Inhibition of gliadin peptides

TECHNICAL FIELD OF THE INVENTION

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

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.

Many inflammatory diseases, including those that are 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

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.

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.

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.

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 are due primarily to TJ rearrangements, and they are believed to underlie many diseases including a large number of inflammatory conditions.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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

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.

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.

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).

AT-1001 is an orally administered octapeptide (Gly Gly Val Leu Val Gin 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).

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

One object of the present invention is to inhibit increased permeability of biological barriers in response to secreted signals.

Another object of the present invention is to provide compounds that inhibit secretion of signals that cause increased permeability of biological barriers.

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).

Another object of the present invention is to provide pharmaceutical compositions that inhibit secretion of signals that cause increased permeability of biological barriers.

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).

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.

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).

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.

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.

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.

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 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.

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.

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.

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.

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

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.

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.

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).

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).

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).

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).

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 PTO treated PBMC supernatant (PBMC-PTG+AT1001).

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).

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).

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+AT11001).

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).

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

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 PTO 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

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 Blackwell 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.

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.

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.

As used herein, "modulate" and all its forms and tenses refer to either increasing or decreasing a particular biochemical or physiological effect.

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.

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

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.

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.

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.

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.

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.

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

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.

Generally, a pharmaceutical composition of the invention will comprise an amount of a compound of the invention in the range of about 1 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.

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.

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.

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).

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.

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.

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.

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.

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 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 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.

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.

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.

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.

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.

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).

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.

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.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome.

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.

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.

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.

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.

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.

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

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.

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.

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, cammunomiein, demethomycin, tetranactin, tranilast, stevastelins, myriocin, gliooxin, FR 651814, SDZ214-104, bredinin, WS9482, mycophenolic acid, mizoribine, 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.

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.

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, deferoxamine, estrogen, azidothymidine (AZT), acyclovir, fameielovir, rimantadine hydrochloride, ganciclovir sodium, 5-aminolevulinic acid, meta-tetrahydroxyphenylchlorin, hexadecafluoro zinc phthalocyanine, tetramethyl hematoporphyrin, and rhodamine 123, and combinations thereof.

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, fimbriac 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.

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).

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.

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, .epsilon.-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.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

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 lipcrdvvlq 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 llqelccqhl 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 llqelccqhl 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 lrplfqlaqg 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 Triticum 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 pqtqqpqqpf 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 mqvdpssqvq 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 gtlvqgqgii 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 ipcrdvvlqq 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 attattavrv 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 qlipcrdvvl 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 aqgsvqpqql 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 qvplmqqqqq 51 fpgqqeqfpp qqpyphqqpf psqqpypqpq pfppqlpypq tqpfppqqpy 101 pqpqpqypqp qqpisqqqaq qqqqqqqtlq qilqqqlipc rdvvlqqhni 151 ahassqvlqq ssyqqlqqlc cqqlfqipeq srcqaihnvv haiilhhhqq 201 qqqqpssqvs yqqpqeqyps gqvsfqssqq npqaqgsvqp qqlpqfqeir 251 nlalqtlpam 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 qllqelccqh lwqipeqsqc qaihnvvhai 201 ilhqqqkqqq qpssqvsfqq plqqyplgqg sfrpsqqnpq aqgsvqpqql 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 gqgtlvqgqg 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

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.

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.

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.

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.

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.

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.

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.

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

Measurement of Trans Epithelial Electric Resistance (TEER) and Epithelial Flux of a Fluorescent Marker Lucifer Yellow

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.

Tight junction agonists and agonists of the Clorf43 and CCDC78 proteins can be identified using the following method, and this method may be easily modified to identify antagonists and inhibitors of the Clorf43 and CCDC78 proteins:

Determination of TEER and Lucifer Yellow Flux

Prepare Modified Hank's Balanced Salt Solution (MHBSS) by obtaining IL 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 (10N).

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.

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.

Using the MilliCell.RTM.-ERS instrument (Millipore), measure and record the transepithelial electrical resistance (TEER) across each filter and record.

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.

Place all plates into incubator set at 37.degree. C. (.+-.0.2), 50 RPM (.+-.5) for a total of 180 minutes.

At t=30, 60, 120 and 180 minutes, measure and record the transepithelial electrical resistance (TEER) across each filter using the MilliCell-ERS instrument.

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.

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.

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.

Analyze each 96-well plate in a Tecan Spectra Fluor Plus using Magellan at 485 and 535 nm.

Materials:

Cells: CaCo-2 cells passage 40-60 grown on Transwell.RTM. plates for 21-28 days

Culture Medium: DMEM supplemented with 10% fetal bovine serum, 1% NEAA, 1% Penn/Strep

Buffers: Hank's Balanced Salt Solution (HBSS) without calcium and magnesit

Flasks: 100.times.20 mm Tissue culture dish Falcon.

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)

The monocytic cell line THP-1 was used to characterize the profile of cytokines whose expression was upregulated on exposure to protease treated gliadin (PTO). THP-1 cells were diluted to 5.times.10.sup.5 cells/ml in RPMI medium supplemented with 10% heat inactivated fetal bovine serum.

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.

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.

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)

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).

Expression of IL-6, IL-8, MIP-1.alpha., and Gro-.alpha. were induced by treatment with LPS and PTO. Expression of these cytokines was not reduced by treatment with peptide GGVLVQPG (SEQ ID NO:1).

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).

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 are 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 TEER Reduced LY SEQ ID NO: Sequence Reduction Permeability 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-Leu-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)Gln-(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 LISTINGS

