the basolateral aspect of these cells.
These permeability changes are inhibited by treatment of the cells
with peptide permeability inhibitors of the invention (FIGS. 5A,
5B, 6A, 6B, 7A, 7B, 8A and 8B). The inventors have also discovered
that specific peptides within the PTG mixture are capable of
crossing epithelial cell monolayers in vitro, and that this peptide
specific mechanism can be inhibited by peptide permeability
inhibitors of the invention (FIGS. 3 and 4).
DEFINITIONS
[0053] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found, for example, in Benjamin Lewin, Genes VII,
published by Oxford University Press, 2000 (ISBN 019879276X);
Kendrew et al. (eds.); The Encyclopedia of Molecular Biology,
published by Blacicwell Publishers, 1994 (ISBN 0632021829); and
Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, published by Wiley, John & Sons,
Inc., 1995 (ISBN 0471186341); and other similar technical
references.
[0054] As used herein, "a" or "an" may mean one or more. As used
herein in the claim(s), when used in conjunction with the word
"comprising", the words "a" or "an" may mean one or more than one.
As used herein "another" may mean at least a second or more.
Furthermore, unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular.
[0055] As used herein, "biological effect" refers to a biochemical
and physiological effect. Biological effect includes, for example,
increases or decreases in the activity of the immune system and any
of its components (including, for example, complement activation),
increases or decreases in receptor binding and increases or
decreases in subsequent downstream effector cellular constituents
(including, for example, growth factor receptor and downstream
effector cellular constituents), increases or decreases in cell
signaling, increases or decreases in gene expression, increases or
decreased in post-translation modification of proteins (including,
for example, phosphorylation), and increases or decreases in
protein activity.
[0056] As used herein, "modulate" and all its forms and tenses
refer to either increasing or decreasing a particular biochemical
or physiological effect.
[0057] As used herein, A "component of the immune system" or an
"immune cell" refers to a component or cell of the immune system
that is involved in enhancing, eliciting, or maintaining an immune
response. The immune system responds to various foreign particles
(including, for example, viruses, bacteria, and allergens) and
non-foreign particles (including, for example, native endogenous
proteins). An immune response includes, for example, antibody
production, chemotaxis, phagocytosis, inflammation, complement
activation, production of cytotoxic molecules (including, for
example, reactive oxygen species and reactive nitrogen species),
cell adhesion, cell infiltration, and production and recruitment of
mediators of any of the foregoing or other immune responses. A
component or cell of the immune system involved in enhancing,
eliciting, or maintaining an immune response includes, for example,
neutrophils, complement proteins (including, for example, C1q, C1r
and C1s), eosinophils, basophils, lymphocytes (including for
example, T cells (including, for example, cytotoxic T cells, memory
T cells, helper T cells, regulatory T cells, natural killer T
cells, and .gamma..delta. T cells) and B cells (including, for
example, plasma B cells, memory B cells, B-1 cells, and B-2
cells)), monocytes, macrophages, dendritic cells (DC), cell
adhesion molecules (including, for example, ICAM and VCAM),
myeloperoxidase, nitric oxide synthase, cyclooxygenase, and
prostaglandin synthase.
[0058] As used herein, "treat" and all its forms and tenses refer
to both therapeutic treatment and prophylactic or preventative
treatment. Those in need of treatment include those already with
the condition or disease as well as those in which the condition or
disease is to be prevented.
Present Invention
[0059] The inventors have identified novel methods and compounds
that inhibit increased permeability of biological barriers in
response to stimuli that are known to induce secretion of
pro-inflammatory cytokines. In specific embodiments the inventors
have identified methods and compounds that inhibit increased
permeability of biological barriers after stimulation by factors
secreted by immune cells on exposure to LPS. In further specific
embodiments the inventors have identified methods and compounds
that inhibit increased permeability of biological barriers after
stimulation by factors secreted by immune cells on exposure to PTG.
Exemplary compounds of the invention that inhibit increased
permeability of biological barriers are presented in Table 20.
[0060] The inventors have also identified novel methods and
compounds that inhibit, reduce and/or prevent translocation of
PTG-derived peptides across biological barriers. In specific
embodiments the inventors have identified methods and compounds
that inhibit, reduce and/or prevent translocation of the peptide
comprising the amino acid sequence PYPQPQLPY (SEQ ID NO:163).
Exemplary compounds of the invention that inhibit, reduce and/or
prevent translocation of PTG-derived peptides across biological
barriers are presented in Table 20.
[0061] Inhibitors of biological barrier permeability may be used in
the practice of the present invention. Such permeability inhibitors
may also be antagonists of mammalian tight junction opening.
Antagonists of mammalian tight junction opening may also be used in
the practice of the present invention. As used herein, permeability
inhibitors prevent, inhibit or reduce the permeability of
biological barriers to macromolecules including, for example,
proteins, peptides and nucleic acids. For example, permeability
inhibitors of the invention may comprise peptide permeability
inhibitors. Examples of peptide permeability inhibitors that may be
used in the practice of the present invention include, but are not
limited to, peptides that comprise an amino acid sequence selected
from the group consisting of: consist of an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1-5, 10-17,
19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91,
92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and
160-162.
[0062] Examples of peptide permeability inhibitors include, but are
not limited to, peptides that consist of an amino acid sequence
selected from the group consisting of SEQ ID NOs:1-162.
[0063] When the permeability inhibitor is a peptide, any length of
peptide may be used. Generally, the size of the peptide antagonist
will range from about 6 to about 100, from about 6 to about 90,
from about 6 to about 80, from about 6 to about 70, from about 6 to
about 60, from about 6 to about 50, from about 6 to about 40, from
about 6 to about 30, from about 6 to about 25, from about 6 to
about 20, from about 6 to about 15, from about 6 to about 14, from
about 6 to about 13, from about 6 to about 12, from about 6 to
about 11, from about 6 to about 10, from about 6 to about 9, or
from about 6 to about 8 amino acids in length. Peptide antagonists
of the invention may be from about 8 to about 100, from about 8 to
about 90, from about 8 to about 80, from about 8 to about 70, from
about 8 to about 60, from about 8 to about 50, from about 8 to
about 40, from about 8 to about 30, from about 8 to about 25, from
about 8 to about 20, from about 8 to about 15, from about 8 to
about 14, from about 8 to about 13, from about 8 to about 12, from
about 8 to about 11, or from about 8 to about 10 amino acids in
length. Peptide antagonists of the invention may be from about 10
to about 100, from about 10 to about 90, from about 10 to about 80,
from about 10 to about 70, from about 10 to about 60, from about 10
to about 50, from about 10 to about 40, from about 10 to about 30,
from about 10 to about 25, from about 10 to about 20, from about 10
to about 15, from about 10 to about 14, from about 10 to about 13,
or from about 10 to about 12 amino acids in length. Peptide
antagonists of the invention may be from about 12 to about 100,
from about 12 to about 90, from about 12 to about 80, from about 12
to about 70, from about 12 to about 60, from about 12 to about 50,
from about 12 to about 40, from about 12 to about 30, from about 12
to about 25, from about 12 to about 20, from about 12 to about 15,
or from about 12 to about 14 amino acids in length. Peptide
antagonists of the invention may be from about 15 to about 100,
from about 15 to about 90, from about 15 to about 80, from about 15
to about 70, from about 15 to about 60, from about 15 to about 50,
from about 15 to about 40, from about 15 to about 30, from about 15
to about 25, from about 15 to about 20, from about 19 to about 15,
from about 15 to about 18, or from about 17 to about 15 amino acids
in length.
[0064] The peptide permeability inhibitors can be chemically
synthesized and purified using well-known techniques, such as
described in High Performance Liquid Chromatography of Peptides and
Proteins: Separation Analysis and Conformation, Eds. Mant et al.,
C.R.C. Press (1991), and a peptide synthesizer, such as Symphony
(Protein Technologies, Inc); or by using recombinant DNA
techniques, i.e., where the nucleotide sequence encoding the
peptide is inserted in an appropriate expression vector, e.g., an
E. coli or yeast expression vector, expressed in the respective
host cell, and purified therefrom using well-known techniques.
Compositions
[0065] Typically, compositions, such as pharmaceutical
compositions, comprise one or more compounds of the invention, and
optionally one or more additional active agents. Compounds of the
invention may be present in an amount sufficient to inhibit the
increased biological barrier permeability in a subject in need
thereof. Compounds of the invention may be present in an amount
sufficient to inhibit, reduce and/or prevent translocation of a
gliadin-derived peptide across a biological barrier in a subject in
need thereof. The amount of a compound of the invention employed in
any given composition may vary according to factors such as the
disease state, age, sex, and weight of the subject. Dosage regimens
may be adjusted to provide the optimum therapeutic response. For
example, a single bolus may be administered, several divided doses
may be administered over time or the dose may be proportionally
reduced or increased as indicated by the exigencies of the
therapeutic situation.
[0066] Generally, a pharmaceutical composition of the invention
will comprise an amount of a compound of the invention in the range
of about 1 .mu.g to about 1 g, preferably about 1 mg to about 1000
mg, from about 10 mg to about 100 mg, from about 10 mg to about 50
mg, or from about 10 mg to about 25 mg of the compound. As used
herein, "about" used to modify a numerical value means within 10%
of the value.
[0067] Compositions of the invention may comprise one or more
compounds of the invention at a level of from about 0.1 wt % to
about 20 wt %, from about 0.1 wt % to about 18 wt %, from about 0.1
wt % to about 16 wt %, from about 0.1 wt % to about 14 wt %, from
about 0.1 wt % to about 12 wt %, from about 0.1 wt % to about 10 wt
%, from about 0.1 wt % to about 8 wt %, from about 0.1 wt % to
about 6 wt %, from about 0.1 wt % to about 4 wt %, from about 0.1
wt % to about 2 wt %, from about 0.1 wt % to about 1 wt %, from
about 0.1 wt % to about 0.9 wt %, from about 0.1 wt % to about 0.8
wt %, from about 0.1 wt % to about 0.7 wt %, from about 0.1 wt % to
about 0.6 wt %, from about 0.1 wt % to about 0.5 wt %, from about
0.1 wt % to about 0.4 wt %, from about 0.1 wt % to about 0.3 wt %,
or from about 0.1 wt % to about 0.2 wt % of the total weight of the
composition. As used herein, "about" used to modify a numerical
value means within 10% of the value. Compositions of the invention
may comprise one or more compounds of the invention at a level of
about 0.1 wt %, about 0.2 wt %, about 0.3 wt %, about 0.4 wt %,
about 0.5 wt %, about 0.6 wt %, about 0.7 wt %, about 0.8 wt %, or
about 0.9 wt % based on the total weight of the composition.
[0068] Compositions of the invention may comprise one or more
compounds of the invention at a level of from about 1 wt % to about
20 wt %, from about 1 wt % to about 18 wt %, from about 1 wt % to
about 16 wt %, from about 1 wt % to about 14 wt %, from about 1 wt
% to about 12 wt %, from about 1 wt % to about 10 wt %, from about
1 wt % to about 9 wt %, from about 1 wt % to about 8 wt %, from
about 1 wt % to about 7 wt %, from about 1 wt % to about 6 wt %,
from about 1 wt % to about 5 wt %, from about 1 wt % to about 4 wt
%, from about 1 wt % to about 3 wt %, or from about 1 wt % to about
2 wt % of the total weight of the composition. As used herein,
"about" used to modify a numerical value means within 10% of the
value. Compositions of the invention may comprise one or more
compounds of the invention at a level of about 1 wt %, about 2 wt
%, about 3 wt %, about 4 wt %, about 5 wt %, about 6 wt %, about 7
wt %, about 8 wt %, or about 9 wt % based on the total weight of
the composition.
[0069] Compositions of the invention, for example, pharmaceutical
compositions comprising one or more compounds of the invention and
one or more additional active agents, may be formulated for
pulmonary delivery (e.g., may be pulmonary dosage forms). Typically
such compositions may be provided as pharmaceutical aerosols, e.g.,
solution aerosols or powder aerosols. Those of skill in the art are
aware of many different methods and devices for the formation of
pharmaceutical aerosols, for example, those disclosed by Sciarra
and Sciarra, Aerosols, in Remington: The Science and Practice of
Pharmacy, 20th Ed., Chapter 50, Gennaro et al. Eds., Lippincott,
Williams and Wilkins Publishing Co., (2000).
[0070] In one embodiment, the dosage forms are in the form of a
powder aerosol (i.e, comprise particles). These are particularly
suitable for use in inhalation delivery systems. Powders may
comprise particles of any size suitable for administration to the
lung.
[0071] Powder formulations may optionally contain at least one
particulate pharmaceutically acceptable carrier known to those of
skill in the art. Examples of suitable pharmaceutical carriers
include, but are not limited to, saccharides, including
monosaccharides, disaccharides, polysaccharides and sugar alcohols
such as arabinose, glucose, fructose, ribose, mannose, sucrose,
trehalose, lactose, maltose, starches, dextran, mannitol or
sorbitol. In one embodiment, a powder formulation may comprise
lactose as a carrier.
[0072] Powder formulations may be contained in any container known
to those in the art. Containers may be capsules of, for example,
gelatin or plastic, or in blisters (e.g. of aluminum or plastic),
for use in a dry powder inhalation device. In some embodiments, the
total weight of the formulation in the container may be from about
5 mg to about 50 mg. In other embodiments, powder formulations may
be contained in a reservoir in a multi-dose dry powder inhalation
device adapted to deliver a suitable amount per actuation.
[0073] Powder formulations typically comprise small particles.
Suitable particles can be prepared using any means known in the
art, for example, by grinding in an airjet mill, ball mill or
vibrator mill, sieving, microprecipitation, spray-drying,
lyophilisation or controlled crystallisation. Typically, particles
will be about 10 microns or less in diameter. Particles for use in
the compositions of the invention may have a diameter of from about
0.1 microns to about 10 microns, from about 0.1 microns to about 9
microns, from about 0.1 microns to about 8 microns, from about 0.1
microns to about 7 microns, from about 0.1 microns to about 6
microns, from about 0.1 microns to about 5 microns, from about 0.1
microns to about 4 microns, from about 0.1 microns to about 3
microns, from about 0.1 microns to about 2 microns, from about 0.1
microns to about 1 micron, from about 0.1 microns to about 0.5
microns, from about 1 micron to about 10 microns, from about 1
micron to about 9 microns, from about 1 micron to about 8 microns,
from about 1 micron to about 7 microns, from about 1 micron to
about 6 microns, from about 1 micron to about 5 microns, from about
1 micron to about 4 microns, from about 1 micron to about 3
microns, from about 1 micron to about 2 microns, from about 2
microns to about 10 microns, from about 2 microns to about 9
microns, from about 2 microns to about 8 microns, from about 2
microns to about 7 microns, from about 2 microns to about 6
microns, from about 2 microns to about 5 microns, from about 2
microns to about 4 microns, or from about 2 microns to about 3
microns. As used herein, "about" used to modify a numerical value
means within 10% of the value. In some embodiments, particles for
use in the invention may be about 1 micron, about 2 microns, about
3 microns, about 4 microns, about 5 microns, about 6 microns, about
7 microns, about 8 microns, about 9 microns, or about 10 microns in
diameter.
[0074] In one embodiment, the dosage forms are in the form of a
solution aerosol (i.e., comprise droplets). Typically, droplets
will be about 10 microns or less in diameter. Droplets for use in
the compositions of the invention may have a diameter of from about
0.1 microns to about 10 microns, from about 0.1 microns to about 9
microns, from about 0.1 microns to about 8 microns, from about 0.1
microns to about 7 microns, from about 0.1 microns to about 6
microns, from about 0.1 microns to about 5 microns, from about 0.1
microns to about 4 microns, from about 0.1 microns to about 3
microns, from about 0.1 microns to about 2 microns, from about 0.1
microns to about 1 micron, from about 0.1 microns to about 0.5
microns, from about micron to about 10 microns, from about 1 micron
to about 9 microns, from about 1 micron to about 8 microns, from
about 1 micron to about 7 microns, from about 1 micron to about 6
microns, from about 1 micron to about 5 microns, from about 1
micron to about 4 microns, from about 1 micron to about 3 microns,
from about 1 micron to about 2 microns, from about 2 microns to
about 10 microns, from about 2 microns to about 9 microns, from
about 2 microns to about 8 microns, from about 2 microns to about 7
microns, from about 2 microns to about 6 microns, from about 2
microns to about 5 microns, from about 2 microns to about 4
microns, or from about 2 microns to about 3 microns. As used
herein, "about" used to modify a numerical value means within 10%
of the value. In some embodiments, particles and/or droplets for
use in the invention may be about 1 micron, about 2 microns, about
3 microns, about 4 microns, about 5 microns, about 6 microns, about
7 microns, about 8 microns, about 9 microns, or about 10 microns in
diameter.
