Celiac Sprue and/or dermatitis herpetiformis are treated by interfering with HLA binding of immunogenic gluten peptides. The antigenicity of gluten oligopeptides and the ill effects caused by an immune response thereto are decreased by administration of an HLA-binding peptide inhibitor. Such inhibitors are analogs of immunogenic gluten peptides and (i) retain the to bind tightly to HLA molecules; (ii) retain the protcolytic stability of these peptides; but (iii) are unable to T cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for treating Celiac Sprue and/or dermatitis herpetiformis and the symptoms thereof by administration of an HLA-binding peptide inhibitor to the patient. In one embodiment, the HLA-binding peptide inhibitor employed in the method is an analog of an immunogenic gluten peptide, where an immunogenic gluten peptide is altered by the replacement of one or more amino acids, where the replacement may be another naturally occurring amino acid, non-naturally occurring amino acids, modified amino acids, amino acid mimetics, and the like. Analogs of immunogenic gluten peptides that (i) retain the ability to bind tightly to HLA molecules; (ii) retain the proteolytic stability of these peptides; but (iii) are unable to activate disease-specific or other T cells, are useful agents to treat Celiac Sprue.

In another aspect, the present invention provides novel HLA-binding peptide inhibitors and methods for treating Celiac Sprue and/or dermatitis herpetiformis by administering those compounds.

In another aspect, the invention provides pharmaceutical formulations comprising an HLA-binding peptide inhibitor and a pharmaceutically acceptable carrier. In one embodiment, such formulations comprise an enteric coating that allows delivery of the active agent to the intestine, and the agents are stabilized to resist digestion or acid-catalyzed modification in acidic stomach conditions. In another embodiment, the formulation also comprises one or more glutenases, as described in U.S. Provisional Application No. 60/392,782 filed Jun. 28, 2002; and U.S. Provisional Application No. 60/428,033, filed Nov. 20, 2002, both of which are incorporated herein by reference. The invention also provides methods for the administration of enteric formulations of one or more HLA-binding peptide inhibitors to treat Celiac Sprue.

In another aspect, the invention provides methods for screening candidate compounds to determine their suitability for use in the subject methods, by assessing the ability of a candidate agent for its ability to bind to HLA molecules, and/or to inhibit the activity of T cells reactive against gluten antigens.

Methods and compositions are provided for modeling the structure of a soluble (extracellular) domain of human HLA-DQ2 bound to an immunodominant gluten epitope, and for identifying molecules that will compete with the gluten peptide for MHC binding. In one embodiment, the methods of the invention utilize structural modeling, and the identification and design of molecules having a particular structure. The structural data provided herein is used for the rational design of drugs that affect immune system activation in Celiac Sprue and/or dermatitis herpetiformis. Analysis of the crystal structure in conjunction with sequence data identifies residues in the immunogenic gluten peptide that are important for interaction with the MHC molecule, and those that are accessible for interaction with the T cell antigen receptor. This information provides a basis for rational drug design.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Celiac Sprue and/or dermatitis herpetiformis are treated by interfering with HLA binding of immunogenic gluten peptides. Therapeutic benefit can be enhanced in some individuals by increasing the digestion of gluten oligopeptides, whether by pretreatment of foodstuffs to be ingested or by administration of an enzyme capable of digesting the gluten oligopeptides, together with administration of an HLA-binding peptide inhibitor. Gluten oligopeptides are highly resistant to cleavage by gastric and pancreatic peptidases such as pepsin, trypsin, chymotrypsin, and the like, and their prolonged presence in the digestive tract can induce an autoimmune response. The antigenicity of gluten oligopeptides and the ill effects caused by an immune response thereto can be decreased by administration of an HLA-binding peptide inhibitor. Such inhibitors are analogs of immunogenic gluten peptides and (i) retain the ability to bind tightly to HLA molecules; (ii) retain the proteolytic stability of these peptides; but (iii) are unable to activate disease-specific or other T cells.