1

18318PRTArtificial SequencePeptide permeability inhibitor 1Gly Gly Val Leu Val Gln Pro Gly1 528PRTArtificial SequencePeptide permeability inhibitor 2Ala Gly Val Leu Val Gln Pro Gly1 538PRTArtificial SequencePeptide permeability inhibitor 3Gly Ala Val Leu Val Gln Pro Gly1 548PRTArtificial SequencePeptide permeability inhibitor 4Gly Gly Ala Leu Val Gln Pro Gly1 558PRTArtificial SequencePeptide permeability inhibitor 5Gly Gly Val Ala Val Gln Pro Gly1 568PRTArtificial SequencePeptide permeability inhibitor 6Gly Gly Val Leu Ala Gln Pro Gly1 578PRTArtificial SequencePeptide permeability inhibitor 7Gly Gly Val Leu Val Ala Pro Gly1 588PRTArtificial SequencePeptide permeability inhibitor 8Gly Gly Val Leu Val Gln Ala Gly1 598PRTArtificial SequencePeptide permeability inhibitor 9Gly Gly Val Leu Val Gln Pro Ala1 5108PRTArtificial SequencePeptide permeability inhibitor 10Gly Asp Val Leu Val Gln Pro Gly1 5118PRTArtificial SequencePeptide permeability inhibitor 11Gly Glu Val Leu Val Gln Pro Gly1 5128PRTArtificial SequencePeptide permeability inhibitor 12Gly Gln Val Leu Val Gln Pro Gly1 5138PRTArtificial SequencePeptide permeability inhibitor 13Gly Phe Val Leu Val Gln Pro Gly1 5148PRTArtificial SequencePeptide permeability inhibitor 14Gly His Val Leu Val Gln Pro Gly1 5158PRTArtificial SequencePeptide permeability inhibitor 15Gly Arg Val Leu Val Gln Pro Gly1 5168PRTArtificial SequencePeptide permeability inhibitor 16Gly Lys Val Leu Val Gln Pro Gly1 5178PRTArtificial SequencePeptide permeability inhibitor 17Gly Ile Val Leu Val Gln Pro Gly1 5188PRTArtificial SequencePeptide permeability inhibitor 18Gly Trp Val Leu Val Gln Pro Gly1 5198PRTArtificial SequencePeptide permeability inhibitor 19Gly Pro Val Leu Val Gln Pro Gly1 5208PRTArtificial SequencePeptide permeability inhibitor 20Gly Val Val Leu Val Gln Pro Gly1 5218PRTArtificial SequencePeptide permeability inhibitor 21Gly Leu Val Leu Val Gln Pro Gly1 5228PRTArtificial SequencePeptide permeability inhibitor 22Gly Asn Val Leu Val Gln Pro Gly1 5238PRTArtificial SequencePeptide permeability inhibitor 23Gly Thr Val Leu Val Gln Pro Gly1 5248PRTArtificial SequencePeptide permeability inhibitor 24Gly Gly Gly Leu Val Gln Pro Gly1 5258PRTArtificial SequencePeptide permeability inhibitor 25Gly Gly Leu Leu Val Gln Pro Gly1 5268PRTArtificial SequencePeptide permeability inhibitor 26Gly Gly Ile Leu Val Gln Pro Gly1 5278PRTArtificial SequencePeptide permeability inhibitor 27Gly Gly Phe Leu Val Gln Pro Gly1 5288PRTArtificial SequencePeptide permeability inhibitor 28Gly Gly Arg Leu Val Gln Pro Gly1 5298PRTArtificial SequencePeptide permeability inhibitor 29Gly Gly Asp Leu Val Gln Pro Gly1 5308PRTArtificial SequencePeptide permeability inhibitor 30Gly Gly Gln Leu Val Gln Pro Gly1 5318PRTArtificial SequencePeptide permeability inhibitor 31Gly Gly His Leu Val Gln Pro Gly1 5328PRTArtificial SequencePeptide permeability inhibitor 32Gly Gly Met Leu Val Gln Pro Gly1 5338PRTArtificial SequencePeptide permeability inhibitor 33Gly Gly Ser Leu Val Gln Pro Gly1 5348PRTArtificial SequencePeptide permeability inhibitor 34Gly Gly Thr Leu Val Gln Pro Gly1 5358PRTArtificial SequencePeptide permeability inhibitor 35Gly Gly Pro Leu Val Gln Pro Gly1 5368PRTArtificial SequencePeptide permeability inhibitor 36Gly Gly Val Gly Val Gln Pro Gly1 5378PRTArtificial SequencePeptide permeability inhibitor 37Gly Gly Val Val Val Gln Pro Gly1 5388PRTArtificial SequencePeptide permeability inhibitor 38Gly Gly Val Ile Val Gln Pro Gly1 5398PRTArtificial SequencePeptide permeability inhibitor 39Gly Gly Val Phe Val Gln Pro Gly1 5408PRTArtificial SequencePeptide permeability inhibitor 40Gly Gly Val Arg Val Gln Pro Gly1 5418PRTArtificial SequencePeptide permeability inhibitor 41Gly Gly Val Asp Val Gln Pro Gly1 5428PRTArtificial SequencePeptide permeability inhibitor 42Gly Gly Val Gln Val Gln Pro Gly1 5438PRTArtificial SequencePeptide permeability inhibitor 43Gly Gly Val His Val Gln Pro Gly1 5448PRTArtificial SequencePeptide permeability inhibitor 44Gly Gly Val Met Val Gln Pro Gly1 5458PRTArtificial SequencePeptide permeability inhibitor 45Gly Gly Val Ser Val Gln Pro Gly1 5468PRTArtificial SequencePeptide permeability inhibitor 46Gly Gly Val Thr Val Gln Pro Gly1 5478PRTArtificial SequencePeptide permeability inhibitor 47Gly Gly Val Pro Val Gln Pro Gly1 5488PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Ala may be D-Ala 48Ala Gly Val Leu Val Gln Pro Gly1 5498PRTArtificial SequencePeptide permeability inhibitor 49Asp Gly Val Leu Val Gln Pro Gly1 5508PRTArtificial SequencePeptide permeability inhibitor 50Glu Gly Val Leu Val Gln Pro Gly1 5518PRTArtificial SequencePeptide permeability inhibitor 51Gln Gly Val Leu Val Gln Pro Gly1 5528PRTArtificial SequencePeptide permeability inhibitor 52Phe Gly Val Leu Val Gln Pro Gly1 5538PRTArtificial SequencePeptide permeability inhibitor 53His Gly Val Leu Val Gln Pro Gly1 5548PRTArtificial SequencePeptide permeability inhibitor 54Arg Gly Val Leu Val Gln Pro Gly1 5558PRTArtificial SequencePeptide permeability inhibitor 55Lys Gly Val Leu Val Gln Pro Gly1 5568PRTArtificial SequencePeptide permeability inhibitor 56Ile Gly Val Leu Val Gln Pro Gly1 5578PRTArtificial SequencePeptide permeability inhibitor 57Trp Gly Val Leu Val Gln Pro Gly1 5588PRTArtificial SequencePeptide permeability inhibitor 58Pro Gly Val Leu Val Gln Pro Gly1 5598PRTArtificial SequencePeptide permeability inhibitor 59Val Gly Val Leu Val Gln Pro Gly1 5608PRTArtificial SequencePeptide permeability inhibitor 60Leu Gly Val Leu Val Gln Pro Gly1 5618PRTArtificial SequencePeptide permeability inhibitor 61Thr Gly Val Leu Val Gln Pro Gly1 5628PRTArtificial SequencePeptide permeability inhibitor 62Asn Gly Val Leu Val Gln Pro Gly1 5638PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Phe may be D-Phe 63Phe Gly Val Leu Val Gln Pro Gly1 5648PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be cyclohexyl-Alamisc_feature(5)..(5)Xaa may be Val or any other natural or non-natural amino acid 64Xaa Gly Val Leu Xaa Gln Pro Gly1 5658PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be Methioninedioxide 65Xaa Gly Val Leu Val Gln Pro Gly1 5667PRTArtificial SequencePeptide permeability inhibitor 66Gly Val Leu Val Gln Pro Gly1 5676PRTArtificial SequencePeptide permeability inhibitor 67Val Leu Val Gln Pro Gly1 5685PRTArtificial SequencePeptide permeability inhibitor 68Leu Val Gln Pro Gly1 5694PRTArtificial SequencePeptide permeability inhibitor 69Val Gln Pro Gly1703PRTArtificial SequencePeptide permeability inhibitor 70Gln Pro Gly1717PRTArtificial SequencePeptide permeability inhibitor 71Gly Gly Val Leu Val Gln Pro1 5726PRTArtificial SequencePeptide permeability inhibitor 72Gly Gly Val Leu Val Gln1 5735PRTArtificial SequencePeptide permeability inhibitor 73Gly Gly Val Leu Val1 5744PRTArtificial SequencePeptide permeability inhibitor 74Gly Gly Val Leu1753PRTArtificial SequencePeptide permeability inhibitor 75Gly Gly Val1769PRTArtificial SequencePeptide permeability inhibitormisc_feature(4)..(4)Val may be D-Val 76Gly Gly Asp Val Leu Val Gln Pro Gly1 5778PRTArtificial SequencePeptide permeability inhibitormisc_feature(4)..(4)Leu may be D-Leu 77Gly Gly Val Leu Val Gln Pro Gly1 5788PRTArtificial SequencePeptide permeability inhibitormisc_feature(5)..