[0075] The compositions of the invention may be formulated for
enteric delivery, for example, may comprise one or more coatings
including, for example, a delayed release coating containing one or
more enteric agents. A delayed release coating is typically
substantially stable in gastric fluid and substantially unstable
(e.g., dissolves rapidly or is physically unstable) in intestinal
fluid, thus providing for substantial release of the compounds of
the invention and/or additional active agent from the composition
in the duodenum or the jejunum.
[0076] The term "stable in gastric fluid" refers to a composition
that releases 30% or less by weight of the total compound of the
invention and/or additional active agent in the composition in
gastric fluid with a pH of 5 or less, or simulated gastric fluid
with a pH of 5 or less, in approximately sixty minutes. Examples of
simulated gastric fluid and simulated intestinal fluid include, but
are not limited to, those disclosed in the 2005 Pharmacopeia
23NF/28USP in Test Solutions at page 2858 and/or other simulated
gastric fluids and simulated intestinal fluids known to those of
skill in the art, for example, simulated gastric fluid and/or
intestinal fluid prepared without enzymes.
[0077] Compositions of the of the invention may release from about
0% to about 30%, from about 0% to about 25%, from about 0% to about
20%, from about 0% to about 15%, from about 0% to about 10%, from
about 5% to about 30%, from about 5% to about 25%, from about 5% to
about 20%, from about 5% to about 15%, from about 5% to about 10%
by weight of the total compound of the invention and/or additional
active agent in the composition in gastric fluid with a pH of 5 or
less, or simulated gastric fluid with a pH of 5 or less, in
approximately sixty minutes. As used herein, "about" used to modify
a numerical value means within 10% of the value. Compositions of
the invention may release about 1%, about 2%, about 3%, about 4%,
about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by
weight of the total compound of the invention in the composition in
gastric fluid with a pH of 5 or less, or simulated gastric fluid
with a pH of 5 or less, in approximately sixty minutes.
[0078] The term "unstable in intestinal fluid" refers to a
composition that releases 70% or more by weight of the total amount
of the compound of the invention and/or additional active agent in
the composition in intestinal fluid or simulated intestinal fluid
in approximately sixty minutes. The term "unstable in near neutral
to alkaline environments" refers to a composition that releases 70%
or more by weight of the total amount of the compound of the
invention and/or additional active agent in the composition in
intestinal fluid with a pH of 5 or greater, or simulated intestinal
fluid with a pH of 5 or greater, in approximately ninety minutes.
For example, a composition that is unstable in near neutral or
alkaline environments may release 70% or more by weight of a
compound of the invention and/or additional active agent in a fluid
having a pH greater than about 5 (e.g., a fluid having a pH of from
about 5 to about 14, from about 6 to about 14, from about 7 to
about 14, from about 8 to about 14, from about 9 to about 14, from
about 10 to about 14, or from about 11 to about 14) in from about 5
minutes to about 90 minutes, from about 10 minutes to about 90
minutes, from about 15 minutes to about 90 minutes, from about 20
minutes to about 90 minutes, from about 25 minutes to about 90
minutes, from about 30 minutes to about 90 minutes, from about 5
minutes to about 60 minutes, from about 10 minutes to about 60
minutes, from about 15 minutes to about 60 minutes, from about 20
minutes to about 60 minutes, from about 25 minutes to about 60
minutes, or from about 30 minutes to about 60 minutes. As used
herein, "about" used to modify a numerical value means within 10%
of the value.
[0079] Compositions of the invention may be formulated for
transcutaneous delivery (e.g., may be transcutaneous dosage forms).
Typically such compositions may be provided as topical solutions
and/or gels. Those of skill in the art are aware of many different
methods and devices for the formation of topical medications, for
example, those disclosed by Block, Medicated Topicals, in
Remington: The Science and Practice of Pharmacy, 20th Ed., Chapter
44, Gennaro et al. Eds., Lippincott, Williams and Wilkins
Publishing Co. (2000).
[0080] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0081] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
adsorb.
[0082] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome.
[0083] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used. In another embodiment, polymeric materials can be
used. In yet another embodiment, a controlled release system can be
placed in proximity of the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose. Other
controlled release systems are well known in the art.
[0084] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0085] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0086] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0087] The amount of the compound of the invention that will be
effective in the treatment, inhibition and/or prevention of a
disease or disorder associated with increased biological barrier
permeability can be determined by standard clinical techniques. The
amount of the compound of the invention that will be effective in
the treatment, inhibition and/or prevention of a disease or
disorder associated with translocation of one or more
gliadin-derived peptides across a biological barrier can be
determined by standard clinical techniques. In addition, in vitro
assays may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the formulation will
also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0088] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
Additional Active Agents
[0089] In addition to one or more compounds of the invention,
compositions of the invention may further comprise one or more
additional active agents, e.g., therapeutic agents, immunogenic
agents and/or imaging agents.
[0090] Additional therapeutic agents that can be used in the
compositions of the invention include agents that act on any organ
of the body, such as heart, brain, intestine, or kidneys. Suitable
additional therapeutic agents include, but are not limited to,
glucose metabolism agents (e.g., insulin), antibiotics,
antineoplastics, antihypertensives, antiepileptics, central nervous
system agents, anti-inflammatory agents and immune system
suppressants.
[0091] Additional therapeutic agents that can be used in the
compositions of the invention include immunosuppressive agents.
Such immunosuppressants used in the method and composition of the
invention can be any agent which tends to attenuate the activity of
the humoral or cellular immune systems. In particular, in one
aspect the invention comprises compositions wherein the
immunosuppressant is selected from the group consisting of
cyclosporin A, FK506, prednisone, methylprednisolone,
cyclophosphamide, thalidomide, azathioprine, and daclizumab,
physalin B, physalin F, physalin G, seco-steroids purified from
Physalis angulata L., 15-deoxyspergualin (DSG, 15-dos), MMF,
rapamycin and its derivatives, CCI-779, FR 900520, FR 900523,
NK86-1086, depsidomycin, kanglemycin-C, spergualin,
prodigiosin25-c, cammunomicin, demethomycin, tetranactin,
tranilast, stevastelins, myriocin, gliooxin, FR 651814, SDZ214-104,
bredinin, WS9482, mycophenolic acid, mimoribine, misoprostol, OKT3,
anti-IL-2 receptor antibodies, azasporine, leflunomide, mizoribine,
azaspirane (SKF 105685), paclitaxel, altretamine, busulfan,
chlorambucil, ifosfamide, mechlorethamine, melphalan, thiotepa,
cladribine, fluorouracil, floxuridine, gemcitabine, thioguanine,
pentostatin, methotrexate, 6-mercaptopurine, cytarabine,
carmustine, lomustine, streptozotocin, carboplatin, cisplatin,
oxaliplatin, iproplatin, tetraplatin, lobaplatin, JM216, JM335,
fludarabine, aminoglutethimide, flutamide, goserelin, leuprolide,
megestrol acetate, cyproterone acetate, tamoxifen, anastrozole,
bicalutamide, dexamethasone, diethylstilbestrol, bleomycin,
dactinomycin, daunorubicin, doxirubicin, idarubicin, mitoxantrone,
losoxantrone, mitomycin-c, plicamycin, paclitaxel, docetaxel,
topotecan, irinotecan, 9-amino camptothecan, 9-nitro camptothecan,
GS-211, etoposide, teniposide, vinblastine, vincristine,
vinorelbine, procarbazine, asparaginase, pegaspargase, octreotide,
estramustine, and hydroxyurea, and combinations thereof. In one
more particular aspect, the immunosuppressant is cyclosporin A.
[0092] Furthermore, the additional therapeutic agent can be
selected from the group consisting of a chemotherapeutic, a gene
therapy vector, a growth factor, a contrast agent, an angiogenesis
factor, a radionuclide, an anti-infection agent, an anti-tumor
compound, a receptor-bound agent, a hormone, a steroid, a protein,
a complexing agent, a polymer, a thrombin inhibitor, an
antithrombogenic agent, a tissue plasminogen activator, a
thrombolytic agent, a fibrinolytic agent, a vasospasm inhibitor, a
calcium channel blocker, a nitrate, a nitric oxide promoter, a
vasodilator, an antihypertensive agent, an antimicrobial agent, an
antibiotic, a glycoprotein IIb/IIIa inhibitor, an inhibitor of
surface glycoprotein receptors, an antiplatelet agent, an
antimitotic, a microtubule inhibitor, a retinoid, an antisecretory
agent, an actin inhibitor, a remodeling inhibitor, an antisense
nucleotide, an agent for molecular genetic intervention, an
antimetabolite, an antiproliferative agent, an anti-cancer agent, a
dexamethasone derivative, an anti-inflammatory steroid, a
non-steroidal anti-inflammatory agent, an immunosuppressive agent,
a PDGF antagonist, a growth hormone antagonist, a growth factor
antibody, an anti-growth factor antibody, a growth factor
antagonist, a dopamine agonist, a radiotherapeutic agent, an
iodine-containing compound, a barium-containing compound, a heavy
metal functioning as a radiopaque agent, a peptide, a protein, an
enzyme, an extracellular matrix component, a cellular component, an
angiotensin converting enzyme inhibitor, a 21-aminosteroid, a free
radical scavenger, an iron chelator, an antioxidant, a sex hormone,
an antipolymerase, an antiviral agent, an IgG2 Kappa antibody
against Pseudomonas aeruginosa exotoxin A and reactive with A431
epidermoid carcinoma cells, monoclonal antibody against the
noradrenergic enzyme dopamine beta-hydroxylase conjugated to
saporin or other antibody targeted therapy agents, gene therapy
agents, a prodrug, a photodynamic therapy agent, and an agent for
treating benign prostatic hyperplasia (BHP), a .sup.14C-, .sup.3H-,
.sup.131I-, .sup.32P- or .sup.36S-radiolabelled form or other
radiolabelled form of any of the foregoing, and combinations
thereof.
[0093] More particularly, the additional therapeutic agent can be
selected from the group consisting of parathyroid hormone, heparin,
human growth hormone, covalent heparin, hirudin, hirulog,
argatroban, D-phenylalanyl-L-poly-L-arginyl chloromethyl ketone,
urokinase, streptokinase, nitric oxide, triclopidine, aspirin,
colchicine, dimethyl sulfoxide, cytochalasin, deoxyribonucleic
acid, methotrexate, tamoxifen citrate, dexamethasone, dexamethasone
sodium phosphate, dexamethasone, acetate, cyclosporin, trapidal,
angiopeptin, angiogenin, dopamine, .sup.60Co, .sup.192Ir, .sup.32P,
.sup.111In, .sup.90Y, .sup.99mTc, pergolide mesylate, bromocriptine
mesylate, gold, tantalum, platinum, tungsten, captopril, enalapril,
ascorbic acid, .alpha.-tocopherol, superoxide dismutase,
dcferoxamine, estrogen, azidothymidine (AZT), acyclovir,
famciclovir, rimantadine hydrochloride, ganciclovir sodium,
5-aminolevulinic acid, meta-tetrahydroxyphenylchlorin,
hexadecafluoro zinc phthalocyanine, tetramethyl hematoporphyrin,
and rhodamine 123, and combinations thereof.
[0094] Compositions of the invention may comprise one or more
immunogenic agents, for example, antigens. Examples of antigens
that can be used in the compositions of the invention (e.g.,
immunogenic and/or vaccine compositions) include peptides,
proteins, microorganisms (e.g., attenuated and/or recombinant
microorganisms), cells (e.g., cancer cells and/or recombinant
cells) and viruses (e.g., attenuated and/or recombinant viruses).
Examples of peptide antigens include the B subunit of the
heat-labile enterotoxin of enterotoxigenic E. coli, the B subunit
of cholera toxin, capsular antigens of enteric pathogens, fimbriae
or pili of enteric pathogens, HIV surface antigens, cancer antigens
(e.g., cancer cells comprising antigens, isolated antigens, etc.),
dust allergens, and acari allergens. Other immunogenic compounds as
are known in the art can also be used.
[0095] Examples of attenuated microorganisms and viruses that can
be used in the compositions of the invention (e.g., vaccine
compositions) include those of enterotoxigenic Escherichia coli,
enteropathogenic Escherichia coli, Vibrio cholerae, Shigella
flexneri, Salmonella typhi and rotavirus (Fasano et al, In: Le
Vaccinazioni in Pediatria, Eds. Vierucci et al, CSH, Milan, pages
109-121 (1991); Guandalini et al, In: Management of Digestive and
Liver Disorders in Infants and Children, Elsevior, Eds. Butz et al,
Amsterdam, Chapter 25 (1993); Levine et al, Sem. Ped. Infect. Dis.,
5.243-250 (1994); and Kaper et al, Clin. Micrbiol. Rev., 8:48-86
(1995), each of which is incorporated by reference herein in its
entirety).
[0096] Any antigen capable of inducing a protective immune response
may be used in the vaccine compositions of the invention. Examples
of suitable antigens include, but are not limited to, measles virus
antigens, mumps virus antigens, rubella virus antigens,
Corynebacterium diphtheriae antigens, Bordetella pertussis
antigens, Clostridium tetani antigens, Bacillus anthracis antigens,
Haemophilus influenzae antigens, smallpox virus antigens, and
influenza virus antigens.
[0097] Compositions of the invention may further comprise one or
more protease inhibitors. Any protease inhibitor can be used,
including, but not limited to, a proteinase, peptidase,
endopeptidase, or exopeptidase inhibitor. A cocktail of inhibitors
can also be used. Alternatively, the protease inhibitors can be
selected from the group consisting of bestatin,
L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine,
ethylenediaminetetra-acetic acid (EDTA),
phenylmethylsulfonylfluoride (PMSF), aprotinin, amyloid protein
precursor (APP), amyloid beta precursor protein,
.alpha.1-proteinase inhibitor, collagen VI, bovine pancreatic
trypsin inhibitor (BPTI), 4-(2-aminoethyl)-benzenesulfonyl fluoride
(AEBSF), antipain, benzamidine, chymostatin, c-aminocaproate,
N-ethylmaleimide, leupeptin, pepstatin A, phosphoramidon, and
combinations thereof. Novel protease inhibitors can also be used.
Indeed, protease inhibitors can be specifically designed or
selected to decrease the proteolysis of the tight junction agonist
and/or the therapeutic agent.
[0098] Compositions of the invention may also comprise one or more
pharmaceutically acceptable excipients. Suitable excipients
include, but are not limited to, buffers, buffer salts, bulking
agents, salts, surface active agents, acids, bases, sugars,
binders, and the like.
Methods of Treatment
[0099] Compounds and pharmaceutical compositions of the invention
can be used for treating, ameliorating, and/or preventing a
disease. Any disease may be treated using the compositions of the
invention by selection of an appropriate active agent, e.g.,
therapeutic and/or immunogenic agent. In one embodiment, the
present invention provides a method of treating diabetes response
in a subject (e.g., a mammal such as a human) by administering a
composition comprising one or more compounds of the invention
together with one or more insulins and/or derivatives thereof. In
another embodiment, the invention provides a method of suppressing
an excessive or undesirable immune response in a subject (e.g., a
mammal such as a human) by administering a composition comprising
one or more compounds of the invention together with one or more
immune-suppressive drugs that may include, for example, cyclosporin
A.
[0100] Examples of diseases that can be treated using the
compositions of the invention include, but are not limited to,
cancer, autoimmune diseases, vascular disease, bacterial
infections, gastritis, gastric cancer, collagenous colitis,
inflammatory bowel disease, necrotizing enterocolitis,
osteoporosis, systemic lupus erythematosus, food allergy, asthma,
celiac disease and irritable bowel syndrome. For example, to treat
inflammatory bowel disease, a composition comprising one or more
compounds of the invention may be administered to the subject
(e.g., a mammal such as a human) in need thereof.