Methods and compositions are provided for the administration of one or more HLA-binding peptide inhibitors to a patient suffering from Celiac Sprue and/or dermatitis herpetiformis. In some embodiments and for some individuals, the methods of the invention remove the requirement that abstention from ingestion of glutens be maintained to keep the disease in remission. The compositions of the invention include formulations of tTGase inhibitors that comprise an enteric coating that allows delivery of the agents to the intestine in an active form; the agents are stabilized to resist digestion or alternative chemical transformations in acidic stomach conditions. In another embodiment, food is pretreated or combined with glutenase, or a glutenase is co-administered (whether in time or in a formulation of the invention) with an HLA-binding peptide inhibitor of the invention.

The subject methods are useful for both prophylactic and therapeutic purposes. Thus, as used herein, the term "treating" is used to refer to both prevention of disease, and treatment of a pre-existing condition. The treatment of ongoing disease, to stabilize or improve the clinical symptoms of the patient, is a particularly important benefit provided by the present invention. Such treatment is desirably performed prior to loss of function in the affected tissues; consequently, the prophylactic therapeutic benefits provided by the invention are also important. Evidence of therapeutic effect may be any diminution in the severity of disease, particularly diminution of the severity of such symptoms as fatigue, chronic diarrhea, malabsorption of nutrients, weight loss, abdominal distension, and anemia. Other disease indicia include the presence of antibodies specific for glutens, antibodies specific for tissue transglutaminase, the presence of pro-inflammatory T cells and cytokines, and degradation of the villus structure of the small intestine. Application of the methods and compositions of the invention can result in the improvement of any and all of these disease indicia of Celiac Sprue.

Patients that can benefit from the present invention include both adults and children. Children in particular benefit from prophylactic treatment, as prevention of early exposure to toxic gluten peptides can prevent development of the disease into its more severe forms. Children suitable for prophylaxis in accordance with the methods of the invention can be identified by genetic testing for predisposition, e.g. by HLA typing; by family history, and by other methods known in the art. As is known in the art for other medications, and in accordance with the teachings herein, dosages of the HLA-binding peptide inhibitors of the invention can be adjusted for pediatric use.

Because most proteases and peptidases are unable to hydrolyze the amide bonds of proline residues, the abundance of proline residues in gliadins and related proteins from wheat, rye and barley can constitute a major digestive obstacle for the enzymes involved. This leads to an increased concentration of relatively stable gluten derived oligopeptides in the gut. These stable gluten derived oligopeptides, called "immunogenic oligopeptides" herein, bind to MHC molecules, including HLA HLA-DQ2 or DQ8 molecules, to stimulate an immune response that results in the autoimmune disease aspects of Celiac Sprue. In some cases the enzyme tissue transglutaminase selectively deamidates certain glutamine residues in these peptides, thereby enhancing their potency for the DQ2 ligand binding pocket.

HLA-binding peptide inhibitors of the present invention are analogs of immunogenic gluten oligopeptides that (i) retain the ability to bind tightly to HLA molecules; (ii) retain the proteolytic stability of these peptides; but (iii) are unable to activate disease-specific or other T cells. The inhibitor may comprise oligomers of analogs. Multivalent gluten derived epitopes have markedly enhanced immunogenicity. Consequently, multivalent oligopeptides analogs can also be expected to have increased potency for MHC molecules. In addition, these longer peptides can be more resistant toward intestinal brush border proteolysis.