(5)Val may be D-Val 78Gly Gly Val Leu Val Gln Pro Gly1 5798PRTArtificial SequencePeptide permeability inhibitormisc_feature(6)..(6)Gln may be D-Gln 79Gly Gly Val Leu Val Gln Pro Gly1 5808PRTArtificial SequencePeptide permeability inhibitormisc_feature(7)..(7)Pro may be D-Pro 80Gly Gly Val Leu Val Gln Pro Gly1 5818PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Promisc_feature(3)..(3)Gln may be D-Glnmisc_feature(4)..(4)Val may be D-Valmisc_feature(5)..(5)Leu may be D-Leumisc_feature(6)..(6)Val may be D-Val 81Gly Pro Gln Val Leu Val Gly Gly1 5828PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Promisc_feature(3)..(3)Gln may be D-Glnmisc_feature(4)..(4)Val may be D-Valmisc_feature(5)..(5)Leu may be D-Leu 82Gly Pro Gln Val Leu Val Gly Gly1 5838PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Promisc_feature(3)..(3)Gln may be D-Glnmisc_feature(4)..(4)Val may be D-Valmisc_feature(6)..(6)Val may be D-Val 83Gly Pro Gln Val Leu Val Gly Gly1 5848PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Promisc_feature(3)..(3)Gln may be D-Glnmisc_feature(5)..(5)Leu may be D-Leumisc_feature(6)..(6)Val may be D-Val 84Gly Pro Gln Val Leu Val Gly Gly1 5858PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Promisc_feature(4)..(4)Val may be D-Valmisc_feature(5)..(5)Leu may be D-Leumisc_feature(6)..(6)Val may be D-Val 85Gly Pro Gln Val Leu Val Gly Gly1 5868PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Gln may be D-Glnmisc_feature(4)..(4)Val may be D-Valmisc_feature(5)..(5)Leu may be D-Leumisc_feature(6)..(6)Val may be D-Val 86Gly Pro Gln Val Leu Val Gly Gly1 5878PRTArtificial SequencePeptide permeability inhibitor 87Gly Pro Gln Val Leu Val Gly Gly1 5888PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Pro 88Gly Pro Gln Val Leu Val Gly Gly1 5898PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Gln may be D-Gln 89Gly Pro Gln Val Leu Val Gly Gly1 5908PRTArtificial SequencePeptide permeability inhibitormisc_feature(4)..(4)Val may be D-Val 90Gly Pro Gln Val Leu Val Gly Gly1 5918PRTArtificial SequencePeptide permeability inhibitormisc_feature(5)..(5)Leu may be D-Leu 91Gly Pro Gln Val Leu Val Gly Gly1 5928PRTArtificial SequencePeptide permeability inhibitormisc_feature(6)..(6)Val may be D-Val 92Gly Pro Gln Val Leu Val Gly Gly1 5938PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Val may be D-Valmisc_feature(4)..(4)Leu may be D-Leumisc_feature(5)..(5)Val may be D-Valmisc_feature(6)..(6)Gln may be D-Glnmisc_feature(7)..(7)Pro may be D-Pro 93Gly Gly Val Leu Val Gln Pro Gly1 5948PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Val may be D-Valmisc_feature(4)..(4)Leu may be D-Leumisc_feature(5)..(5)Val may be D-Valmisc_feature(6)..(6)Gln may be D-Gln 94Gly Gly Val Leu Val Gln Pro Gly1 5958PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Val may be D-Valmisc_feature(4)..(4)Leu may be D-Leumisc_feature(5)..(5)Val may be D-Valmisc_feature(7)..(7)Pro may be D-Pro 95Gly Gly Val Leu Val Gln Pro Gly1 5968PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Val may be D-Valmisc_feature(4)..(4)Leu may be D-Leumisc_feature(6)..(6)Gln may be D-Glnmisc_feature(7)..(7)Pro may be D-Pro 96Gly Gly Val Leu Val Gln Pro Gly1 5978PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Val may be D-Valmisc_feature(5)..(5)Val may be D-Valmisc_feature(6)..(6)Gln may be D-Glnmisc_feature(7)..(7)Pro may be D-Pro 97Gly Gly Val Leu Val Gln Pro Gly1 5988PRTArtificial SequencePeptide permeability inhibitormisc_feature(4)..(4)Leu may be D-Leumisc_feature(5)..(5)Val may be D-Valmisc_feature(6)..(6)Gln may be D-Glnmisc_feature(7)..(7)Pro may be D-Pro 98Gly Gly Val Leu Val Gln Pro Gly1 5998PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Phe may be D-Phe 99Gly Phe Val Leu Val Gln Pro Gly1 51003PRTArtificial SequencePeptide permeability inhibitor 100Ala Pro Gly11013PRTArtificial SequencePeptide permeability inhibitor 101Gln Ala Gly11023PRTArtificial SequencePeptide permeability inhibitor 102Gln Pro Ala11033PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Gln may be D-Gln 103Gln Pro Gly11043PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Pro 104Gln Pro Gly11053PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Gln may be D-Glnmisc_feature(2)..(2)Pro may be D-Pro 105Gln Pro Gly11063PRTArtificial SequencePeptide permeability inhibitor 106Gly Pro Gln11073PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Pro 107Gly Pro Gln11083PRTArtificial SequencePeptide permeability inhibitormisc_feature(3)..(3)Gln may be D-Gln 108Gly Pro Gln11093PRTArtificial SequencePeptide permeability inhibitormisc_feature(2)..(2)Pro may be D-Promisc_feature(3)..(3)Gln may be D-Gln 109Gly Pro Gln11103PRTArtificial SequencePeptide permeability inhibitor 110Ala Pro Gly11113PRTArtificial SequencePeptide permeability inhibitor 111His Pro Gly11123PRTArtificial SequencePeptide permeability inhibitor 112Asp Pro Gly11133PRTArtificial SequencePeptide permeability inhibitor 113Arg Pro Gly11143PRTArtificial SequencePeptide permeability inhibitor 114Phe Pro Gly11153PRTArtificial SequencePeptide permeability inhibitor 115Gly Pro Gly11163PRTArtificial SequencePeptide permeability inhibitor 116Glu Pro Gly11173PRTArtificial SequencePeptide permeability inhibitor 117Lys Pro Gly11183PRTArtificial SequencePeptide permeability inhibitor 118Leu Pro Gly11193PRTArtificial SequencePeptide permeability inhibitor 119Met Pro Gly11203PRTArtificial SequencePeptide permeability inhibitor 120Asn Pro Gly11213PRTArtificial SequencePeptide permeability inhibitor 121Ser Pro Gly11223PRTArtificial SequencePeptide permeability inhibitor 122Tyr Pro Gly11233PRTArtificial SequencePeptide permeability inhibitor 123Thr Pro Gly11243PRTArtificial SequencePeptide permeability inhibitor 124Ile Pro Gly11253PRTArtificial SequencePeptide permeability inhibitor 125Trp Pro Gly11263PRTArtificial SequencePeptide permeability inhibitor 126Pro Pro Gly11273PRTArtificial SequencePeptide permeability inhibitor 127Val Pro Gly11283PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 128Xaa Pro Gly11293PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 129Xaa Val Gly11303PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 130Xaa Gln Gly11313PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 131Xaa Ser Gly11323PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 132Xaa Lys Gly11333PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 133Xaa Phe Gly11343PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 134Xaa Glu Gly11353PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 135Xaa Thr Gly11363PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 136Xaa Ile Gly11373PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 137Xaa Tyr Gly11383PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 138Xaa His Gly11393PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 139Xaa Asn Gly11403PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 140Xaa Arg Gly11413PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 141Xaa Gly Gly11423PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 142Xaa Trp Gly11433PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 143Xaa Asp