[0101] In another example, to treat cancer of the colon or rectal
area, a composition comprising a therapeutically effective amount
of Erbitux.RTM. (Cetuximab) together with a GM-CSF and/or IL-16
inhibiting amount of one or more compounds of the invention may be
administered to the subject (e.g., a mammal such as a human) in
need thereof. In another example, to treat breast cancer, a
composition comprising a therapeutically effective amount of
Herceptin.RTM. (Trastuzumab) together with a GM-CSF and/or IL-16
inhibiting amount of one or more compounds of the invention may be
administered to the subject (e.g., a mammal such as a human) in
need thereof. In another example, to treat various types of cancer,
a composition comprising a therapeutically effective amount of
Avastin.RTM. (Bevacizumab) together with a GM-CSF and/or IL-16
inhibiting amount of one or more compounds of the invention may be
administered to the subject (e.g., a mammal such as a human) in
need thereof. Another example involves treatment of osteoporosis by
administration of a composition comprising one or more compounds of
the invention together with a therapeutically effective amount of
Fosamax.RTM. (Alendronate) to the subject in need thereof. Another
example involves treatment of transplant rejection by
administration of a composition comprising one or compounds of the
invention together with a therapeutically effective amount of
Cyclosporin A to the subject in need thereof. Another example
involves treatment of anemia by administration of a composition
comprising one or more compounds of the invention together with a
therapeutically effective amount of erythropoietin to the subject
in need thereof. Another example involves treatment of hemophilia
by administration of a composition comprising one or more compounds
of the invention together with a therapeutically effective amount
of Factor VIII to the subject in need thereof.
[0102] In some embodiments, compositions of the invention (e.g.,
pharmaceutical compositions) may be given repeatedly over a
protracted period, i.e., may be chronically administered.
Typically, compositions may be administered one or more times each
day in an amount suitable to prevent, reduce the likelihood of an
attack of, or reduce the severity of an attack of the underlying
disease condition (e.g., diabetes, cancer, transplant rejection,
etc). Such compositions may be administered chronically, for
example, one or more times daily over a plurality of days.
[0103] In some embodiments, compositions of the invention (e.g.,
pharmaceutical compositions) may be used to treat acute attacks of
the underlying disease (e.g., diabetes, cancer, transplant
rejection, etc). Typically, embodiments of this type will require
administration of the compositions of the invention to a subject
undergoing an attack in an amount suitable to reduce the severity
of the attack. One or more administrations may be used.
[0104] In some embodiments, compounds of the invention may be used
in the manufacture of compositions and pharmaceutical compositions
for use in the methods described above.
[0105] While the invention has been described with reference to
certain particular embodiments thereof, those skilled in the art
will appreciate that various modifications may be made without
departing from the spirit and scope of the invention. The scope of
the appended claims is not to be limited to the specific
embodiments described.
Methods of Screening
[0106] Screening for inhibitors of gliadin-derived peptide
translocation across biological barriers can be accomplished by a
variety of techniques. Likewise, screening for inhibitors of
PTG-induced factors that increase biological barrier permeability
can be accomplished by a variety of techniques. Gliadin-derived
peptide binding to test compounds (inhibitor candidates) can be
directly measured, or inhibition of binding of gliadin-derived
peptides to a cell preparation can be measured. Gliadin-derived
peptides can be labeled to facilitate measurement of binding.
Assays may be in cell-free systems or in cell-based systems. Any
binding assay format can be used, including formats where the
receptor is attached to a solid support, either directly or
indirectly.
[0107] Test compounds which can be tested are any compounds. The
compounds may be tested as single compounds or in combinations of
compounds. The compounds may be structurally identified or of
unknown structure. The compounds may be novel or previously known.
The compounds may be natural products or synthetic.
[0108] According to one embodiment of the invention the test
compounds are fragments of gliadin. Gliadin is a family of proteins
which are produced by wheat and other grains. Examples of gliadins
are gliadin alpha, gamma, and omega. Gliadins are the aqueous
alcohol-soluble storage proteins in the seed. There is great
heterogeneity even within a single class of gliadins. At least six,
seven, eight, nine, ten, eleven, fifteen, twenty, thirty,
thirty-five, fifty, or seventy-five amino acid residues may be used
in fragments of gliadin as test compounds. Fragments include any
molecule which is less than full length. Fragments may be, e.g.,
synthesized or the result of proteolytic degradation. The following
tables provide the sequences of a representative number of
gliadins.
TABLE-US-00001 TABLE 1 Amino acid sequence of alpha-gliadin from
Triticum aestivum (NCBI accession no. CAB76964, (SEQ ID NO: 165)) 1
mvrvpvpqlq pqnpsqqqpq eqvplvqqqq fpgqqqpfpp qqpypqpqpf 51
psqqpylqlq pfpqpqlpyp qpqlpypqpq lpypqpqpfr pqqpypqsqp 101
qysqpqqpis qqqqqqqqqq qqkqqqqqqq qilqqilqqq liperdvvlq 151
qhsiaygssq vlqqstyqlv qqlccqqlwq ipeqsrcqai hnvvhaiilh 201
qqqqqqqqqq qqplsqvsfq qpqqqypsgq gsfqpsqqnp qaqgsvqpqq 251
lpqfeeirnl aletlpamcn vyippyctia pvgifgtnyr
TABLE-US-00002 TABLE 2 Amino acid sequence of alpha-gliadin
precursor from Triticum turgidum subsp. durum (NCBI accession no.
CA135909, (SEQ ID NO: 166)) 1 mktflilall aivattatta vrvpvpqlqr
qnpsqqqpqe qvplvqqqqf 51 lgqqqpfppq qpypqpqpfp sqqpylqlqp
fpqpqlpysq pqpfrpqqpy 101 pqpqprysqp qqpisqqqqq qhqqhqqhhq
eqqilqqilq qqlipcmdvv 151 lqqhniahrr sqvlqqstyq llgelccqhl
wqipeqsqcq aihnvvhaii 201 phqqqkqqqq pssqfsfqqp lqqyplgqgs
frpsqqnpqa qgsvqpqqlp 251 qfeeirnlal qtlpamcnvy ippyctiapf
gifgtn
TABLE-US-00003 TABLE 3 Amino acid sequence of alpha/beta-gliadin
precursor from Triticum aestivum (NCBI accession no. AAA34280, (SEQ
ID NO: 167)) 1 mktflilvll aivattatta vrfpvpqlqp qnpsqqqpqe
qvplvqqqqf 51 lgqqqpfppq qpypqpqpfp sqlpylqlqp fpqpqlpysq
pqpfrpqqpy 101 pqpqpqysqp qqpisqqqqq qqqqqqqqqq qqqilqqilq
qqlipcmdvv 151 lqqhniahgr sqvlqqstyq llgelccqhl wqipeqsqcq
aihnvvhaii 201 lhqqqkqqqq pssqvsfqqp lqqyplgqgs frpsqqnpqa
qgsvqpqqlp 251 qfeeirnlal qtlpamcnvy ippyctiapf gifgtn
TABLE-US-00004 TABLE 4 Amino acid sequence of Gamma-gliadin
precursor from Triticum aestivum (NCBI accession no. P21292, (SEQ
ID NO: 168)) 1 mktlliltil amattiatan mqvdpsgqvq wpqqqpfpqp
qqpfcqqpqr 51 tipqphqtfh hqpqqtfpqp qqtyphqpqq qfpqtqqpqq
pfpqpqqtfp 101 qqpqlpfpqq pqqpfpqpqq pqqpfpqsqq pqqpfpqpqq
qfpqpqqpqq 151 sfpqqqqpai qsflqqqmnp cknfllqqcn hvslvsslvs
iilprsdcqv 201 mqqqccqqla qipqqlqcaa ihsvahsiim qqeqqqgvpi
lrplfglaqg 251 lgiiqpqqpa qlegirslvl ktlptmcnvy vppdcstinv
pyanidagig 301 gq
TABLE-US-00005 TABLE 5 Amino acid sequence of Gamma-gliadin B
precursor from Triticum aestivum (NCBI accession no. P06659, (SEQ
ID NO: 169)) 1 mktlliltil amaitiatan mqadpsgqvq wpqqqpflqp
hqpfsqqpqq 51 ifpqpqqtfp hqpqqqfpqp qqpqqqflqp rqpfpqqpqq
pypqqpqqpf 101 pqtqqpqqpf pqskqpqqpf pqpqqpqqsf pqqqpsliqq
slqqqlnpck 151 nfllqqckpv slvsslwsii lppsdcqvmr qqccqqlaqi
pqqlqcaaih 201 svvhsiimqq eqqeqlqgvq ilvplsqqqq vgqgilvqgq
giiqpqqpaq 251 levirslvlq tlptmcnvyv ppycstirap fasivasigg q
TABLE-US-00006 TABLE 6 Amino acid sequence of Gamma-gliadin
(Gliadin B-III) from Tritieum aestivum (NCBI accession no. P04730,
(SEQ ID NO: 170)) 1 pqqpfplqpq qsflwqsqqp flqqpqqpsp qpqqvvqiis
patpttipsa 51 gkptsapfpq qqqqhqqlaq qqipvvqpsi lqqlnpckvf
lqqqcspvam 101 pqrlarsqml qqsschvmqq qccqqlpqip qqsryqaira
iiysiilqeq 151 qqvqgsiqsq qqqpqqlgqc vsqpqqqsqq qlgqqpqqqq
laqgtflqph 201 qiaqlevmts ialrilptmc svnvplyrtt tsvpfgvgtg vgay
TABLE-US-00007 TABLE 7 Amino acid sequence of Gamma-gliadin
precursor from Triticum aestivum (NCBI accession no. P08453, (SEQ
ID NO: 171)) 1 mktlliltil amaitigtan iqvdpsgqvq wlqqqlvpql
qqplsqqpqq 51 tfpqpqqtfp hqpqqqvpqp qqpqqpflqp qqpfpqqpqq
pfpqtqqpqq 101 pfpqqpqqpf pfpqqpqqpf pqqpqqpfpq tqqpqqpfpq
lqqpqqpfpq 151 pqqqlpqpqq pqqsfpqqqr pfiqpslqqq lnpcknillq
qskpaslvss 201 lwsiiwpqsd cqvmrqqccq qlaqipqqlq caaihsvvhs
iimqqqqqqq 251 qqqgidiflp lsqheqvgqg slvqgqgiiq pqqpaqleai
rslvlqtlps 301 mcnvyvppec simrapfasi vagiggq
TABLE-US-00008 TABLE 8 Amino acid sequence of Gamma-gliadin B-I
precursor from Triticum aestivum (NCBI accession no. P04729, (SEQ
ID NO: 172)) 1 mktflvfali avvatsaiaq metscisgle rpwqqqplpp
qqsfsqqppf 51 sqqqqqplpq qpsfsqqqpp fsqqqpilsq qppfsqqqqp
vlpqqspfsq 101 qqqlvlppqq qqqqlvqqqi pivqpsvlqq lnpckvflqq
qcspvampqr 151 larsqmwqqs schvmqqqcc qqlqqipeqs ryeairaiiy
siilqeqqqg 201 fvqpqqqqpq qsgqgvsqsq qqsqqqlgqc sfqqpqqqlg
qqpqqqqqqq 251 vlqgtflqph qiahleavts ialrtlptmc svnvplysat
tsvpfgvgtg 301 vgay
TABLE-US-00009 TABLE 9 Amino acid sequence of Gamma-gliadin
precursor from Triticum aestivum (NCBI accession no. P08079, (SEQ
ID NO: 173)) 1 mktlliltil amaitigtan mqvdpssqvg wpqqqpvpqp
hqpfsqqpqq 51 tfpqpqqtfp hqpqqqfpqp qqpqqqflqp qqpfpqqpqq
pypqqpqqpf 101 pqtqqpqqlf pqsqqpqqqf sqpqqqfpqp qqpqqsfpqq
qppfiqpslq 151 qqvnpcknfl lqqckpvslv sslwsmiwpq sdcqvmrqqc
cqqlaqipqq 201 lqcaaihtii hsiimqqeqq eqqqgmhill plyqqqqvgq
gtivqgqgii 251 q
TABLE-US-00010 TABLE 10 Amino acid sequence of Alpha/beta-gliadin
MM1 precursor (Prolamin) from Triticum aestivum (NCBI accession no.
P18573, (SEQ ID NO: 174)) 1 mktflilall aivattaria vrvpvpqlqp
qnpsqqqpqe qvplvqqqqf 51 pgqqqpfppq qpypqpqpfp sqqpylqlqp
fpqpqlpypq pqlpypqpql 101 pypqpqpfrp qqpypqsqpq ysqpqqpisq
qqqqqqqqqq qkqqqqqqqq 151 ilqqilqqql iperdvvlqq hsiaygssqv
lqqstyqlvq qlccqqlwqi 201 peqsrcqaih nvvhaiilhq qqqqqqqqqq
qplsqvsfqq pqqqypsgqg 251 sfqpsqqnpq aqgsvqpqql pqfeeirnla
letlpamcnv yippyctiap 301 vgifgtn
TABLE-US-00011 TABLE 11 Amino acid sequence of Alpha/beta-gliadin
clone PTO-A10 (Prolamin) from Triticum aestivum (NCBI accession no.
P04728, (SEQ ID NO: 175)) 1 pqpqpqysqp qqpisqqqqq qqqqqqqqqq
eqqilqqilq qqlipcmdvv 51 lqqhniahgr sqvlqqstyq llqelccqhl
wqipeqsqcq aihnvvhaii 101 lhqqqqkqqq qpssqfsfqq plqqyplgqg
sfrpsqqnpq aqgsvqpqql 151 pqfeirnlal qtlpamcnvy ippyctiapf
gifgtn
TABLE-US-00012 TABLE 12 Amino acid sequence of Alpha/beta-gliadin
clone PW8142 precursor (Prolamin) from Triticum aestivum (NCBI
accession no. P04727, (SEQ ID NO: 176)) 1 mktflilalv attattavry
pvpqlqpknp sqqqpqeqvp lvqqqqfpgq 51 qqqfppqqpy pqpqpfpsqq
pylqlqpfpq pqpflpqlpy pqpqsfppqq 101 pypqqrpkyl qpqqpisqqq
aqqqqqqqqq qqqqqqqqil qqilqqqlip 151 crdvvlqqhn iahassqvlq
qstyqllqql ccqqllqipe qsrcqaihnv 201 vhaiimhqqe qqqqlqqqqq
qqlqqqqqqq qqqqqpssqv sfqqpqqqyp 251 ssqgsfqpsq qnpqaqgsvq
pqqlpqfaei rnlalqtlpa mcnvyipphc 301 sttiapfgif gtn
TABLE-US-00013 TABLE 13 Amino acid sequence of Alpha/beta-gliadin
clone PW1215 precursor (Prolamin) from Triticum aestivum (NCBI
accession no. P04726, (SEQ ID NO: 177)) 1 mktflilall aivattatta
vrvpvpqpqp qnpsqpqpqg qvplvqqqqf 51 pgqqqqfppq qpypqpqpfp
sqqpylqlqp fpqpqpfppq lpypqpppfs 101 pqqpypqpqp qypqpqqpis
qqqaqqqqqq qqqqqqqqqq qqilqqilqq 151 qliperdvvl qqhniahars
qvlqqstyqp lqqlccqqlw qipeqsrcqa 201 ihnvvhaiil hqqqrqqqps
sqvslqqpqq qypsgqgffq psqqnpqaqg 251 svqpqqlpqf eeirnlalqt
lprmcnvyip pycsttiapf gifgtn
TABLE-US-00014 TABLE 14 Amino acid sequence of Alpha/beta-gliadin
A-IV precursor (Prolamin) from Triticum aestivum (NCBI accession
no. P04724, (SEQ ID NO: 178)) 1 mktflilalr aivattatia vrvpvpqlqp
qnpsqqqpqk qvplvqqqqf 51 pgqqqpfppq qpypqqqpfp sqqpymqlqp
fpqpqlpypq pqlpypqpqp 101 frpqqsypqp qpqysqpqqp isqqqqqqqq
qqqqqqqilq qilqqqlipc 151 rdvvlqqhsi ahgssqvlqq styqlvqqfc
cqqlwqipeq srcqaihnvv 201 haiilhqqqq qqqqqqqqqq qplsqvcfqq
sqqqypsgqg sfqpsqqnpq 251 aggsvqpqql pqfeeirnla letlpamcnv
yippyctiap vgifgtn
TABLE-US-00015 TABLE 15 Amino acid sequence of Alpha/beta-gliadin
A-III precursor (Prolamin) from Triticum aestivum (NCBI accession
no. P04723, (SEQ ID NO: 179)) 1 mktflilall aivattatsa vrvpvpqlqp
qnpsqqqpqe qvplmqqqqg 51 fpgqqeqfpp qqpyphqqpf psqqpypqpq
pfppqlpypq tqpfppqqpy 101 pqpqpqypqp qqpisqqqaq qqqqqqqtlq
gilqqqlipc rdvvlqqhni 151 ahassqvlqq ssyqqlqqlc cqqlfqipeq
srcgaihnvv haiilhhhqq 201 qqqqpssqvs yqqpgegyps gqvsfqssqq
npgaggsvqp qqlpqfgeir 251 nlalgtlpam cnvyippycs ttiapfgifg tn
TABLE-US-00016 TABLE 16 Amino acid sequence of Alpha/beta-gliadin
A-II precursor (Prolamin) from Triticum aestivum (NCBI accession
no. P04722, (SEQ ID NO: 180)) 1 mktfpilall aivattatta vrvpvpqlql
qnpsqqqpqe qvplvqeqqf 51 qgqqqpfppq qpypqpqpfp sqqpylqlqp
fpqpqlpypq pqpfrpqqpy 101 pqpqpqysqp qqpisqqqqq qqqqqqqqqq
ilqqilqqql ipcrdvvlqq 151 hniahgssqv lqestyqlvq qlccqqlwqi
peqsrcqaih nvvhaiilhq 201 qhhhhqqqqq qqqqqplsqv sfqqpqqqyp
sgqgffqpsq qnpqaqgsfq 251 pqqlpqfeei rnlalqtlpa mcnvyippyc
tiapfgifgt n
TABLE-US-00017 TABLE 17 Amino acid sequence of Alpha/beta-gliadin
A-I precursor (Prolamin) from Triticum aestivum (NCBI accession no.
P04721, (SEQ ID NO: 181)) 1 mktflilall aivattatta vrvpvpqlqp
qnpsqqqpqe qvplvqqqqf 51 lgqqqpfppq qpypqpqpfp sqqpylqlqp
flqpqlpysq pqpfrpqqpy 101 pqpqpqysqp qqpisqqqqq qqqqqqqqqq
qqqqiiqqil qqqlipcmdv 151 vlqqhnivhg ksqvlqqsty qllgelccqh
lwqipeqsqc qaihnvvhai 201 ilhqqqkqqq qpssqvsfqq plqqyplgqg
sfrpsqqnpq aggsvqpqql 251 pqfeeirnla rk
TABLE-US-00018 TABLE 18 Amino acid sequence of gamma gliadin from
Triticum aestivum (NCBI accession no. AAQ63860, (SEQ ID NO: 182)) 1
mniqvdpssq vpwpqqqpfp qphqpfsqqp qqtfpqpqqt fphqpqqqfs 51
qpqqpqqqfi qpqqpfpqqp qqtypqrpqq pfpqtqqpqq pfpqsqqpqq 101
pfpqpqqqfp qpqqpqqsfp qqqpsliqqs lqqqlnpckn fllqqckpvs 151
lvsslwsmil prsdcqvmrq qccqqlaqip qqlqcaaihs ivhsiimqqe 201
qqeqrqgvqi lvplsqqqqv gqgtivqgqg iiqpqqpaql evirslvlqt 251
latmcnvyvp pycstirapf asivagiggq yr
TABLE-US-00019 TABLE 19 Amino acid sequence of Omega-gliadin from
Triticum monococcum (NCBI accession no. P02865, (SEQ ID NO: 183)) 1
arqlnpsdqe lqspqqlypq qpypqqpy
[0109] Inhibitors of gliadin-derived peptide translocation across
biological barriers are useful for treating diseases characterized
by inflammation, including autoimmune diseases and particularly
including celiac disease. Inhibitors of PTG-induced factors that
increase biological barrier permeability are useful for treating
diseases characterized by inflammation, including autoimmune
diseases and particularly including celiac disease.
[0110] Activity of inhibitors of gliadin-derived peptide
translocation and/or inhibitors of PTG-induced permeability can be
measured by any means known in the art. Signaling events which can
be determined include decrease in TEER, increase in LY
permeability, increase in cytokine release, microglial recruitment,
tyrosine kinase phosphorylation and chemotaxis, and increase in
MMP-2 and MMP-9 gelatinolytic activity in cell-conditioned
media.
[0111] The invention provides methods of identifying agents,
compounds or lead compounds for agents active in inhibiting
PTG-induced alterations in biological barrier permeability and/or
peptide translocation. Generally, screening methods of the
invention involve assaying for compounds which modulate the
interaction of one or more gliadin fragments with one or more cells
(e.g., epithelial cells, immune cells). A wide variety of assays
for binding agents is provided including labeled in vitro
protein-ligand binding assays, cell based assays, immunoassays,
etc. A wide variety of formats may be used, including
co-immunoprecipitation, 2-hybrid transactivation, fluorescent
polarization, NMR, fluorescent resonance energy transfer (FRET),
transcriptional activation, etc. For example, a wide variety of
NMR-based methods are available to rapidly screen libraries of
small compounds for binding to protein targets (Hajduk, P. J., et
al. Quarterly Reviews of Biophysics, 1999. 32 (3): 211-40). In some
embodiments, methods of the invention may be automated (e.g., high
throughput screening) and may be used to screen chemical libraries
for lead compounds. Identified compounds may be used to treat
diseases involving increased biological barrier permeability
including, for example, celiac disease, inflammatory bowel diseases
and autoimmune diseases. Compounds identified by the methods of the
invention may be further optimized to modulate biological barrier
modulation, for example, may be derivatized. Multiple iterations of
screening and derivatization may be employed to optimize the
modulation of biological barrier permeability.
[0112] In vitro ligand binding assays employ a mixture of
components including one or more gliadin-derived peptides or
fragments and one or more gliadin binding components.
Gliadin-derived peptides or fragments may be provided as fusion
proteins (e.g., with purification tags such as 6-His). Assay
mixtures typically further comprise a compound to be tested for
inhibitory activity. Compounds to be tested may be of any kind
known to those skilled in the art, for example, may be organic
compounds, peptides, proteins, nucleic acids, lipids, carbohydrates
and mixtures thereof. A variety of other reagents may also be
included in the mixture including, but not limited to, salts,
buffers, neutral proteins, e.g. albumin, detergents, protease
inhibitors, nuclease inhibitors, antimicrobial agents, etc.
[0113] In general, assay mixtures may be incubated under conditions
in which, but for the presence of the compound to be tested,
gliadin-derived peptides or fragments specifically bind the gliadin
binding components with a reference binding affinity. The mixture
components can be added in any order that provides for the
requisite bindings and incubations may be performed at any
temperature which facilitates optimal binding. Incubation periods
are likewise selected for optimal binding. In some embodiments,
incubation periods may be minimized to facilitate rapid,
high-throughput screening.
[0114] After incubation, the effect of the compound to be tested on
the gliadin binding may be detected by any convenient way. For
example, the gliadin-derived peptide or fragment or the gliadin
binding component may be immobilized, and the other labeled; then
in a solid-phase format, any of a variety of methods may be used to
detect the label depending on the nature of the label and other
assay components, e.g. through optical or electron density,
radiative emissions, nonradiative energy transfers, etc. or
indirectly detected with antibody conjugates, etc.
[0115] A difference in the binding affinity of the gliadin-derived
peptide or fragment and the gliadin binding component in the
absence of the compound to be tested as compared with the binding
affinity in the presence of the compound to be tested indicates
that the compound modulates the binding of the gliadin-derived
peptide or fragment and the gliadin binding component. A
difference, as used herein, is statistically significant and
preferably represents at least a 50%, 60%, 70%, 80%, or 90%
difference.
[0116] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
[0117] Measurement Of Trans Epithelial Electric Resistance (TEER)
And Epithelial Flux Of A Fluorescent Marker Lucifer Yellow
[0118] CaCo2 cells form monolayers that exhibit tight junctions
between adjacent cells. Agonists of tight junctions can be
identified by their ability to enhance the flux of compounds (e.g.
ions, Lucifer Yellow) through a cell monolayer that comprises tight
junctions; or by their ability to reduce TEER across a cell
monolayer that comprises tight junctions. Treatment of CaCo2
monolayers with peptide tight junction agonist compounds leads to
enhancement of Lucifer Yellow permeability through CaCo2 monolayers
compared to vehicle alone. Treatment of CaCo2 monolayers with
peptide tight junction agonist compounds leads to a decrease in
TEER across CaCo2 monolayers compared to vehicle alone.
[0119] Tight junction agonists and agonists of the C1orf43 and
CCDC78 proteins can be identified using the following method, and
this method may be easily modified to identify antagonists and
inhibitors of the C1orf43 and CCDC78 proteins:
[0120] Determination of TEER and Lucifer Yellow Flux
[0121] Prepare Modified Hank's Balanced Salt Solution (MHBSS) by
obtaining 1 L bottle of HBSS removing 10 ml of HBSS and replacing
it with 10 ml HEPES buffer pH 7.0. Adjust pH to 7.4.+-.0.1 using
concentrated NaOH (ION).
[0122] Remove CaCo-2 cells from incubator, grown on 12-well, 3.0
.mu.M, polycarbonate Transwell.RTM. filters (Corning) and record
passage#, date cells seeded and age in days.
[0123] Aspirate cell culture medium from both the apical (AP) and
basolateral (BL) compartments, replacing with 0.5 ml and 1.5 ml of
MHBSS, respectively. Incubate cells at 37.degree. C. for 30
minutes.
[0124] Using the MilliCell.RTM.-ERS instrument (Millipore), measure
and record the transepithelial electrical resistance (TEER) across
each filter and record.
[0125] Aspirate solution from the apical compartment of each filter
(n=3 per condition) and replace with 0.5 ml of control and test
solutions containing Lucifer Yellow and test compound if
appropriate.
[0126] Place all plates into incubator set at 37.degree. C.
(.+-.0.2), 50 RPM (.+-.5) for a total of 180 minutes.
[0127] At t=30, 60, 120 and 180 minutes, measure and record the
transepithelial electrical resistance (TEER) across each filter
using the MilliCell-ERS instrument.
[0128] At t=60, 120 and 180 minutes remove 100 .mu.l from each
basolateral compartment and place it in a 96-well plate for Lucifer
Yellow analysis, replace with 100 .mu.l of MHBSS.
[0129] Make a Lucifer Yellow standard curve with the following
dilutions (7500 .mu.M, 3750 .mu.M, 750 .mu.M, 375 .mu.M, 75 .mu.M,
37.5 .mu.M, 7.5 .mu.M, 3.75 .mu.M, 0.75 .mu.M) and pipette 100
.mu.L of each into a 96-well plate except for the first three
standards mentioned above which require a 1:10 dilutions prior to
transferring to the 96-well plate.
[0130] Harvest the remaining start solutions and what is left in
each apical compartment into 1.5 ml vials. Freeze at -20.degree. C.
for future analysis.
[0131] Analyze each 96-well plate in a Tecan Spectra Fluor Plus
using Magellan at 485 and 535 nm.
[0132] Materials:
[0133] Cells: CaCo-2 cells passage 40-60 grown on Transwell.RTM.
plates for 21-28 days
[0134] Culture Medium: DMEM supplemented with 10% fetal bovine
serum, 1% NEAA, 1% Penn/Strep
[0135] Buffers: Hank's Balanced Salt Solution (HBSS) without
calcium and magnesium
[0136] Flasks: 100.times.20 mm Tissue culture dish Falcon.
[0137] Plates: 12 well polycarbonate Transwell.RTM. filters; 0.3 uM
pore size
Example 2
Identification of Cytokines Upregulated on Treatment of THP-1 Cells
by PT-Gliadin (PTG)
[0138] The monocytic cell line THP-1 was used to characterize the
profile of cytokines whose expression was upregulated on exposure
to protease treated gliadin (PTG). THP-1 cells were diluted to
5.times.10.sup.5 cells/ml in RPMI medium supplemented with 10% heat
inactivated fetal bovine serum.
[0139] 5.times.10.sup.5 (1 ml) cells were plated in each well of a
12 well plate, and cells were incubated at 37.degree. C. overnight.
Test compounds (PTG 1 mg/ml; LPS 1 .mu.g/ml) were added to the
cultures, and incubation was continued a further 18 hours at
37.degree. C.
[0140] Culture supernatants were harvested, and
cytokines/chemokines were measured in each sample using a
nitrocellulose membrane based proteomic profiler assay (R&D
Systems). Assays were performed in triplicate. The cytokines
screened in this assay included C5a, CD40 ligand, G-CSF, GM-CSF,
GRO-.alpha./CXCL1, I-309/CCL1, ICAM-1, IFN.gamma., IL-1.alpha.,
IL-1.beta., IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70,
IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32.alpha.,
IP-10/CXCL10, I-TAC/CXCL11, MCP-1/CCL2, MIF, MIP-1.alpha./CCL3,
MIP-1.beta./CCL3, RANTES/CCL5, SDF-1/CXCL12, Serpin-E1/PAI-1,
TNF.alpha., and TREM-1.
[0141] After 6 hours of PTG exposure THP-1 cells demonstrated
increased expression of the cytokines IL-8, MIP-1.alpha.,
MIP-1.beta., TNF-.alpha. and Gro-.alpha.. After 24 hours of
exposure to PTG increased expression of RANTES and MIF were also
observed.
Example 3
Identification of Cytokines Upregulated on Treatment of PBMCs by
PT-Gliadin (PTG)
[0142] Peripheral blood mononuclear cells were isolated from
donated human blood samples using methods known in the art, and
these PBMCs were used to characterize the profile of cytokines
whose expression was upregulated on exposure to protease treated
gliadin (PTG). PBMCs were suspended in RPMI medium supplemented
with 5% heat inactivated human AB serum, and 2.times.10.sup.5 cells
were plated in each well of a 96 well plate. Cells were incubated
at 37.degree. C. with PTG (1 mg/ml) or LPS (1 .mu.g/ml) in the
presence or absence of test compounds being examined for the
ability to suppress cytokine production. Supernatant samples were
harvested following treatment, and cytokines were assayed by ELISA
(R&D Systems).
[0143] Expression of IL-6, IL-8, MIP-1.alpha., and Gro-.alpha. were
induced by treatment with LPS and PTG. Expression of these
cytokines was not reduced by treatment with peptide GGVLVQPG (SEQ
ID NO:1).
[0144] Increased expression of GM-CSF and IL-16 was induced by
exposure to LPS and PTG. This increased expression of these
cytokines was inhibited by treatment with peptide GGVLVQPG (SEQ ID
NO:1).