Gly11443PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 144Xaa Met Gly11453PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 145Xaa Leu Gly11463PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 146Xaa Pro Gln11473PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 147Xaa Pro Asn11483PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 148Xaa Pro Gln11493PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 149Xaa Pro Ser11503PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 150Xaa Pro Pro11513PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 151Xaa Pro Trp11523PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 152Xaa Pro Asp11533PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 153Xaa Pro His11543PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 154Xaa Pro Leu11553PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 155Xaa Pro Arg11563PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 156Xaa Pro Val11573PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 157Xaa Pro Lys11583PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 158Xaa Pro Glu11593PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 159Xaa Pro Phe11603PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 160Xaa Pro Ile11613PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 161Xaa Pro Met11623PRTArtificial SequencePeptide permeability inhibitormisc_feature(1)..(1)Xaa may be L-pyroglutamic acid 162Xaa Pro Tyr11639PRTTriticum sp. 163Pro Tyr Pro Gln Pro Gln Leu Pro Tyr1 516413PRTTriticum sp. 164Leu Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr1 5 10165290PRTTriticum aestivum 165Met Val Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn Pro Ser Gln1 5 10 15Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln Gln Phe Pro 20 25 30Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro Gln Pro Gln 35 40 45Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro Phe Pro Gln 50 55 60Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln65 70 75 80Leu Pro Tyr Pro Gln Pro Gln Pro Phe Arg Pro Gln Gln Pro Tyr Pro 85 90 95Gln Ser Gln Pro Gln Tyr Ser Gln Pro Gln Gln Pro Ile Ser Gln Gln 100 105 110Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Lys Gln Gln Gln Gln Gln 115 120 125Gln Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys 130 135 140Arg Asp Val Val Leu Gln Gln His Ser Ile Ala Tyr Gly Ser Ser Gln145 150 155 160Val Leu Gln Gln Ser Thr Tyr Gln Leu Val Gln Gln Leu Cys Cys Gln 165 170 175Gln Leu Trp Gln Ile Pro Glu Gln Ser Arg Cys Gln Ala Ile His Asn 180 185 190Val Val His Ala Ile Ile Leu His Gln Gln Gln Gln Gln Gln Gln Gln 195 200 205Gln Gln Gln Gln Pro Leu Ser Gln Val Ser Phe Gln Gln Pro Gln Gln 210 215 220Gln Tyr Pro Ser Gly Gln Gly Ser Phe Gln Pro Ser Gln Gln Asn Pro225 230 235 240Gln Ala Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Glu 245 250 255Ile Arg Asn Leu Ala Leu Glu Thr Leu Pro Ala Met Cys Asn Val Tyr 260 265 270Ile Pro Pro Tyr Cys Thr Ile Ala Pro Val Gly Ile Phe Gly Thr Asn 275 280 285Tyr Arg 290166286PRTTriticum turgidum subsp. durum 166Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Leu Gln Arg Gln Asn 20 25 30Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln 35 40 45Gln Phe Leu Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60Gln Pro Gln Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro65 70 75 80Phe Pro Gln Pro Gln Leu Pro Tyr Ser Gln Pro Gln Pro Phe Arg Pro 85 90 95Gln Gln Pro Tyr Pro Gln Pro Gln Pro Arg Tyr Ser Gln Pro Gln Gln 100 105 110Pro Ile Ser Gln Gln Gln Gln Gln Gln His Gln Gln His Gln Gln His 115 120 125His Gln Glu Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile 130 135 140Pro Cys Met Asp Val Val Leu Gln Gln His Asn Ile Ala His Arg Arg145 150 155 160Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu Leu Gln Glu Leu Cys 165 170 175Cys Gln His Leu Trp Gln Ile Pro Glu Gln Ser Gln Cys Gln Ala Ile 180 185 190His Asn Val Val His Ala Ile Ile Pro His Gln Gln Gln Lys Gln Gln 195 200 205Gln Gln Pro Ser Ser Gln Phe Ser Phe Gln Gln Pro Leu Gln Gln Tyr 210 215 220Pro Leu Gly Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn Pro Gln Ala225 230 235 240Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Glu Ile Arg 245 250 255Asn Leu Ala Leu Gln Thr Leu Pro Ala Met Cys Asn Val Tyr Ile Pro 260 265 270Pro Tyr Cys Thr Ile Ala Pro Phe Gly Ile Phe Gly Thr Asn 275 280 285167286PRTTriticum aestivum 167Met Lys Thr Phe Leu Ile Leu Val Leu Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Thr Ala Val Arg Phe Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln 35 40 45Gln Phe Leu Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60Gln Pro Gln Pro Phe Pro Ser Gln Leu Pro Tyr Leu Gln Leu Gln Pro65 70 75 80Phe Pro Gln Pro Gln Leu Pro Tyr Ser Gln Pro Gln Pro Phe Arg Pro 85 90 95Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln 100 105 110Pro Ile Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 115 120 125Gln Gln Gln Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile 130 135 140Pro Cys Met Asp Val Val Leu Gln Gln His Asn Ile Ala His Gly Arg145 150 155 160Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu Leu Gln Glu Leu Cys 165 170 175Cys Gln His Leu Trp Gln Ile Pro Glu Gln Ser Gln Cys Gln Ala Ile 180 185 190His Asn Val Val His Ala Ile Ile Leu His Gln Gln Gln Lys Gln Gln 195 200 205Gln Gln Pro Ser Ser Gln Val Ser Phe Gln Gln Pro Leu Gln Gln Tyr 210 215 220Pro Leu Gly Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn Pro Gln Ala225 230 235 240Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Glu Ile Arg 245 250 255Asn Leu Ala Leu Gln Thr Leu Pro Ala Met Cys Asn Val Tyr Ile Pro 260 265 270Pro Tyr Cys Thr Ile Ala Pro Phe Gly Ile Phe Gly Thr Asn 275 280 285168302PRTTriticum aestivum 168Met Lys Thr Leu