[0145] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims. All publications, patents and patent
applications mentioned in this specification are indicative of the
level of skill of those skilled in the art to which this invention
pertains, and arc herein incorporated by reference to the same
extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
TABLE-US-00020 TABLE 20 Peptide permeability inhibitors Prevented
Reduced SEQ TEER LY Per- ID NO: Sequence Reduction meability 1
Gly-Gly-Val-Leu-Val-Gln-Pro-Gly - + 2
Ala-Gly-Val-Leu-Val-Gln-Pro-Gly - + 3
Gly-Ala-Val-Leu-Val-Gln-Pro-Gly - + 4
Gly-Gly-Ala-Leu-Val-Gln-Pro-Gly - + 5
Gly-Gly-Val-Ala-Val-Gln-Pro-Gly - + 6
Gly-Gly-Val-Leu-Ala-Gln-Pro-Gly - - 7
Gly-Gly-Val-Leu-Val-Ala-Pro-Gly - - 8
Gly-Gly-Val-Leu-Val-Gln-Ala-Gly - - 9
Gly-Gly-Val-Leu-Val-Gln-Pro-Ala - - 10
Gly-Asp-Val-Leu-Val-Gln-Pro-Gly + + 11
Gly-Glu-Val-Leu-Val-Gln-Pro-Gly + + 12
Gly-Gln-Val-Leu-Val-Gln-Pro-Gly + + 13
Gly-Phe-Val-Leu-Val-Gln-Pro-Gly + + 14
Gly-His-Val-Leu-Val-Gln-Pro-Gly + + 15
Gly-Arg-Val-Leu-Val-Gln-Pro-Gly + + 16
Gly-Lys-Val-Leu-Val-Gln-Pro-Gly + + 17
Gly-Ile-Val-Leu-Val-Gln-Pro-Gly + + 18
Gly-Trp-Val-Leu-Val-Gln-Pro-Gly - - 19
Gly-Pro-Val-Leu-Val-Gln-Pro-Gly + + 20
Gly-Val-Val-Leu-Val-Gln-Pro-Gly + + 21
Gly-Leu-Val-Leu-Val-Gln-Pro-Gly + + 22
Gly-Asn-Val-Leu-Val-Gln-Pro-Gly + + 23
Gly-Thr-Val-Leu-Val-Gln-Pro-Gly + + 24
Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly - - 25
Gly-Gly-Leu-Leu-Val-Gln-Pro-Gly - - 26
Gly-Gly-Ile-Leu-Val-Gln-Pro-Gly - - 27
Gly-Gly-Phe-Leu-Val-Gln-Pro-Gly + + 28
Gly-Gly-Arg-Leu-Val-Gln-Pro-Gly - - 29
Gly-Gly-Asp-Leu-Val-Gln-Pro-Gly - - 30
Gly-Gly-Gln-Leu-Val-Gln-Pro-Gly - - 31
Gly-Gly-His-Leu-Val-Gln-Pro-Gly - - 32
Gly-Gly-Met-Leu-Val-Gln-Pro-Gly + + 33
Gly-Gly-Ser-Leu-Val-Gln-Pro-Gly - - 34
Gly-Gly-Thr-Leu-Val-Gln-Pro-Gly + + 35
Gly-Gly-Pro-Leu-Val-Gln-Pro-Gly - - 36
Gly-Gly-Val-Gly-Val-Gln-Pro-Gly + + 37
Gly-Gly-Val-Val-Val-Gln-Pro-Gly - - 38
Gly-Gly-Val-Ile-Val-Gln-Pro-Gly - - 39
Gly-Gly-Val-Phe-Val-Gln-Pro-Gly - - 40
Gly-Gly-Val-Arg-Val-Gln-Pro-Gly - - 41
Gly-Gly-Val-Asp-Val-Gln-Pro-Gly - - 42
Gly-Gly-Val-Gln-Val-Gln-Pro-Gly - - 43
Gly-Gly-Val-His-Val-Gln-Pro-Gly - - 44
Gly-Gly-Val-Met-Val-Gln-Pro-Gly - - 45
Gly-Gly-Val-Ser-Val-Gln-Pro-Gly - - 46
Gly-Gly-Val-Thr-Val-Gln-Pro-Gly - - 47
Gly-Gly-Val-Pro-Val-Gln-Pro-Gly - - 48
D-Ala-Gly-Val-Leu-Val-Gln-Pro-Gly + + 49
Asp-Gly-Val-Leu-Val-Gln-Pro-Gly + + 50
Glu-Gly-Val-Leu-Val-Gln-Pro-Gly - - 51
Gln-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 52
Phe-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 53
His-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 54
Arg-Gly-Val-Leu-Val-Gln-Pro-Gly - - 55
Lys-Gly-Val-Lcu-Val-Gln-Pro-Gly + + 56
Ile-Gly-Val-Leu-Val-Gln-Pro-Gly - - 57
Trp-Gly-Val-Leu-Val-Gln-Pro-Gly - - 58
Pro-Gly-Val-Leu-Val-Gln-Pro-Gly + + 59
Val-Gly-Val-Leu-Val-Gln-Pro-Gly - - 60
Leu-Gly-Val-Leu-Val-Gln-Pro-Gly - - 61
Thr-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 62
Asn-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 63
D-Phe-Gly-Val-Leu-Val-Gln-Pro-Gly - - 64
Cha-Gly-Val-Leu-Lav-Gln-Pro-Gly NT NT 65
Met(O)2-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 66
Gly-Val-Leu-Val-Gln-Pro-Gly - - 67 Val-Leu-Val-Gln-Pro-Gly + + 68
Leu-Val-Gln-Pro-Gly + + 69 Val-Gln-Pro-Gly + + 70 Gln-Pro-Gly + +
71 Gly-Gly-Val-Leu-Val-Gln-Pro - + 72 Gly-Gly-Val-Leu-Val-Gln + +
73 Gly-Gly-Val-Leu-Val + + 74 Gly-Gly-Val-Leu + + 75 Gly-Gly-Val +
+ 76 Gly-Gly-D-Val-Leu-Val-Gln-Pro-Gly + + 77
Gly-Gly-Val-D-Leu-Val-Gln-Pro-Gly + + 78
Gly-Gly-Val-Leu-D-Val-Gln-Pro-Gly - - 79
Gly-Gly-Val-Leu-Val-D-Gln-Pro-Gly + + 80
Gly-Gly-Val-Leu-Val-Gln-D-Pro-Gly + + 81
Gly-D-Pro-D-Gln-D-Val-D-Leu-D-Val- + + Gly-Gly 82
Gly-D-Pro-D-Gln-D-Val-D-Leu-Val-Gly- + + Gly 83
Gly-D-Pro-D-Gln-D-Val-Leu-D-Val-Gly- + + Gly 84
Gly-D-Pro-D-Gln-Val-D-Leu-D-Val-Gly- + + Gly 85
Gly-D-Pro-Gln-D-Val-D-Leu-D-Val-Gly- + + Gly 86
Gly-Pro-D-Gln-D-Val-D-Leu-D-Val-Gly- - - Gly 87
Gly-Pro-Gln-Val-Leu-Val-Gly-Gly + + 88
Gly-D-Pro-Gln-Val-Leu-Val-Gly-Gly + + 89
Gly-Pro-D-Gln-Val-Leu-Val-Gly-Gly - - 90
Gly-Pro-Gln-D-Val-Leu-Val-Gly-Gly - - 91
Gly-Pro-Gln-Val-D-Leu-Val-Gly-Gly + + 92
Gly-Pro-Gln-Val-Leu-D-Val-Gly-Gly + + 93
Gly-Gly-D-Val-D-Leu-D-Val-D-Gln-D- Pro-Gly 94
Gly-Gly-D-Val-D-Leu-D-Val-D-Gln-Pro- + - Gly 95
Gly-Gly-D-Val-D-Leu-D-Val-Gln-D-Pro- - - Gly 96
Gly-Gly-D-Val-D-Leu-Val-D-Gln-D-Pro- - - Gly 97
Gly-Gly-D-Val-Leu-D-Val-D-Gln-D-Pro- - - Gly 98
Gly-Gly-Val-D-Leu-D-Val-D-Gln-D-Pro- + + Gly 99
Gly-D-Phe-Val-Leu-Val-Gln-Pro-Gly + + 100 Ala-Pro-Gly + + 101
Gln-Ala-Gly + + 102 Gln-Pro-Ala + + 103 (d)Gln-Pro-Gly + + 104
Gln-(d)Pro-Gly + + 105 (d)G1n-(d)Pro-Gly - - 106 Gly-Pro-Gln + +
107 Gly-(d)Pro-Gln - - 108 Gly-Pro-(d)Gln - - 109 Gly-(d)Pro-(d)Gln
- - 110 Ala-Pro-Gly + + 111 His-Pro-Gly + + 112 Asp-Pro-Gly - - 113
Arg-Pro-Gly + + 114 Phe-Pro-Gly + + 115 Gly-Pro-Gly + + 116
Glu-Pro-Gly + +
117 Lys-Pro-Gly + + 118 Leu-Pro-Gly + + 119 Met-Pro-Gly + + 120
Asn-Pro-Gly + + 121 Ser-Pro-Gly + + 122 Tyr-Pro-Gly + + 123
Thr-Pro-Gly - + 124 Ile-Pro-Gly + + 125 Trp-Pro-Gly + + 126
Pro-Pro-Gly - - 127 Val-Pro-Gly - + 128 Glp-Pro-Gly + + 129
Glp-Val-Gly - - 130 Glp-Gln-Gly - - 131 Glp-Ser-Gly - - 132
Glp-Lys-Gly - - 133 Glp-Phe-Gly - - 134 Glp-Glu-Gly - - 135
Glp-Thr-Gly - - 136 Glp-Ile-Gly - - 137 Glp-Tyr-Gly - - 138
Glp-His-Gly - - 139 Glp-Asn-Gly - - 140 Glp-Arg-Gly - - 141
Glp-Gly-Gly - - 142 Glp-Trp-Gly - - 143 Glp-Asp-Gly - - 144
Glp-Met-Gly - - 145 Glp-Leu-Gly - - 146 Glp-Pro-Gln - - 147
Glp-Pro-Asn + - 148 Glp-Pro-Gln - - 149 Glp-Pro-Ser - - 150
Glp-Pro-Pro + - 151 Glp-Pro-Trp - - 152 Glp-Pro-Asp - - 153
Glp-Pro-His - - 154 Glp-Pro-Leu - - 155 Glp-Pro-Arg - - 156
Glp-Pro-Val - - 157 Glp-Pro-Lys - - 158 Glp-Pro-Glu - - 159
Glp-Pro-Phe - - 160 Glp-Pro-Ile + - 161 Glp-Pro-Met + - 162
Glp-Pro-Tyr + - Met(O)2 = Methioninedioxide, Cha = cyclohexyl-Ala
Sequence CWU 1
1
18318PRTArtificial SequencePeptide permeability inhibitor 1Gly Gly
Val Leu Val Gln Pro Gly 1 5 28PRTArtificial SequencePeptide
permeability inhibitor 2Ala Gly Val Leu Val Gln Pro Gly 1 5
38PRTArtificial SequencePeptide permeability inhibitor 3Gly Ala Val
Leu Val Gln Pro Gly 1 5 48PRTArtificial SequencePeptide
permeability inhibitor 4Gly Gly Ala Leu Val Gln Pro Gly 1 5
58PRTArtificial SequencePeptide permeability inhibitor 5Gly Gly Val
Ala Val Gln Pro Gly 1 5 68PRTArtificial SequencePeptide
permeability inhibitor 6Gly Gly Val Leu Ala Gln Pro Gly 1 5
78PRTArtificial SequencePeptide permeability inhibitor 7Gly Gly Val
Leu Val Ala Pro Gly 1 5 88PRTArtificial SequencePeptide
permeability inhibitor 8Gly Gly Val Leu Val Gln Ala Gly 1 5
98PRTArtificial SequencePeptide permeability inhibitor 9Gly Gly Val
Leu Val Gln Pro Ala 1 5 108PRTArtificial SequencePeptide
permeability inhibitor 10Gly Asp Val Leu Val Gln Pro Gly 1 5
118PRTArtificial SequencePeptide permeability inhibitor 11Gly Glu
Val Leu Val Gln Pro Gly 1 5 128PRTArtificial SequencePeptide
permeability inhibitor 12Gly Gln Val Leu Val Gln Pro Gly 1 5
138PRTArtificial SequencePeptide permeability inhibitor 13Gly Phe
Val Leu Val Gln Pro Gly 1 5 148PRTArtificial SequencePeptide
permeability inhibitor 14Gly His Val Leu Val Gln Pro Gly 1 5
158PRTArtificial SequencePeptide permeability inhibitor 15Gly Arg
Val Leu Val Gln Pro Gly 1 5 168PRTArtificial SequencePeptide
permeability inhibitor 16Gly Lys Val Leu Val Gln Pro Gly 1 5
178PRTArtificial SequencePeptide permeability inhibitor 17Gly Ile
Val Leu Val Gln Pro Gly 1 5 188PRTArtificial SequencePeptide
permeability inhibitor 18Gly Trp Val Leu Val Gln Pro Gly 1 5
198PRTArtificial SequencePeptide permeability inhibitor 19Gly Pro
Val Leu Val Gln Pro Gly 1 5 208PRTArtificial SequencePeptide
permeability inhibitor 20Gly Val Val Leu Val Gln Pro Gly 1 5
218PRTArtificial SequencePeptide permeability inhibitor 21Gly Leu
Val Leu Val Gln Pro Gly 1 5 228PRTArtificial SequencePeptide
permeability inhibitor 22Gly Asn Val Leu Val Gln Pro Gly 1 5
238PRTArtificial SequencePeptide permeability inhibitor 23Gly Thr
Val Leu Val Gln Pro Gly 1 5 248PRTArtificial SequencePeptide
permeability inhibitor 24Gly Gly Gly Leu Val Gln Pro Gly 1 5
258PRTArtificial SequencePeptide permeability inhibitor 25Gly Gly
Leu Leu Val Gln Pro Gly 1 5 268PRTArtificial SequencePeptide
permeability inhibitor 26Gly Gly Ile Leu Val Gln Pro Gly 1 5
278PRTArtificial SequencePeptide permeability inhibitor 27Gly Gly
Phe Leu Val Gln Pro Gly 1 5 288PRTArtificial SequencePeptide
permeability inhibitor 28Gly Gly Arg Leu Val Gln Pro Gly 1 5
298PRTArtificial SequencePeptide permeability inhibitor 29Gly Gly
Asp Leu Val Gln Pro Gly 1 5 308PRTArtificial SequencePeptide
permeability inhibitor 30Gly Gly Gln Leu Val Gln Pro Gly 1 5
318PRTArtificial SequencePeptide permeability inhibitor 31Gly Gly
His Leu Val Gln Pro Gly 1 5 328PRTArtificial SequencePeptide
permeability inhibitor 32Gly Gly Met Leu Val Gln Pro Gly 1 5
338PRTArtificial SequencePeptide permeability inhibitor 33Gly Gly
Ser Leu Val Gln Pro Gly 1 5 348PRTArtificial SequencePeptide
permeability inhibitor 34Gly Gly Thr Leu Val Gln Pro Gly 1 5
358PRTArtificial SequencePeptide permeability inhibitor 35Gly Gly
Pro Leu Val Gln Pro Gly 1 5 368PRTArtificial SequencePeptide
permeability inhibitor 36Gly Gly Val Gly Val Gln Pro Gly 1 5
378PRTArtificial SequencePeptide permeability inhibitor 37Gly Gly
Val Val Val Gln Pro Gly 1 5 388PRTArtificial SequencePeptide
permeability inhibitor 38Gly Gly Val Ile Val Gln Pro Gly 1 5
398PRTArtificial SequencePeptide permeability inhibitor 39Gly Gly
Val Phe Val Gln Pro Gly 1 5 408PRTArtificial SequencePeptide
permeability inhibitor 40Gly Gly Val Arg Val Gln Pro Gly 1 5
418PRTArtificial SequencePeptide permeability inhibitor 41Gly Gly
Val Asp Val Gln Pro Gly 1 5 428PRTArtificial SequencePeptide
permeability inhibitor 42Gly Gly Val Gln Val Gln Pro Gly 1 5
438PRTArtificial SequencePeptide permeability inhibitor 43Gly Gly
Val His Val Gln Pro Gly 1 5 448PRTArtificial SequencePeptide
permeability inhibitor 44Gly Gly Val Met Val Gln Pro Gly 1 5
458PRTArtificial SequencePeptide permeability inhibitor 45Gly Gly
Val Ser Val Gln Pro Gly 1 5 468PRTArtificial SequencePeptide
permeability inhibitor 46Gly Gly Val Thr Val Gln Pro Gly 1 5
478PRTArtificial SequencePeptide permeability inhibitor 47Gly Gly
Val Pro Val Gln Pro Gly 1 5 488PRTArtificial SequencePeptide
permeability inhibitor 48Ala Gly Val Leu Val Gln Pro Gly 1 5
498PRTArtificial SequencePeptide permeability inhibitor 49Asp Gly
Val Leu Val Gln Pro Gly 1 5 508PRTArtificial SequencePeptide
permeability inhibitor 50Glu Gly Val Leu Val Gln Pro Gly 1 5
518PRTArtificial SequencePeptide permeability inhibitor 51Gln Gly
Val Leu Val Gln Pro Gly 1 5 528PRTArtificial SequencePeptide
permeability inhibitor 52Phe Gly Val Leu Val Gln Pro Gly 1 5
538PRTArtificial SequencePeptide permeability inhibitor 53His Gly
Val Leu Val Gln Pro Gly 1 5 548PRTArtificial SequencePeptide
permeability inhibitor 54Arg Gly Val Leu Val Gln Pro Gly 1 5
558PRTArtificial SequencePeptide permeability inhibitor 55Lys Gly
Val Leu Val Gln Pro Gly 1 5 568PRTArtificial SequencePeptide
permeability inhibitor 56Ile Gly Val Leu Val Gln Pro Gly 1 5
578PRTArtificial SequencePeptide permeability inhibitor 57Trp Gly
Val Leu Val Gln Pro Gly 1 5 588PRTArtificial SequencePeptide
permeability inhibitor 58Pro Gly Val Leu Val Gln Pro Gly 1 5