Leu Ile Leu Thr Ile Leu Ala Met Ala Thr Thr Ile1 5 10 15Ala Thr Ala Asn Met Gln Val Asp Pro Ser Gly Gln Val Gln Trp Pro 20 25 30Gln Gln Gln Pro Phe Pro Gln Pro Gln Gln Pro Phe Cys Gln Gln Pro 35 40 45Gln Arg Thr Ile Pro Gln Pro His Gln Thr Phe His His Gln Pro Gln 50 55 60Gln Thr Phe Pro Gln Pro Gln Gln Thr Tyr Pro His Gln Pro Gln Gln65 70 75 80Gln Phe Pro Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln 85 90 95Gln Thr Phe Pro Gln Gln Pro Gln Leu Pro Phe Pro Gln Gln Pro Gln 100 105 110Gln Pro Phe Pro Gln Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln Ser 115 120 125Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Gln Phe Pro Gln 130 135 140Pro Gln Gln Pro Gln Gln Ser Phe Pro Gln Gln Gln Gln Pro Ala Ile145 150 155 160Gln Ser Phe Leu Gln Gln Gln Met Asn Pro Cys Lys Asn Phe Leu Leu 165 170 175Gln Gln Cys Asn His Val Ser Leu Val Ser Ser Leu Val Ser Ile Ile 180 185 190Leu Pro Arg Ser Asp Cys Gln Val Met Gln Gln Gln Cys Cys Gln Gln 195 200 205Leu Ala Gln Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Val 210 215 220Ala His Ser Ile Ile Met Gln Gln Glu Gln Gln Gln Gly Val Pro Ile225 230 235 240Leu Arg Pro Leu Phe Gln Leu Ala Gln Gly Leu Gly Ile Ile Gln Pro 245 250 255Gln Gln Pro Ala Gln Leu Glu Gly Ile Arg Ser Leu Val Leu Lys Thr 260 265 270Leu Pro Thr Met Cys Asn Val Tyr Val Pro Pro Asp Cys Ser Thr Ile 275 280 285Asn Val Pro Tyr Ala Asn Ile Asp Ala Gly Ile Gly Gly Gln 290 295 300169291PRTTriticum aestivum 169Met Lys Thr Leu Leu Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile1 5 10 15Ala Thr Ala Asn Met Gln Ala Asp Pro Ser Gly Gln Val Gln Trp Pro 20 25 30Gln Gln Gln Pro Phe Leu Gln Pro His Gln Pro Phe Ser Gln Gln Pro 35 40 45Gln Gln Ile Phe Pro Gln Pro Gln Gln Thr Phe Pro His Gln Pro Gln 50 55 60Gln Gln Phe Pro Gln Pro Gln Gln Pro Gln Gln Gln Phe Leu Gln Pro65 70 75 80Arg Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Tyr Pro Gln Gln Pro 85 90 95Gln Gln Pro Phe Pro Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln 100 105 110Ser Lys Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Pro Gln Gln 115 120 125Ser Phe Pro Gln Gln Gln Pro Ser Leu Ile Gln Gln Ser Leu Gln Gln 130 135 140Gln Leu Asn Pro Cys Lys Asn Phe Leu Leu Gln Gln Cys Lys Pro Val145 150 155 160Ser Leu Val Ser Ser Leu Trp Ser Ile Ile Leu Pro Pro Ser Asp Cys 165 170 175Gln Val Met Arg Gln Gln Cys Cys Gln Gln Leu Ala Gln Ile Pro Gln 180 185 190Gln Leu Gln Cys Ala Ala Ile His Ser Val Val His Ser Ile Ile Met 195 200 205Gln Gln Glu Gln Gln Glu Gln Leu Gln Gly Val Gln Ile Leu Val Pro 210 215 220Leu Ser Gln Gln Gln Gln Val Gly Gln Gly Ile Leu Val Gln Gly Gln225 230 235 240Gly Ile Ile Gln Pro Gln Gln Pro Ala Gln Leu Glu Val Ile Arg Ser 245 250 255Leu Val Leu Gln Thr Leu Pro Thr Met Cys Asn Val Tyr Val Pro Pro 260 265 270Tyr Cys Ser Thr Ile Arg Ala Pro Phe Ala Ser Ile Val Ala Ser Ile 275 280 285Gly Gly Gln 290170244PRTTriticum aestivum 170Pro Gln Gln Pro Phe Pro Leu Gln Pro Gln Gln Ser Phe Leu Trp Gln1 5 10 15Ser Gln Gln Pro Phe Leu Gln Gln Pro Gln Gln Pro Ser Pro Gln Pro 20 25 30Gln Gln Val Val Gln Ile Ile Ser Pro Ala Thr Pro Thr Thr Ile Pro 35 40 45Ser Ala Gly Lys Pro Thr Ser Ala Pro Phe Pro Gln Gln Gln Gln Gln 50 55 60His Gln Gln Leu Ala Gln Gln Gln Ile Pro Val Val Gln Pro Ser Ile65 70 75 80Leu Gln Gln Leu Asn Pro Cys Lys Val Phe Leu Gln Gln Gln Cys Ser 85 90 95Pro Val Ala Met Pro Gln Arg Leu Ala Arg Ser Gln Met Leu Gln Gln 100 105 110Ser Ser Cys His Val Met Gln Gln Gln Cys Cys Gln Gln Leu Pro Gln 115 120 125Ile Pro Gln Gln Ser Arg Tyr Gln Ala Ile Arg Ala Ile Ile Tyr Ser 130 135 140Ile Ile Leu Gln Glu Gln Gln Gln Val Gln Gly Ser Ile Gln Ser Gln145 150 155 160Gln Gln Gln Pro Gln Gln Leu Gly Gln Cys Val Ser Gln Pro Gln Gln 165 170 175Gln Ser Gln Gln Gln Leu Gly Gln Gln Pro Gln Gln Gln Gln Leu Ala 180 185 190Gln Gly Thr Phe Leu Gln Pro His Gln Ile Ala Gln Leu Glu Val Met 195 200 205Thr Ser Ile Ala Leu Arg Ile Leu Pro Thr Met Cys Ser Val Asn Val 210 215 220Pro Leu Tyr Arg Thr Thr Thr Ser Val Pro Phe Gly Val Gly Thr Gly225 230 235 240Val Gly Ala Tyr171327PRTTriticum aestivum 171Met Lys Thr Leu Leu Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile1 5 10 15Gly Thr Ala Asn Ile Gln Val Asp Pro Ser Gly Gln Val Gln Trp Leu 20 25 30Gln Gln Gln Leu Val Pro Gln Leu Gln Gln Pro Leu Ser Gln Gln Pro 35 40 45Gln Gln Thr Phe Pro Gln Pro Gln Gln Thr Phe Pro His Gln Pro Gln 50 55 60Gln Gln Val Pro Gln Pro Gln Gln Pro Gln Gln Pro Phe Leu Gln Pro65 70 75 80Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln Thr Gln 85 90 95Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln 100 105 110Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Phe 115 120 125Pro Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Leu Gln Gln Pro 130 135 140Gln Gln Pro Phe Pro Gln Pro Gln Gln Gln Leu Pro Gln Pro Gln Gln145 150 155 160Pro Gln Gln Ser Phe Pro Gln Gln Gln Arg Pro Phe Ile Gln Pro Ser 165 170 175Leu Gln Gln Gln Leu Asn Pro Cys Lys Asn Ile Leu Leu Gln Gln Ser 180 185 190Lys Pro Ala Ser Leu Val Ser Ser Leu Trp Ser Ile Ile Trp Pro Gln 195 200 205Ser Asp Cys Gln Val Met Arg Gln Gln Cys Cys Gln Gln Leu Ala Gln 210 215 220Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Val Val His Ser225 230 235 240Ile Ile Met Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gly Ile Asp 245 250 255Ile Phe Leu Pro Leu Ser Gln His Glu Gln Val Gly Gln Gly Ser Leu 260 265 270Val Gln Gly Gln Gly Ile Ile Gln Pro Gln Gln Pro Ala Gln Leu Glu 275 280 285Ala Ile Arg Ser Leu Val Leu Gln Thr Leu Pro Ser Met Cys Asn Val 290 295 300Tyr Val Pro Pro Glu Cys Ser Ile Met Arg Ala Pro Phe Ala Ser Ile305 310 315 320Val Ala Gly Ile Gly Gly Gln 325172304PRTTriticum aestivum 172Met Lys Thr Phe Leu Val Phe Ala Leu Ile Ala Val Val Ala Thr Ser1 5 10 15Ala Ile Ala Gln Met Glu Thr

Ser Cys Ile Ser Gly Leu Glu Arg Pro 20 25 30Trp Gln Gln Gln Pro Leu Pro Pro Gln Gln Ser Phe Ser Gln Gln Pro 35 40 45Pro Phe Ser Gln Gln Gln Gln Gln Pro Leu Pro Gln Gln Pro Ser Phe 50 55 60Ser Gln Gln Gln Pro Pro Phe Ser Gln Gln Gln Pro Ile Leu Ser Gln65 70 75 80Gln Pro Pro Phe Ser Gln Gln Gln Gln Pro Val Leu Pro Gln Gln Ser 85 90 95Pro Phe Ser Gln Gln Gln Gln Leu Val Leu Pro Pro Gln Gln Gln Gln 100 105 110Gln Gln Leu Val Gln Gln Gln Ile Pro Ile Val Gln Pro Ser Val Leu 115 120 125Gln Gln Leu Asn Pro Cys Lys Val Phe Leu Gln Gln Gln Cys Ser Pro 130 135 140Val Ala Met Pro Gln Arg Leu Ala Arg Ser Gln Met Trp Gln Gln Ser145 150 155 160Ser Cys His Val Met Gln Gln Gln Cys Cys Gln Gln Leu Gln Gln Ile 165 170 175Pro Glu Gln Ser Arg Tyr Glu Ala Ile Arg Ala Ile Ile Tyr Ser Ile 180 185 190Ile Leu Gln Glu Gln Gln Gln Gly Phe Val Gln Pro Gln Gln Gln Gln 195 200 205Pro Gln Gln Ser Gly Gln Gly Val Ser Gln Ser Gln Gln Gln Ser Gln 210 215 220Gln Gln Leu Gly Gln Cys Ser Phe Gln Gln Pro Gln Gln Gln Leu Gly225 230 235 240Gln Gln Pro Gln Gln Gln Gln Gln Gln Gln Val Leu Gln Gly Thr Phe 245 250 255Leu Gln Pro His Gln Ile Ala His Leu Glu Ala Val Thr Ser Ile Ala 260 265 270Leu Arg Thr Leu Pro Thr Met Cys Ser Val Asn Val Pro Leu Tyr Ser 275 280 285Ala Thr Thr Ser Val Pro Phe Gly Val Gly Thr Gly Val Gly Ala Tyr 290 295 300173251PRTTriticum aestivum 173Met Lys Thr Leu Leu Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile1 5 10 15Gly Thr Ala Asn Met Gln Val Asp Pro Ser Ser Gln Val Gln Trp Pro 20 25 30Gln Gln Gln Pro Val Pro Gln Pro His Gln Pro Phe Ser Gln Gln Pro 35 40 45Gln Gln Thr Phe Pro Gln Pro Gln Gln Thr Phe Pro His Gln Pro Gln 50 55 60Gln Gln Phe Pro Gln Pro Gln Gln Pro Gln Gln Gln Phe Leu Gln Pro65 70 75 80Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Tyr Pro Gln Gln Pro 85 90 95Gln Gln Pro Phe Pro Gln Thr Gln Gln Pro Gln Gln Leu Phe Pro Gln 100 105 110Ser Gln Gln Pro Gln Gln Gln Phe Ser Gln Pro Gln Gln Gln Phe Pro 115 120 125Gln Pro Gln Gln Pro Gln Gln Ser Phe Pro Gln Gln Gln Pro Pro Phe 130 135 140Ile Gln Pro Ser Leu Gln Gln Gln Val Asn Pro Cys Lys Asn Phe Leu145 150 155 160Leu Gln Gln Cys Lys Pro Val Ser Leu Val Ser Ser Leu Trp Ser Met 165 170 175Ile Trp Pro Gln Ser Asp Cys Gln Val Met Arg Gln Gln Cys Cys Gln 180 185 190Gln Leu Ala Gln Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Thr 195 200 205Ile Ile His Ser Ile Ile Met Gln Gln Glu Gln Gln Glu Gln Gln Gln 210 215 220Gly Met His Ile Leu Leu Pro Leu Tyr Gln Gln Gln Gln Val Gly Gln225 230 235 240Gly Thr Leu Val Gln Gly Gln Gly Ile Ile Gln 245 250174307PRTTriticum aestivum 174Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Arg Ile Ala Val Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln 35 40 45Gln Phe Pro Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60Gln Pro Gln Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro65 70 75 80Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro 85 90 95Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe Arg Pro Gln Gln 100 105 110Pro Tyr Pro Gln Ser Gln Pro Gln Tyr Ser Gln Pro Gln Gln Pro Ile 115 120 125Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Lys Gln Gln 130 135 140Gln Gln Gln Gln Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu145 150 155 160Ile Pro Cys Arg Asp Val Val Leu Gln Gln His Ser Ile Ala Tyr Gly 165 170 175Ser Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu Val Gln Gln Leu 180 185 190Cys Cys Gln Gln Leu Trp Gln Ile Pro Glu Gln Ser Arg Cys Gln Ala 195 200 205Ile His Asn Val Val His Ala Ile Ile Leu His Gln Gln Gln Gln Gln 210 215 220Gln Gln Gln Gln Gln Gln Gln Pro Leu Ser Gln Val Ser Phe Gln Gln225 230 235 240Pro Gln Gln Gln Tyr Pro Ser Gly Gln Gly Ser Phe Gln Pro Ser Gln 245 250 255Gln Asn Pro Gln Ala Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln 260 265 270Phe Glu Glu Ile Arg Asn Leu Ala Leu Glu Thr Leu Pro Ala Met Cys 275 280 285Asn Val Tyr Ile Pro Pro Tyr Cys Thr Ile Ala Pro Val Gly Ile Phe 290 295 300Gly Thr Asn305175186PRTTriticum aestivum 175Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln Pro Ile Ser Gln1 5 10 15Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu Gln 20 25 30Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys Met Asp 35 40 45Val Val Leu Gln Gln His Asn Ile Ala His Gly Arg Ser Gln Val Leu 50 55 60Gln Gln Ser Thr Tyr Gln Leu Leu Gln Glu Leu Cys Cys Gln His Leu65 70 75 80Trp Gln Ile Pro Glu Gln Ser Gln Cys Gln Ala Ile His Asn Val Val 85 90 95His Ala Ile Ile Leu His Gln Gln Gln Gln Lys Gln Gln Gln Gln Pro 100 105 110Ser Ser Gln Phe Ser Phe Gln Gln Pro Leu Gln Gln Tyr Pro Leu Gly 115 120 125Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser 130 135 140Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Ile Arg Asn Leu Ala Leu145 150 155 160Gln Thr Leu Pro Ala Met Cys Asn Val Tyr Ile Pro Pro Tyr Cys Thr 165 170 175Ile Ala Pro Phe Gly Ile Phe Gly Thr Asn 180 185176313PRTTriticum aestivum 176Met Lys Thr Phe Leu Ile Leu Ala Leu Val Ala Thr Thr Ala Thr Thr1 5 10 15Ala Val Arg Val Pro Val Pro Gln Leu Gln Pro Lys Asn Pro Ser Gln 20 25 30Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln Gln Phe Pro 35 40 45Gly Gln Gln Gln Gln Phe Pro Pro Gln Gln Pro Tyr Pro Gln Pro Gln 50 55 60Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro Phe Pro Gln65 70 75 80Pro Gln Pro Phe Leu Pro Gln Leu Pro Tyr Pro Gln Pro Gln Ser Phe 85 90 95Pro Pro Gln Gln Pro Tyr Pro Gln Gln Arg Pro Lys Tyr Leu Gln Pro 100 105 110Gln Gln Pro Ile Ser Gln Gln Gln Ala Gln Gln Gln Gln Gln Gln Gln 115 120 125Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ile Leu Gln Gln Ile Leu 130 135 140Gln Gln Gln Leu Ile Pro Cys Arg Asp Val Val Leu Gln Gln His Asn145 150 155 160Ile Ala His Ala Ser Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu 165 170 175Leu Gln Gln Leu Cys Cys Gln Gln Leu Leu Gln Ile Pro Glu Gln Ser 180 185 190Arg