598PRTArtificial SequencePeptide permeability inhibitor 59Val Gly
Val Leu Val Gln Pro Gly 1 5 608PRTArtificial SequencePeptide
permeability inhibitor 60Leu Gly Val Leu Val Gln Pro Gly 1 5
618PRTArtificial SequencePeptide permeability inhibitor 61Thr Gly
Val Leu Val Gln Pro Gly 1 5 628PRTArtificial SequencePeptide
permeability inhibitor 62Asn Gly Val Leu Val Gln Pro Gly 1 5
638PRTArtificial SequencePeptide permeability inhibitor 63Phe Gly
Val Leu Val Gln Pro Gly 1 5 648PRTArtificial SequencePeptide
permeability inhibitor 64Xaa Gly Val Leu Xaa Gln Pro Gly 1 5
658PRTArtificial SequencePeptide permeability inhibitor 65Xaa Gly
Val Leu Val Gln Pro Gly 1 5 667PRTArtificial SequencePeptide
permeability inhibitor 66Gly Val Leu Val Gln Pro Gly 1 5
676PRTArtificial SequencePeptide permeability inhibitor 67Val Leu
Val Gln Pro Gly 1 5 685PRTArtificial SequencePeptide permeability
inhibitor 68Leu Val Gln Pro Gly 1 5 694PRTArtificial
SequencePeptide permeability inhibitor 69Val Gln Pro Gly 1
703PRTArtificial SequencePeptide permeability inhibitor 70Gln Pro
Gly 1 717PRTArtificial SequencePeptide permeability inhibitor 71Gly
Gly Val Leu Val Gln Pro 1 5 726PRTArtificial SequencePeptide
permeability inhibitor 72Gly Gly Val Leu Val Gln 1 5
735PRTArtificial SequencePeptide permeability inhibitor 73Gly Gly
Val Leu Val 1 5 744PRTArtificial SequencePeptide permeability
inhibitor 74Gly Gly Val Leu 1 753PRTArtificial SequencePeptide
permeability inhibitor 75Gly Gly Val 1 769PRTArtificial
SequencePeptide permeability inhibitor 76Gly Gly Asp Val Leu Val
Gln Pro Gly 1 5 778PRTArtificial SequencePeptide permeability
inhibitor 77Gly Gly Val Leu Val Gln Pro Gly 1 5 788PRTArtificial
SequencePeptide permeability inhibitor 78Gly Gly Val Leu Val Gln
Pro Gly 1 5 798PRTArtificial SequencePeptide permeability inhibitor
79Gly Gly Val Leu Val Gln Pro Gly 1 5 808PRTArtificial
SequencePeptide permeability inhibitor 80Gly Gly Val Leu Val Gln
Pro Gly 1 5 818PRTArtificial SequencePeptide permeability inhibitor
81Gly Pro Gln Val Leu Val Gly Gly 1 5 828PRTArtificial
SequencePeptide permeability inhibitor 82Gly Pro Gln Val Leu Val
Gly Gly 1 5 838PRTArtificial SequencePeptide permeability inhibitor
83Gly Pro Gln Val Leu Val Gly Gly 1 5 848PRTArtificial
SequencePeptide permeability inhibitor 84Gly Pro Gln Val Leu Val
Gly Gly 1 5 858PRTArtificial SequencePeptide permeability inhibitor
85Gly Pro Gln Val Leu Val Gly Gly 1 5 868PRTArtificial
SequencePeptide permeability inhibitor 86Gly Pro Gln Val Leu Val
Gly Gly 1 5 878PRTArtificial SequencePeptide permeability inhibitor
87Gly Pro Gln Val Leu Val Gly Gly 1 5 888PRTArtificial
SequencePeptide permeability inhibitor 88Gly Pro Gln Val Leu Val
Gly Gly 1 5 898PRTArtificial SequencePeptide permeability inhibitor
89Gly Pro Gln Val Leu Val Gly Gly 1 5 908PRTArtificial
SequencePeptide permeability inhibitor 90Gly Pro Gln Val Leu Val
Gly Gly 1 5 918PRTArtificial SequencePeptide permeability inhibitor
91Gly Pro Gln Val Leu Val Gly Gly 1 5 928PRTArtificial
SequencePeptide permeability inhibitor 92Gly Pro Gln Val Leu Val
Gly Gly 1 5 938PRTArtificial SequencePeptide permeability inhibitor
93Gly Gly Val Leu Val Gln Pro Gly 1 5 948PRTArtificial
SequencePeptide permeability inhibitor 94Gly Gly Val Leu Val Gln
Pro Gly 1 5 958PRTArtificial SequencePeptide permeability inhibitor
95Gly Gly Val Leu Val Gln Pro Gly 1 5 968PRTArtificial
SequencePeptide permeability inhibitor 96Gly Gly Val Leu Val Gln
Pro Gly 1 5 978PRTArtificial SequencePeptide permeability inhibitor
97Gly Gly Val Leu Val Gln Pro Gly 1 5 988PRTArtificial
SequencePeptide permeability inhibitor 98Gly Gly Val Leu Val Gln
Pro Gly 1 5 998PRTArtificial SequencePeptide permeability inhibitor
99Gly Phe Val Leu Val Gln Pro Gly 1 5 1003PRTArtificial
SequencePeptide permeability inhibitor 100Ala Pro Gly 1
1013PRTArtificial SequencePeptide permeability inhibitor 101Gln Ala
Gly 1 1023PRTArtificial SequencePeptide permeability inhibitor
102Gln Pro Ala 1 1033PRTArtificial SequencePeptide permeability
inhibitor 103Gln Pro Gly 1 1043PRTArtificial SequencePeptide
permeability inhibitor 104Gln Pro Gly 1 1053PRTArtificial
SequencePeptide permeability inhibitor 105Gln Pro Gly 1
1063PRTArtificial SequencePeptide permeability inhibitor 106Gly Pro
Gln 1 1073PRTArtificial SequencePeptide permeability inhibitor
107Gly Pro Gln 1 1083PRTArtificial SequencePeptide permeability
inhibitor 108Gly Pro Gln 1 1093PRTArtificial SequencePeptide
permeability inhibitor 109Gly Pro Gln 1 1103PRTArtificial
SequencePeptide permeability inhibitor 110Ala Pro Gly 1
1113PRTArtificial SequencePeptide permeability inhibitor 111His Pro
Gly 1 1123PRTArtificial SequencePeptide permeability inhibitor
112Asp Pro Gly 1 1133PRTArtificial SequencePeptide permeability
inhibitor 113Arg Pro Gly 1 1143PRTArtificial SequencePeptide
permeability inhibitor 114Phe Pro Gly 1 1153PRTArtificial
SequencePeptide permeability inhibitor 115Gly Pro Gly 1
1163PRTArtificial SequencePeptide permeability inhibitor 116Glu Pro
Gly 1 1173PRTArtificial SequencePeptide permeability inhibitor
117Lys Pro Gly 1 1183PRTArtificial SequencePeptide permeability
inhibitor 118Leu Pro Gly 1 1193PRTArtificial SequencePeptide
permeability inhibitor 119Met Pro Gly 1 1203PRTArtificial
SequencePeptide permeability inhibitor 120Asn Pro Gly 1
1213PRTArtificial SequencePeptide permeability inhibitor 121Ser Pro
Gly 1 1223PRTArtificial SequencePeptide permeability inhibitor
122Tyr Pro Gly 1 1233PRTArtificial SequencePeptide permeability
inhibitor 123Thr Pro Gly 1 1243PRTArtificial SequencePeptide
permeability inhibitor 124Ile Pro Gly 1 1253PRTArtificial
SequencePeptide permeability inhibitor 125Trp Pro Gly 1
1263PRTArtificial SequencePeptide permeability inhibitor 126Pro Pro
Gly 1 1273PRTArtificial SequencePeptide permeability inhibitor
127Val Pro Gly 1 1283PRTArtificial SequencePeptide permeability
inhibitor 128Xaa Pro Gly 1 1293PRTArtificial SequencePeptide
permeability inhibitor 129Xaa Val Gly 1 1303PRTArtificial
SequencePeptide permeability inhibitor 130Xaa Gln Gly 1
1313PRTArtificial SequencePeptide permeability inhibitor 131Xaa Ser
Gly 1 1323PRTArtificial SequencePeptide permeability inhibitor
132Xaa Lys Gly 1 1333PRTArtificial SequencePeptide permeability
inhibitor 133Xaa Phe Gly 1 1343PRTArtificial SequencePeptide
permeability inhibitor 134Xaa Glu Gly 1 1353PRTArtificial
SequencePeptide permeability inhibitor 135Xaa Thr Gly 1
1363PRTArtificial SequencePeptide permeability inhibitor 136Xaa Ile
Gly 1 1373PRTArtificial SequencePeptide permeability inhibitor
137Xaa Tyr Gly 1 1383PRTArtificial SequencePeptide permeability
inhibitor 138Xaa His Gly 1 1393PRTArtificial SequencePeptide
permeability inhibitor 139Xaa Asn Gly 1 1403PRTArtificial
SequencePeptide permeability inhibitor 140Xaa Arg Gly 1
1413PRTArtificial SequencePeptide permeability inhibitor 141Xaa Gly
Gly 1 1423PRTArtificial SequencePeptide permeability inhibitor
142Xaa Trp Gly 1 1433PRTArtificial SequencePeptide permeability
inhibitor 143Xaa Asp Gly 1 1443PRTArtificial SequencePeptide
permeability inhibitor 144Xaa Met Gly 1 1453PRTArtificial
SequencePeptide permeability inhibitor 145Xaa Leu Gly 1
1463PRTArtificial SequencePeptide permeability inhibitor 146Xaa Pro
Gln 1 1473PRTArtificial SequencePeptide permeability inhibitor
147Xaa Pro Asn 1 1483PRTArtificial SequencePeptide permeability
inhibitor 148Xaa Pro Gln 1 1493PRTArtificial SequencePeptide
permeability inhibitor 149Xaa Pro Ser 1 1503PRTArtificial
SequencePeptide permeability inhibitor 150Xaa Pro Pro 1
1513PRTArtificial SequencePeptide permeability inhibitor 151Xaa Pro
Trp 1 1523PRTArtificial SequencePeptide permeability inhibitor
152Xaa Pro Asp 1 1533PRTArtificial SequencePeptide permeability
inhibitor 153Xaa Pro His 1 1543PRTArtificial SequencePeptide
permeability inhibitor 154Xaa Pro Leu 1 1553PRTArtificial
SequencePeptide permeability inhibitor 155Xaa Pro Arg 1
1563PRTArtificial SequencePeptide permeability inhibitor 156Xaa Pro
Val 1 1573PRTArtificial SequencePeptide permeability inhibitor
157Xaa Pro Lys 1 1583PRTArtificial SequencePeptide permeability
inhibitor 158Xaa Pro Glu 1 1593PRTArtificial SequencePeptide
permeability inhibitor 159Xaa Pro Phe 1 1603PRTArtificial
SequencePeptide permeability inhibitor 160Xaa Pro Ile 1
1613PRTArtificial SequencePeptide permeability inhibitor 161Xaa Pro
Met 1 1623PRTArtificial SequencePeptide permeability inhibitor
162Xaa Pro Tyr 1 1639PRTTriticum sp. 163Pro Tyr Pro Gln Pro Gln Leu
Pro Tyr 1 5 16413PRTTriticum sp. 164Leu Gly Gln Gln Gln Pro Phe Pro
Pro Gln Gln Pro Tyr 1 5 10 165290PRTTriticum aestivum 165Met Val
Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn Pro Ser Gln 1 5 10 15
Gln
Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln Gln Phe Pro 20 25
30 Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro Gln Pro Gln
35 40 45 Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro Phe
Pro Gln 50 55 60 Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr
Pro Gln Pro Gln 65 70 75 80 Leu Pro Tyr Pro Gln Pro Gln Pro Phe Arg
Pro Gln Gln Pro Tyr Pro 85 90 95 Gln Ser Gln Pro Gln Tyr Ser Gln
Pro Gln Gln Pro Ile Ser Gln Gln 100 105 110 Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Lys Gln Gln Gln Gln Gln 115 120 125 Gln Gln Gln Ile
Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys 130 135 140 Arg Asp
Val Val Leu Gln Gln His Ser Ile Ala Tyr Gly Ser Ser Gln 145 150 155
160 Val Leu Gln Gln Ser Thr Tyr Gln Leu Val Gln Gln Leu Cys Cys Gln
165 170 175 Gln Leu Trp Gln Ile Pro Glu Gln Ser Arg Cys Gln Ala Ile
His Asn 180 185 190 Val Val His Ala Ile Ile Leu His Gln Gln Gln Gln
Gln Gln Gln Gln 195 200 205 Gln Gln Gln Gln Pro Leu Ser Gln Val Ser
Phe Gln Gln Pro Gln Gln 210 215 220 Gln Tyr Pro Ser Gly Gln Gly Ser
Phe Gln Pro Ser Gln Gln Asn Pro 225 230 235 240 Gln Ala Gln Gly Ser
Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Glu 245 250 255 Ile Arg Asn
Leu Ala Leu Glu Thr Leu Pro Ala Met Cys Asn Val Tyr 260 265 270 Ile
Pro Pro Tyr Cys Thr Ile Ala Pro Val Gly Ile Phe Gly Thr Asn 275 280
285 Tyr Arg 290 166286PRTTriticum turgidum subsp. durum 166Met Lys
Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr 1 5 10 15
Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Leu Gln Arg Gln Asn 20
25 30 Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln
Gln 35 40 45 Gln Phe Leu Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln
Pro Tyr Pro 50 55 60 Gln Pro Gln Pro Phe Pro Ser Gln Gln Pro Tyr
Leu Gln Leu Gln Pro 65 70 75 80 Phe Pro Gln Pro Gln Leu Pro Tyr Ser
Gln Pro Gln Pro Phe Arg Pro 85 90 95 Gln Gln Pro Tyr Pro Gln Pro
Gln Pro Arg Tyr Ser Gln Pro Gln Gln 100 105 110 Pro Ile Ser Gln Gln
Gln Gln Gln Gln His Gln Gln His Gln Gln His 115 120 125 His Gln Glu
Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile 130 135 140 Pro
Cys Met Asp Val Val Leu Gln Gln His Asn Ile Ala His Arg Arg 145 150
155 160 Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu Leu Gln Glu Leu
Cys 165 170 175 Cys Gln His Leu Trp Gln Ile Pro Glu Gln Ser Gln Cys
Gln Ala Ile 180 185 190 His Asn Val Val His Ala Ile Ile Pro His Gln
Gln Gln Lys Gln Gln 195 200 205 Gln Gln Pro Ser Ser Gln Phe Ser Phe
Gln Gln Pro Leu Gln Gln Tyr 210 215 220 Pro Leu Gly Gln Gly Ser Phe
Arg Pro Ser Gln Gln Asn Pro Gln Ala 225 230 235 240 Gln Gly Ser Val
Gln Pro Gln Gln Leu Pro Gln Phe Glu Glu Ile Arg 245 250 255 Asn Leu