Cys Gln Ala Ile His Asn Val Val His Ala Ile Ile Met His Gln 195 200 205Gln Glu Gln Gln Gln Gln Leu Gln Gln Gln Gln Gln Gln Gln Leu Gln 210 215 220Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Ser Ser Gln Val225 230 235 240Ser Phe Gln Gln Pro Gln Gln Gln Tyr Pro Ser Ser Gln Gly Ser Phe 245 250 255Gln Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser Val Gln Pro Gln 260 265 270Gln Leu Pro Gln Phe Ala Glu Ile Arg Asn Leu Ala Leu Gln Thr Leu 275 280 285Pro Ala Met Cys Asn Val Tyr Ile Pro Pro His Cys Ser Thr Thr Ile 290 295 300Ala Pro Phe Gly Ile Phe Gly Thr Asn305 310177296PRTTriticum aestivum 177Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Pro Gln Pro Gln Asn 20 25 30Pro Ser Gln Pro Gln Pro Gln Gly Gln Val Pro Leu Val Gln Gln Gln 35 40 45Gln Phe Pro Gly Gln Gln Gln Gln Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60Gln Pro Gln Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro65 70 75 80Phe Pro Gln Pro Gln Pro Phe Pro Pro Gln Leu Pro Tyr Pro Gln Pro 85 90 95Pro Pro Phe Ser Pro Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr 100 105 110Pro Gln Pro Gln Gln Pro Ile Ser Gln Gln Gln Ala Gln Gln Gln Gln 115 120 125Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ile Leu 130 135 140Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys Arg Asp Val Val Leu145 150 155 160Gln Gln His Asn Ile Ala His Ala Arg Ser Gln Val Leu Gln Gln Ser 165 170 175Thr Tyr Gln Pro Leu Gln Gln Leu Cys Cys Gln Gln Leu Trp Gln Ile 180 185 190Pro Glu Gln Ser Arg Cys Gln Ala Ile His Asn Val Val His Ala Ile 195 200 205Ile Leu His Gln Gln Gln Arg Gln Gln Gln Pro Ser Ser Gln Val Ser 210 215 220Leu Gln Gln Pro Gln Gln Gln Tyr Pro Ser Gly Gln Gly Phe Phe Gln225 230 235 240Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser Val Gln Pro Gln Gln 245 250 255Leu Pro Gln Phe Glu Glu Ile Arg Asn Leu Ala Leu Gln Thr Leu Pro 260 265 270Arg Met Cys Asn Val Tyr Ile Pro Pro Tyr Cys Ser Thr Thr Ile Ala 275 280 285Pro Phe Gly Ile Phe Gly Thr Asn 290 295178297PRTTriticum aestivum 178Met Lys Thr Phe Leu Ile Leu Ala Leu Arg Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Ile Ala Val Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30Pro Ser Gln Gln Gln Pro Gln Lys Gln Val Pro Leu Val Gln Gln Gln 35 40 45Gln Phe Pro Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60Gln Gln Gln Pro Phe Pro Ser Gln Gln Pro Tyr Met Gln Leu Gln Pro65 70 75 80Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro 85 90 95Gln Pro Gln Pro Phe Arg Pro Gln Gln Ser Tyr Pro Gln Pro Gln Pro 100 105 110Gln Tyr Ser Gln Pro Gln Gln Pro Ile Ser Gln Gln Gln Gln Gln Gln 115 120 125Gln Gln Gln Gln Gln Gln Gln Gln Gln Ile Leu Gln Gln Ile Leu Gln 130 135 140Gln Gln Leu Ile Pro Cys Arg Asp Val Val Leu Gln Gln His Ser Ile145 150 155 160Ala His Gly Ser Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu Val 165 170 175Gln Gln Phe Cys Cys Gln Gln Leu Trp Gln Ile Pro Glu Gln Ser Arg 180 185 190Cys Gln Ala Ile His Asn Val Val His Ala Ile Ile Leu His Gln Gln 195 200 205Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Leu Ser 210 215 220Gln Val Cys Phe Gln Gln Ser Gln Gln Gln Tyr Pro Ser Gly Gln Gly225 230 235 240Ser Phe Gln Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser Val Gln 245 250 255Pro Gln Gln Leu Pro Gln Phe Glu Glu Ile Arg Asn Leu Ala Leu Glu 260 265 270Thr Leu Pro Ala Met Cys Asn Val Tyr Ile Pro Pro Tyr Cys Thr Ile 275 280 285Ala Pro Val Gly Ile Phe Gly Thr Asn 290 295179282PRTTriticum aestivum 179Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Ser Ala Val Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Met Gln Gln Gln 35 40 45Gln Gln Phe Pro Gly Gln Gln Glu Gln Phe Pro Pro Gln Gln Pro Tyr 50 55 60Pro His Gln Gln Pro Phe Pro Ser Gln Gln Pro Tyr Pro Gln Pro Gln65 70 75 80Pro Phe Pro Pro Gln Leu Pro Tyr Pro Gln Thr Gln Pro Phe Pro Pro 85 90 95Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Pro Gln Pro Gln Gln 100 105 110Pro Ile Ser Gln Gln Gln Ala Gln Gln Gln Gln Gln Gln Gln Gln Thr 115 120 125Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys Arg Asp Val Val 130 135 140Leu Gln Gln His Asn Ile Ala His Ala Ser Ser Gln Val Leu Gln Gln145 150 155 160Ser Ser Tyr Gln Gln Leu Gln Gln Leu Cys Cys Gln Gln Leu Phe Gln 165 170 175Ile Pro Glu Gln Ser Arg Cys Gln Ala Ile His Asn Val Val His Ala 180 185 190Ile Ile Leu His His His Gln Gln Gln Gln Gln Gln Pro Ser Ser Gln 195 200 205Val Ser Tyr Gln Gln Pro Gln Glu Gln Tyr Pro Ser Gly Gln Val Ser 210 215 220Phe Gln Ser Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser Val Gln Pro225 230 235 240Gln Gln Leu Pro Gln Phe Gln Glu Ile Arg Asn Leu Ala Leu Gln Thr 245 250 255Leu Pro Ala Met Cys Asn Val Tyr Ile Pro Pro Tyr Cys Ser Thr Thr 260 265 270Ile Ala Pro Phe Gly Ile Phe Gly Thr Asn 275 280180291PRTTriticum aestivum 180Met Lys Thr Phe Pro Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Leu Gln Leu Gln Asn 20 25 30Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Glu Gln 35 40 45Gln Phe Gln Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60Gln Pro Gln Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro65 70 75 80Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe Arg Pro 85 90 95Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln 100 105 110Pro Ile Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 115 120 125Gln Gln Ile Leu Gln Gln Ile Leu Gln Gln Gln Leu Ile Pro Cys Arg 130 135 140Asp Val Val Leu Gln Gln His Asn Ile Ala His Gly Ser Ser Gln Val145 150 155 160Leu Gln Glu Ser Thr Tyr Gln Leu Val Gln Gln Leu Cys Cys Gln Gln 165 170 175Leu Trp Gln Ile Pro Glu Gln Ser Arg Cys Gln Ala Ile His Asn Val 180 185 190Val His Ala Ile Ile Leu His Gln Gln His His His His Gln Gln Gln 195 200 205Gln Gln Gln Gln Gln Gln Gln Pro Leu Ser Gln Val Ser Phe Gln Gln 210 215 220Pro Gln Gln Gln Tyr Pro Ser Gly Gln Gly Phe Phe Gln Pro Ser Gln225 230

235 240Gln Asn Pro Gln Ala Gln Gly Ser Phe Gln Pro Gln Gln Leu Pro Gln 245 250 255Phe Glu Glu Ile Arg Asn Leu Ala Leu Gln Thr Leu Pro Ala Met Cys 260 265 270Asn Val Tyr Ile Pro Pro Tyr Cys Thr Ile Ala Pro Phe Gly Ile Phe 275 280 285Gly Thr Asn 290181262PRTTriticum aestivum 181Met Lys Thr Phe Leu Ile Leu Ala Leu Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Leu Gln Pro Gln Asn 20 25 30Pro Ser Gln Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln 35 40 45Gln Phe Leu Gly Gln Gln Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60Gln Pro Gln Pro Phe Pro Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro65 70 75 80Phe Leu Gln Pro Gln Leu Pro Tyr Ser Gln Pro Gln Pro Phe Arg Pro 85 90 95Gln Gln Pro Tyr Pro Gln Pro Gln Pro Gln Tyr Ser Gln Pro Gln Gln 100 105 110Pro Ile Ser Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 115 120 125Gln Gln Gln Gln Gln Gln Ile Ile Gln Gln Ile Leu Gln Gln Gln Leu 130 135 140Ile Pro Cys Met Asp Val Val Leu Gln Gln His Asn Ile Val His Gly145 150 155 160Lys Ser Gln Val Leu Gln Gln Ser Thr Tyr Gln Leu Leu Gln Glu Leu 165 170 175Cys Cys Gln His Leu Trp Gln Ile Pro Glu Gln Ser Gln Cys Gln Ala 180 185 190Ile His Asn Val Val His Ala Ile Ile Leu His Gln Gln Gln Lys Gln 195 200 205Gln Gln Gln Pro Ser Ser Gln Val Ser Phe Gln Gln Pro Leu Gln Gln 210 215 220Tyr Pro Leu Gly Gln Gly Ser Phe Arg Pro Ser Gln Gln Asn Pro Gln225 230 235 240Ala Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Phe Glu Glu Ile 245 250 255Arg Asn Leu Ala Arg Lys 260182282PRTTriticum aestivum 182Met Asn Ile Gln Val Asp Pro Ser Ser Gln Val Pro Trp Pro Gln Gln1 5 10 15Gln Pro Phe Pro Gln Pro His Gln Pro Phe Ser Gln Gln Pro Gln Gln 20 25 30Thr Phe Pro Gln Pro Gln Gln Thr Phe Pro His Gln Pro Gln Gln Gln 35 40 45Phe Ser Gln Pro Gln Gln Pro Gln Gln Gln Phe Ile Gln Pro Gln Gln 50 55 60Pro Phe Pro Gln Gln Pro Gln Gln Thr Tyr Pro Gln Arg Pro Gln Gln65 70 75 80Pro Phe Pro Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Ser Gln 85 90 95Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Gln Phe Pro Gln Pro 100 105 110Gln Gln Pro Gln Gln Ser Phe Pro Gln Gln Gln Pro Ser Leu Ile Gln 115 120 125Gln Ser Leu Gln Gln Gln Leu Asn Pro Cys Lys Asn Phe Leu Leu Gln 130 135 140Gln Cys Lys Pro Val Ser Leu Val Ser Ser Leu Trp Ser Met Ile Leu145 150 155 160Pro Arg Ser Asp Cys Gln Val Met Arg Gln Gln Cys Cys Gln Gln Leu 165 170 175Ala Gln Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser Ile Val 180 185 190His Ser Ile Ile Met Gln Gln Glu Gln Gln Glu Gln Arg Gln Gly Val 195 200 205Gln Ile Leu Val Pro Leu Ser Gln Gln Gln Gln Val Gly Gln Gly Thr 210 215 220Leu Val Gln Gly Gln Gly Ile Ile Gln Pro Gln Gln Pro Ala Gln Leu225 230 235 240Glu Val Ile Arg Ser Leu Val Leu Gln Thr Leu Ala Thr Met Cys Asn 245 250 255Val Tyr Val Pro Pro Tyr Cys Ser Thr Ile Arg Ala Pro Phe Ala Ser 260 265 270Ile Val Ala Gly Ile Gly Gly Gln Tyr Arg 275 28018328PRTTriticum monococcum 183Ala Arg Gln Leu Asn Pro Ser Asp Gln Glu Leu Gln Ser Pro Gln Gln1 5 10 15Leu Tyr Pro Gln Gln Pro Tyr Pro Gln Gln Pro Tyr 20 25