Ala Leu Gln Thr Leu Pro Ala Met Cys Asn Val Tyr Ile Pro 260 265 270
Pro Tyr Cys Thr Ile Ala Pro Phe Gly Ile Phe Gly Thr Asn 275 280 285
167286PRTTriticum aestivum 167Met Lys Thr Phe Leu Ile Leu Val Leu
Leu Ala Ile Val Ala Thr Thr 1 5 10 15 Ala Thr Thr Ala Val Arg Phe
Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30 Pro Ser Gln Gln Gln
Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln 35 40 45 Gln Phe Leu
Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60 Gln
Pro Gln Pro Phe Pro Ser Gln Leu Pro Tyr Leu Gln Leu Gln Pro 65 70
75 80 Phe Pro Gln Pro Gln Leu Pro Tyr Ser Gln Pro Gln Pro Phe Arg
Pro 85 90 95 Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Ser Gln
Pro Gln Gln 100 105 110 Pro Ile Ser Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln 115 120 125 Gln Gln Gln Gln Gln Ile Leu Gln Gln
Ile Leu Gln Gln Gln Leu Ile 130 135 140 Pro Cys Met Asp Val Val Leu
Gln Gln His Asn Ile Ala His Gly Arg 145 150 155 160 Ser Gln Val Leu
Gln Gln Ser Thr Tyr Gln Leu Leu Gln Glu Leu Cys 165 170 175 Cys Gln
His Leu Trp Gln Ile Pro Glu Gln Ser Gln Cys Gln Ala Ile 180 185 190
His Asn Val Val His Ala Ile Ile Leu His Gln Gln Gln Lys Gln Gln 195
200 205 Gln Gln Pro Ser Ser Gln Val Ser Phe Gln Gln Pro Leu Gln Gln
Tyr 210 215 220 Pro Leu Gly Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn
Pro Gln Ala 225 230 235 240 Gln Gly Ser Val Gln Pro Gln Gln Leu Pro
Gln Phe Glu Glu Ile Arg 245 250 255 Asn Leu Ala Leu Gln Thr Leu Pro
Ala Met Cys Asn Val Tyr Ile Pro 260 265 270 Pro Tyr Cys Thr Ile Ala
Pro Phe Gly Ile Phe Gly Thr Asn 275 280 285 168302PRTTriticum
aestivum 168Met Lys Thr Leu Leu Ile Leu Thr Ile Leu Ala Met Ala Thr
Thr Ile 1 5 10 15 Ala Thr Ala Asn Met Gln Val Asp Pro Ser Gly Gln
Val Gln Trp Pro 20 25 30 Gln Gln Gln Pro Phe Pro Gln Pro Gln Gln
Pro Phe Cys Gln Gln Pro 35 40 45 Gln Arg Thr Ile Pro Gln Pro His
Gln Thr Phe His His Gln Pro Gln 50 55 60 Gln Thr Phe Pro Gln Pro
Gln Gln Thr Tyr Pro His Gln Pro Gln Gln 65 70 75 80 Gln Phe Pro Gln
Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln 85 90 95 Gln Thr
Phe Pro Gln Gln Pro Gln Leu Pro Phe Pro Gln Gln Pro Gln 100 105 110
Gln Pro Phe Pro Gln Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln Ser 115
120 125 Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Gln Phe Pro
Gln 130 135 140 Pro Gln Gln Pro Gln Gln Ser Phe Pro Gln Gln Gln Gln
Pro Ala Ile 145 150 155 160 Gln Ser Phe Leu Gln Gln Gln Met Asn Pro
Cys Lys Asn Phe Leu Leu 165 170 175 Gln Gln Cys Asn His Val Ser Leu
Val Ser Ser Leu Val Ser Ile Ile 180 185 190 Leu Pro Arg Ser Asp Cys
Gln Val Met Gln Gln Gln Cys Cys Gln Gln 195 200 205 Leu Ala Gln Ile
Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Val 210 215 220 Ala His
Ser Ile Ile Met Gln Gln Glu Gln Gln Gln Gly Val Pro Ile 225 230 235
240 Leu Arg Pro Leu Phe Gln Leu Ala Gln Gly Leu Gly Ile Ile Gln Pro
245 250 255 Gln Gln Pro Ala Gln Leu Glu Gly Ile Arg Ser Leu Val Leu
Lys Thr 260 265 270 Leu Pro Thr Met Cys Asn Val Tyr Val Pro Pro Asp
Cys Ser Thr Ile 275 280 285 Asn Val Pro Tyr Ala Asn Ile Asp Ala Gly
Ile Gly Gly Gln 290 295 300 169291PRTTriticum aestivum 169Met Lys
Thr Leu Leu Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile 1 5 10 15
Ala Thr Ala Asn Met Gln Ala Asp Pro Ser Gly Gln Val Gln Trp Pro 20
25 30 Gln Gln Gln Pro Phe Leu Gln Pro His Gln Pro Phe Ser Gln Gln
Pro 35 40 45 Gln Gln Ile Phe Pro Gln Pro Gln Gln Thr Phe Pro His
Gln Pro Gln 50 55 60 Gln Gln Phe Pro Gln Pro Gln Gln Pro Gln Gln
Gln Phe Leu Gln Pro 65 70 75 80 Arg Gln Pro Phe Pro Gln Gln Pro Gln
Gln Pro Tyr Pro Gln Gln Pro 85 90 95 Gln Gln Pro Phe Pro Gln Thr
Gln Gln Pro Gln Gln Pro Phe Pro Gln 100 105 110 Ser Lys Gln Pro Gln
Gln Pro Phe Pro Gln Pro Gln Gln Pro Gln Gln 115 120 125 Ser Phe Pro
Gln Gln Gln Pro Ser Leu Ile Gln Gln Ser Leu Gln Gln 130 135 140 Gln
Leu Asn Pro Cys Lys Asn Phe Leu Leu Gln Gln Cys Lys Pro Val 145 150
155 160 Ser Leu Val Ser Ser Leu Trp Ser Ile Ile Leu Pro Pro Ser Asp
Cys 165 170 175 Gln Val Met Arg Gln Gln Cys Cys Gln Gln Leu Ala Gln
Ile Pro Gln 180 185 190 Gln Leu Gln Cys Ala Ala Ile His Ser Val Val
His Ser Ile Ile Met 195 200 205 Gln Gln Glu Gln Gln Glu Gln Leu Gln
Gly Val Gln Ile Leu Val Pro 210 215 220 Leu Ser Gln Gln Gln Gln Val
Gly Gln Gly Ile Leu Val Gln Gly Gln 225 230 235 240 Gly Ile Ile Gln
Pro Gln Gln Pro Ala Gln Leu Glu Val Ile Arg Ser 245 250 255 Leu Val
Leu Gln Thr Leu Pro Thr Met Cys Asn Val Tyr Val Pro Pro 260 265 270
Tyr Cys Ser Thr Ile Arg Ala Pro Phe Ala Ser Ile Val Ala Ser Ile 275
280 285 Gly Gly Gln 290 170244PRTTriticum aestivum 170Pro Gln Gln
Pro Phe Pro Leu Gln Pro Gln Gln Ser Phe Leu Trp Gln 1 5 10 15 Ser
Gln Gln Pro Phe Leu Gln Gln Pro Gln Gln Pro Ser Pro Gln Pro 20 25
30 Gln Gln Val Val Gln Ile Ile Ser Pro Ala Thr Pro Thr Thr Ile Pro
35 40 45 Ser Ala Gly Lys Pro Thr Ser Ala Pro Phe Pro Gln Gln Gln
Gln Gln 50 55 60 His Gln Gln Leu Ala Gln Gln Gln Ile Pro Val Val
Gln Pro Ser Ile 65 70 75 80 Leu Gln Gln Leu Asn Pro Cys Lys Val Phe
Leu Gln Gln Gln Cys Ser 85 90 95 Pro Val Ala Met Pro Gln Arg Leu
Ala Arg Ser Gln Met Leu Gln Gln 100 105 110 Ser Ser Cys His Val Met
Gln Gln Gln Cys Cys Gln Gln Leu Pro Gln 115 120 125 Ile Pro Gln Gln
Ser Arg Tyr Gln Ala Ile Arg Ala Ile Ile Tyr Ser 130 135 140 Ile Ile
Leu Gln Glu Gln Gln Gln Val Gln Gly Ser Ile Gln Ser Gln 145 150 155
160 Gln Gln Gln Pro Gln Gln Leu Gly Gln Cys Val Ser Gln Pro Gln Gln
165 170 175 Gln Ser Gln Gln Gln Leu Gly Gln Gln Pro Gln Gln Gln Gln
Leu Ala 180 185 190 Gln Gly Thr Phe Leu Gln Pro His Gln Ile Ala Gln
Leu Glu Val Met 195 200 205 Thr Ser Ile Ala Leu Arg Ile Leu Pro Thr
Met Cys Ser Val Asn Val 210 215 220 Pro Leu Tyr Arg Thr Thr Thr Ser
Val Pro Phe Gly Val Gly Thr Gly 225 230 235 240 Val Gly Ala Tyr
171327PRTTriticum aestivum 171Met Lys Thr Leu Leu Ile Leu Thr Ile
Leu Ala Met Ala Ile Thr Ile 1 5 10 15 Gly Thr Ala Asn Ile Gln Val
Asp Pro Ser Gly Gln Val Gln Trp Leu 20 25 30 Gln Gln Gln Leu Val
Pro Gln Leu Gln Gln Pro Leu Ser Gln Gln Pro 35 40 45 Gln Gln Thr
Phe Pro Gln Pro Gln Gln Thr Phe Pro His Gln Pro Gln 50 55 60 Gln
Gln Val Pro Gln Pro Gln Gln Pro Gln Gln Pro Phe Leu Gln Pro 65 70
75 80 Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln Thr
Gln 85 90 95 Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro
Phe Pro Gln 100 105 110 Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Gln
Pro Gln Gln Pro Phe 115 120 125 Pro Gln Thr Gln Gln Pro Gln Gln Pro
Phe Pro Gln Leu Gln Gln Pro 130 135 140 Gln Gln Pro Phe Pro Gln Pro
Gln Gln Gln Leu Pro Gln Pro Gln Gln 145 150 155 160 Pro Gln Gln Ser
Phe Pro Gln Gln Gln Arg Pro Phe Ile Gln Pro Ser 165 170 175 Leu Gln
Gln Gln Leu Asn Pro Cys Lys Asn Ile Leu Leu Gln Gln Ser 180 185 190
Lys Pro Ala Ser Leu Val Ser Ser Leu Trp Ser Ile Ile Trp Pro Gln 195
200 205 Ser Asp Cys Gln Val Met Arg Gln Gln Cys Cys Gln Gln Leu Ala
Gln 210 215 220 Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Val
Val His Ser 225 230 235 240 Ile Ile Met Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gly Ile Asp 245 250 255 Ile Phe Leu Pro Leu Ser Gln His
Glu Gln Val Gly Gln Gly Ser Leu 260 265 270 Val Gln Gly Gln Gly Ile
Ile Gln Pro Gln Gln Pro Ala Gln Leu Glu 275 280 285 Ala Ile Arg Ser
Leu Val Leu Gln Thr Leu Pro Ser Met Cys Asn Val 290 295 300 Tyr Val
Pro Pro Glu Cys Ser Ile Met Arg Ala Pro Phe Ala Ser Ile 305 310 315
320 Val Ala Gly Ile Gly Gly Gln 325 172304PRTTriticum aestivum
172Met Lys Thr Phe Leu Val Phe Ala Leu Ile Ala Val Val Ala Thr Ser
1 5 10 15 Ala Ile Ala Gln Met Glu Thr Ser Cys Ile Ser Gly Leu Glu
Arg Pro 20 25 30 Trp Gln Gln Gln Pro Leu Pro Pro Gln Gln Ser Phe
Ser Gln Gln Pro 35 40 45 Pro Phe Ser Gln Gln Gln Gln Gln Pro Leu
Pro Gln Gln Pro Ser Phe 50 55 60 Ser Gln Gln Gln Pro Pro Phe Ser
Gln Gln Gln Pro Ile Leu Ser Gln 65 70 75 80 Gln Pro Pro Phe Ser Gln
Gln Gln Gln Pro Val Leu Pro Gln Gln Ser 85 90 95 Pro Phe Ser Gln
Gln Gln Gln Leu Val Leu Pro Pro Gln Gln Gln Gln 100 105 110 Gln Gln
Leu Val Gln Gln Gln Ile Pro Ile Val Gln Pro Ser Val Leu 115 120 125
Gln Gln Leu Asn Pro Cys Lys Val Phe Leu Gln Gln Gln Cys Ser Pro 130
135 140 Val Ala Met Pro Gln Arg Leu Ala Arg Ser Gln Met Trp Gln Gln
Ser 145 150 155 160 Ser Cys His Val Met Gln Gln Gln Cys Cys Gln Gln
Leu Gln Gln Ile 165 170 175 Pro Glu Gln Ser Arg Tyr Glu Ala Ile Arg
Ala Ile Ile Tyr Ser Ile 180 185 190 Ile Leu Gln Glu Gln Gln Gln Gly
Phe Val Gln Pro Gln Gln Gln Gln 195 200 205 Pro Gln Gln Ser Gly Gln
Gly Val Ser Gln Ser Gln Gln Gln Ser Gln 210 215 220 Gln Gln Leu Gly
Gln Cys Ser Phe Gln Gln Pro Gln Gln Gln Leu Gly 225 230 235 240 Gln
Gln Pro Gln Gln Gln Gln Gln Gln Gln Val Leu Gln Gly Thr Phe 245 250
255 Leu Gln Pro His Gln Ile Ala His Leu Glu Ala Val Thr Ser Ile
Ala 260 265 270 Leu Arg Thr Leu Pro Thr Met Cys Ser Val Asn Val Pro
Leu Tyr Ser 275 280 285 Ala Thr Thr Ser Val Pro Phe Gly Val Gly Thr
Gly Val Gly Ala Tyr 290 295 300 173251PRTTriticum aestivum 173Met
Lys Thr Leu Leu Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile 1 5 10
15 Gly Thr Ala Asn Met Gln Val Asp Pro Ser Ser Gln Val Gln Trp Pro
20 25 30 Gln Gln Gln Pro Val Pro Gln Pro His Gln Pro Phe Ser Gln
Gln Pro 35 40 45 Gln Gln Thr Phe Pro Gln Pro Gln Gln Thr Phe Pro
His Gln Pro Gln 50 55 60 Gln Gln Phe Pro Gln Pro Gln Gln Pro Gln
Gln Gln Phe Leu Gln Pro 65 70 75 80 Gln Gln Pro Phe Pro Gln Gln Pro
Gln Gln Pro Tyr Pro Gln Gln Pro 85 90 95 Gln Gln Pro Phe Pro Gln
Thr Gln Gln Pro Gln Gln Leu Phe Pro Gln 100 105 110 Ser Gln Gln Pro
Gln Gln Gln Phe Ser Gln Pro Gln Gln Gln Phe Pro 115 120 125 Gln Pro
Gln Gln Pro Gln Gln Ser Phe Pro Gln Gln Gln Pro Pro Phe 130 135 140
Ile Gln Pro Ser Leu Gln Gln Gln Val Asn Pro Cys Lys Asn Phe Leu 145
150 155 160 Leu Gln Gln Cys Lys Pro Val Ser Leu Val Ser Ser Leu Trp
Ser Met 165 170 175 Ile Trp Pro Gln Ser Asp Cys Gln Val Met Arg Gln
Gln Cys Cys Gln 180 185 190 Gln Leu Ala Gln Ile Pro Gln Gln Leu Gln
Cys Ala Ala Ile His Thr 195 200 205 Ile Ile His Ser Ile Ile Met Gln
Gln Glu Gln Gln Glu Gln Gln Gln 210 215 220 Gly Met His Ile Leu Leu
Pro Leu Tyr Gln Gln Gln Gln Val Gly Gln 225 230 235 240 Gly Thr Leu
Val Gln Gly Gln Gly Ile Ile Gln 245 250 174307PRTTriticum aestivum
174Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr
1 5 10 15 Ala Arg Ile Ala Val Arg Val Pro Val Pro Gln Leu Gln Pro
Gln Asn 20 25 30 Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu
Val Gln Gln Gln 35 40 45 Gln Phe Pro Gly Gln Gln Gln Pro Phe Pro
Pro Gln Gln Pro Tyr Pro 50 55 60 Gln Pro Gln Pro Phe Pro Ser Gln
Gln Pro Tyr Leu Gln Leu Gln Pro 65 70 75 80 Phe Pro Gln Pro Gln Leu
Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro 85 90 95 Gln Pro Gln Leu
Pro Tyr Pro Gln Pro Gln Pro Phe Arg Pro Gln Gln 100 105 110 Pro Tyr
Pro Gln Ser Gln Pro Gln Tyr Ser Gln Pro Gln Gln Pro Ile 115 120 125
Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Lys Gln Gln 130
135 140 Gln Gln Gln Gln Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln
Leu 145 150 155 160 Ile Pro Cys Arg Asp Val Val Leu Gln Gln His Ser
Ile Ala Tyr Gly 165 170 175 Ser Ser Gln Val Leu Gln Gln Ser Thr Tyr
Gln Leu Val Gln Gln Leu 180 185 190 Cys Cys Gln Gln Leu Trp Gln Ile
Pro Glu Gln Ser Arg Cys Gln Ala 195 200 205 Ile His Asn Val Val His
Ala Ile Ile Leu His Gln Gln Gln Gln Gln 210 215 220 Gln Gln Gln Gln
Gln Gln Gln Pro Leu Ser Gln Val Ser Phe Gln Gln 225 230 235 240 Pro
Gln Gln Gln Tyr Pro Ser Gly Gln Gly Ser Phe Gln Pro Ser Gln 245 250
255 Gln Asn Pro Gln Ala Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln
260 265 270 Phe Glu Glu Ile Arg Asn Leu Ala Leu Glu Thr Leu Pro Ala
Met Cys 275 280 285 Asn Val Tyr Ile Pro Pro Tyr Cys Thr Ile Ala Pro
Val Gly Ile Phe 290 295 300 Gly Thr Asn 305 175186PRTTriticum
aestivum 175Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln Pro Ile
Ser Gln 1 5 10 15 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Glu Gln 20 25 30 Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln
Leu Ile Pro Cys Met Asp 35 40 45 Val Val Leu Gln Gln His Asn Ile
Ala His Gly Arg Ser Gln Val Leu 50 55 60 Gln Gln Ser Thr Tyr Gln
Leu Leu Gln Glu Leu Cys Cys Gln His Leu 65 70 75 80 Trp Gln Ile Pro
Glu Gln Ser Gln Cys Gln Ala Ile His Asn Val Val 85 90 95 His Ala
Ile Ile Leu His Gln Gln Gln Gln Lys Gln Gln Gln Gln Pro 100 105 110
Ser Ser Gln Phe Ser Phe Gln Gln Pro Leu Gln Gln Tyr Pro Leu Gly 115
120 125 Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly
Ser 130 135 140 Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Ile Arg Asn
Leu Ala Leu 145 150 155 160 Gln Thr Leu Pro Ala Met Cys Asn Val Tyr
Ile Pro Pro Tyr Cys Thr 165 170 175 Ile Ala Pro Phe Gly Ile Phe Gly
Thr Asn 180 185 176313PRTTriticum aestivum 176Met Lys Thr Phe Leu
Ile Leu Ala Leu Val Ala Thr Thr Ala Thr Thr 1 5 10 15 Ala Val Arg
Val Pro Val Pro Gln Leu Gln Pro Lys Asn Pro Ser Gln 20 25 30 Gln
Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln Gln Phe Pro 35 40
45 Gly Gln Gln Gln Gln Phe Pro Pro Gln Gln Pro Tyr Pro Gln Pro Gln
50 55 60 Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro Phe
Pro Gln 65 70 75 80 Pro Gln Pro Phe Leu Pro Gln Leu Pro Tyr Pro Gln
Pro Gln Ser Phe 85 90 95 Pro Pro Gln Gln Pro Tyr Pro Gln Gln Arg
Pro Lys Tyr Leu Gln Pro 100 105 110 Gln Gln Pro Ile Ser Gln Gln Gln
Ala Gln Gln Gln Gln Gln Gln Gln 115 120 125 Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Ile Leu Gln Gln Ile Leu 130 135 140 Gln Gln Gln Leu
Ile Pro Cys Arg Asp Val Val Leu Gln Gln His Asn 145 150 155 160 Ile
Ala His Ala Ser Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu 165 170
175 Leu Gln Gln Leu Cys Cys Gln Gln Leu Leu Gln Ile Pro Glu Gln Ser
180 185 190 Arg Cys Gln Ala Ile His Asn Val Val His Ala Ile Ile Met
His Gln 195 200 205 Gln Glu Gln Gln Gln Gln Leu Gln Gln Gln Gln Gln
Gln Gln Leu Gln 210 215 220 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Pro Ser Ser Gln Val 225 230 235 240 Ser Phe Gln Gln Pro Gln Gln
Gln Tyr Pro Ser Ser Gln Gly Ser Phe 245 250 255 Gln Pro Ser Gln Gln
Asn Pro Gln Ala Gln Gly Ser Val Gln Pro Gln 260 265 270 Gln Leu Pro
Gln Phe Ala Glu Ile Arg Asn Leu Ala Leu Gln Thr Leu 275 280 285 Pro
Ala Met Cys Asn Val Tyr Ile Pro Pro His Cys Ser Thr Thr Ile 290 295
300 Ala Pro Phe Gly Ile Phe Gly Thr Asn 305 310 177296PRTTriticum
aestivum 177Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala
Thr Thr 1 5 10 15 Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Pro
Gln Pro Gln Asn 20 25 30 Pro Ser Gln Pro Gln Pro Gln Gly Gln Val
Pro Leu Val Gln Gln Gln 35 40 45 Gln Phe Pro Gly Gln Gln Gln Gln
Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60 Gln Pro Gln Pro Phe Pro
Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro 65 70 75 80 Phe Pro Gln Pro
Gln Pro Phe Pro Pro Gln Leu Pro Tyr Pro Gln Pro 85 90 95 Pro Pro
Phe Ser Pro Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr 100 105 110
Pro Gln Pro Gln Gln Pro Ile Ser Gln Gln Gln Ala Gln Gln Gln Gln 115
120 125 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ile
Leu 130 135 140 Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys Arg Asp
Val Val Leu 145 150 155 160 Gln Gln His Asn Ile Ala His Ala Arg Ser
Gln Val Leu Gln Gln Ser 165 170 175 Thr Tyr Gln Pro Leu Gln Gln Leu
Cys Cys Gln Gln Leu Trp Gln Ile 180 185 190 Pro Glu Gln Ser Arg Cys
Gln Ala Ile His Asn Val Val His Ala Ile 195 200 205 Ile Leu His Gln
Gln Gln Arg Gln Gln Gln Pro Ser Ser Gln Val Ser 210 215 220 Leu Gln
Gln Pro Gln Gln Gln Tyr Pro Ser Gly Gln Gly Phe Phe Gln 225 230 235
240 Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser Val Gln Pro Gln Gln
245 250 255 Leu Pro Gln Phe Glu Glu Ile Arg Asn Leu Ala Leu Gln Thr
Leu Pro 260 265 270 Arg Met Cys Asn Val Tyr Ile Pro Pro Tyr Cys Ser
Thr Thr Ile Ala 275 280 285 Pro Phe Gly Ile Phe Gly Thr Asn 290 295
178297PRTTriticum aestivum 178Met Lys Thr Phe Leu Ile Leu Ala Leu
Arg Ala Ile Val Ala Thr Thr 1 5 10 15 Ala Thr Ile Ala Val Arg Val
Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30 Pro Ser Gln Gln Gln
Pro Gln Lys Gln Val Pro Leu Val Gln Gln Gln 35 40 45 Gln Phe Pro
Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60 Gln
Gln Gln Pro Phe Pro Ser Gln Gln Pro Tyr Met Gln Leu Gln Pro 65 70
75 80 Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr
Pro 85 90 95 Gln Pro Gln Pro Phe Arg Pro Gln Gln Ser Tyr Pro Gln
Pro Gln Pro 100 105 110 Gln Tyr Ser Gln Pro Gln Gln Pro Ile Ser Gln
Gln Gln Gln Gln Gln 115 120 125 Gln Gln Gln Gln Gln Gln Gln Gln Gln
Ile Leu Gln Gln Ile Leu Gln 130 135 140 Gln Gln Leu Ile Pro Cys Arg
Asp Val Val Leu Gln Gln His Ser Ile 145 150 155 160 Ala His Gly Ser
Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu Val 165 170 175 Gln Gln
Phe Cys Cys Gln Gln Leu Trp Gln Ile Pro Glu Gln Ser Arg 180 185 190
Cys Gln Ala Ile His Asn Val Val His Ala Ile Ile Leu His Gln Gln 195
200 205 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Leu
Ser 210 215 220 Gln Val Cys Phe Gln Gln Ser Gln Gln Gln Tyr Pro Ser
Gly Gln Gly 225 230 235 240 Ser Phe Gln Pro Ser Gln Gln Asn Pro Gln
Ala Gln Gly Ser Val Gln 245 250 255 Pro Gln Gln Leu Pro Gln Phe Glu
Glu Ile Arg Asn Leu Ala Leu Glu 260 265 270 Thr Leu Pro Ala Met Cys
Asn Val Tyr Ile Pro Pro Tyr Cys Thr Ile 275 280 285 Ala Pro Val Gly
Ile Phe Gly Thr Asn 290 295 179282PRTTriticum aestivum 179Met Lys
Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr 1 5 10 15
Ala Thr Ser Ala Val Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn 20
25 30 Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Met Gln Gln
Gln 35 40 45 Gln Gln Phe Pro Gly Gln Gln Glu Gln Phe Pro Pro Gln
Gln Pro Tyr 50 55 60 Pro His Gln Gln Pro Phe Pro Ser Gln Gln Pro
Tyr Pro Gln Pro Gln 65 70 75 80 Pro Phe Pro Pro Gln Leu Pro Tyr Pro
Gln Thr Gln Pro Phe Pro Pro 85 90 95 Gln Gln Pro Tyr Pro Gln Pro
Gln Pro Gln Tyr Pro Gln Pro Gln Gln 100 105 110 Pro Ile Ser Gln Gln
Gln Ala Gln Gln Gln Gln Gln Gln Gln Gln Thr 115 120 125 Leu Gln Gln
Ile Leu Gln Gln Gln Leu Ile Pro Cys Arg Asp Val Val 130 135 140 Leu
Gln Gln His Asn Ile Ala His Ala Ser Ser Gln Val Leu Gln Gln 145 150
155 160 Ser Ser Tyr Gln Gln Leu Gln Gln Leu Cys Cys Gln Gln Leu Phe
Gln 165 170 175 Ile Pro Glu Gln Ser Arg Cys Gln Ala Ile His Asn Val
Val His Ala 180 185 190 Ile Ile Leu His His His Gln Gln Gln Gln Gln
Gln Pro Ser Ser Gln 195 200 205 Val Ser Tyr Gln Gln Pro Gln Glu Gln
Tyr Pro Ser Gly Gln Val Ser 210 215 220 Phe Gln Ser Ser Gln Gln Asn
Pro Gln Ala Gln Gly Ser Val Gln Pro 225 230 235 240 Gln Gln Leu Pro
Gln Phe Gln Glu Ile Arg Asn Leu Ala Leu Gln Thr 245 250 255 Leu Pro
Ala Met Cys Asn Val Tyr Ile Pro Pro Tyr Cys Ser Thr Thr 260 265 270
Ile Ala Pro Phe Gly Ile Phe Gly Thr Asn 275 280 180291PRTTriticum
aestivum 180Met Lys Thr Phe Pro Ile Leu Ala Leu Leu Ala Ile Val Ala
Thr Thr 1 5 10 15 Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Leu
Gln Leu Gln Asn 20 25 30 Pro Ser Gln Gln Gln Pro Gln Glu Gln Val
Pro Leu Val Gln Glu Gln 35 40 45 Gln Phe Gln Gly Gln Gln Gln Pro
Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60 Gln Pro Gln Pro Phe Pro
Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro 65 70 75 80 Phe Pro Gln Pro
Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe Arg Pro 85 90 95 Gln Gln
Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln 100 105 110
Pro Ile Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 115
120 125 Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys
Arg 130 135 140 Asp Val Val Leu Gln Gln His Asn Ile Ala His Gly Ser
Ser Gln Val 145 150 155 160 Leu Gln Glu Ser Thr Tyr Gln Leu Val Gln
Gln Leu Cys Cys Gln Gln 165 170 175 Leu Trp Gln Ile Pro Glu Gln Ser
Arg Cys Gln Ala Ile His Asn Val 180 185 190 Val His Ala Ile Ile Leu
His Gln Gln His His His His Gln Gln Gln 195 200 205 Gln Gln Gln Gln
Gln Gln Gln Pro Leu Ser Gln Val Ser Phe Gln Gln 210 215 220 Pro Gln
Gln Gln Tyr Pro Ser Gly Gln Gly Phe Phe Gln Pro Ser Gln 225 230 235
240 Gln Asn Pro Gln Ala Gln Gly Ser Phe Gln Pro Gln Gln Leu Pro Gln
245 250 255 Phe Glu Glu Ile Arg Asn Leu Ala Leu Gln Thr Leu Pro Ala
Met Cys 260 265 270 Asn Val Tyr Ile Pro Pro Tyr Cys Thr Ile Ala Pro
Phe Gly Ile Phe 275 280 285 Gly Thr Asn 290 181262PRTTriticum
aestivum 181Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala
Ile Val Ala Thr Thr 1 5 10 15 Ala Thr Thr Ala Val Arg Val Pro Val
Pro Gln Leu Gln Pro Gln Asn 20 25 30 Pro Ser Gln Gln Gln Pro Gln
Glu Gln Val Pro Leu Val Gln Gln Gln 35 40 45 Gln Phe Leu Gly Gln
Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60 Gln Pro Gln
Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro 65 70 75 80 Phe
Leu Gln Pro Gln Leu Pro Tyr Ser Gln Pro Gln Pro Phe Arg Pro 85 90
95 Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln
100 105 110 Pro Ile Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln 115 120 125 Gln Gln Gln Gln Gln Gln Ile Ile Gln Gln Ile Leu
Gln Gln Gln Leu 130 135 140 Ile Pro Cys Met Asp Val Val Leu Gln Gln
His Asn Ile Val His Gly 145 150 155 160 Lys Ser Gln Val Leu Gln Gln
Ser Thr Tyr Gln Leu Leu Gln Glu Leu 165 170 175 Cys Cys Gln His Leu
Trp Gln Ile Pro Glu Gln Ser Gln Cys Gln Ala 180 185 190 Ile His Asn
Val Val His Ala Ile Ile Leu His Gln Gln Gln Lys Gln 195 200 205 Gln
Gln Gln Pro Ser Ser Gln Val Ser Phe Gln Gln Pro Leu Gln Gln 210 215
220 Tyr Pro Leu Gly Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn Pro Gln
225 230 235 240 Ala Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Phe
Glu Glu Ile 245 250 255 Arg Asn Leu Ala Arg Lys 260
182282PRTTriticum aestivum 182Met Asn Ile Gln Val Asp Pro Ser Ser
Gln Val Pro Trp Pro Gln Gln 1 5 10 15 Gln Pro Phe Pro Gln Pro His
Gln Pro Phe Ser Gln Gln Pro Gln Gln 20 25 30 Thr Phe Pro Gln Pro
Gln Gln Thr Phe Pro His Gln Pro Gln Gln Gln 35 40 45 Phe Ser Gln
Pro Gln Gln Pro Gln Gln Gln Phe Ile Gln Pro Gln Gln 50 55 60 Pro
Phe Pro Gln Gln Pro Gln Gln Thr Tyr Pro Gln Arg Pro Gln Gln 65 70
75 80 Pro Phe Pro Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Ser
Gln 85 90 95 Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Gln Phe
Pro Gln Pro 100 105 110 Gln Gln Pro Gln Gln Ser Phe Pro Gln Gln Gln
Pro Ser Leu Ile Gln 115 120 125 Gln Ser Leu Gln Gln Gln Leu Asn Pro
Cys Lys Asn Phe Leu Leu Gln 130 135 140 Gln Cys Lys Pro Val Ser Leu
Val Ser Ser Leu Trp Ser Met Ile Leu 145 150 155 160 Pro Arg Ser Asp
Cys Gln Val Met Arg Gln Gln Cys Cys Gln Gln Leu 165 170 175 Ala Gln
Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Ile Val 180 185 190
His Ser Ile Ile Met Gln Gln Glu Gln Gln Glu Gln Arg Gln Gly Val 195
200 205 Gln Ile Leu Val Pro Leu Ser Gln Gln Gln Gln Val Gly Gln Gly
Thr 210 215 220 Leu Val Gln Gly Gln Gly Ile Ile Gln Pro Gln Gln Pro
Ala Gln Leu 225 230 235 240 Glu Val Ile Arg Ser Leu Val Leu Gln Thr
Leu Ala Thr Met Cys Asn 245 250 255 Val Tyr Val Pro Pro Tyr Cys Ser
Thr Ile Arg Ala Pro Phe Ala Ser 260 265 270 Ile Val Ala Gly Ile Gly
Gly Gln Tyr Arg 275 280 18328PRTTriticum monococcum 183Ala Arg Gln
Leu Asn Pro Ser Asp Gln Glu Leu Gln Ser Pro Gln Gln 1 5 10 15 Leu
Tyr Pro Gln Gln Pro Tyr Pro Gln Gln Pro Tyr 20 25
* * * * *