An immunogenic gluten oligopeptide analog is an analog of a peptide that comprises at least about 8 residues, and may comprise at least about 10 residues; at least about 11 residues, at least about 12 residues, at least about 13 residues, at least about 14 residues, or more, where the term "residue" refers to naturally occurring amino acids, non-naturally occurring amino acids, and amino acid mimetics or derivatives; and where the gluten peptide is altered by the replacement of one or more amino acids. The replacement may be another naturally occurring amino acid, non-naturally occurring amino acids, modified amino acids, amino acid mimetics, and the like; and may further be derivitized to further reduce the affinity of these ligands for disease-specific T cell receptors. The sequence of immunogenic gluten oligopeptides can be determined by one of skill in the art. Immunogenic gliadin oligopeptides are peptides derived during normal human digestion of gliadins and related storage proteins as described above, from dietary cereals, e.g. wheat, rye, barley, and the like. Such oligopeptides act as antigens for T cells in Celiac Sprue. For binding to Class II MHC proteins, immunogenic peptides are usually from about 8 to 20 amino acids in length, more usually from about 10 to 18 amino acids. Such peptides may include PXP motifs, such as the motif PQPQLP. Determination of whether an oligopeptide is immunogenic for a particular patient is readily determined by standard T cell activation and other assays known to those of skill in the art.

Among gluten proteins with potential harmful effect to Celiac Sprue patients are included the storage proteins of wheat, species of which include Triticum aestivum; Triticum aethiopicum; Triticum baeoticum; Triticum militinae; Triticum monococcum; Triticum sinskajae; Triticum timopheevii; Triticum turgidum; Triticum urartu, Triticum vavilovii; Triticum zhukovskyi; etc. A review of the genes encoding wheat storage proteins may be found in Colot (1990) Genet Eng (N Y) 12:225 41. Gliadin is the alcohol-soluble protein fraction of wheat gluten. Gliadins are typically rich in glutamine and proline, particularly in the N-terminal part. For example, the first 100 amino acids of .alpha.- and .gamma.-gliadins contain .about.35% and .about.20% of glutamine and proline residues, respectively. Many wheat gliadins have been characterized, and as there are many strains of wheat and other cereals, it is anticipated that many more sequences will be identified using routine methods of molecular biology. Examples of gliadin sequences include but are not limited to wheat alpha gliadin sequences, for example as provided in Genbank, accession numbers AJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607; AJ133606; AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307; U51306; U51304; U51303; U50984; and U08287. A sequence of wheat omega gliadin is set forth in Genbank accession number AF280605.

Among the immunogenic gluten oligopeptides that may be modified to generate an HLA-binding peptide inhibitor are included the peptide sequence (SEQ ID NO:2) QLQPFPQPELPYP; the sequence (SEQ ID NO:3) PQPELPY; the sequence (SEQ ID NO:4) PFPQPELPYP, (SEQ ID NO:5 PQPELPYPQPQLP, (SEQ ID NO:6) PQQSFPEQQPP, (SEQ ID NO:7) VQGQGIIQPEQPAQ, (SEQ ID NO:8) FPEQPQQPYPQQP, (SEQ ID NO:9) FPQQPEQPYPQQP, (SEQ ID NO:10) FSQPEQEFPQPQ and longer peptides containing such sequences or multiple copies of such sequences. Gliadins, secalins and hordeins contain several (SEQ ID NO:11) PQPQLPY sequences or sequences similar thereto rich in Pro-Gln residues that are high-affinity substrates for tTGase. The tTGase catalyzed deamidation of such sequences increases their affinity for HLA-DQ2, the class II MHC allele present in >90% Celiac Sprue patients. Presentation of these deamidated sequences by DQ2 positive antigen presenting cells effectively stimulates proliferation of gliadin-specific T cells from intestinal biopsies of most Celiac Sprue patients, providing evidence for the proposed mechanism of disease progression in Celiac Sprue.

Analog oligopeptides of the invention comprise at least one difference in amino acid sequence from a native gluten peptide, by the replacement of an amino acid with a different amino acid; a non-naturally occurring amino acid, a peptidomimetics, substituted amino acid, and the like. An L-amino acid from the native peptide may be altered to any other one of the 20 L-amino acids commonly found in proteins, any one of the corresponding D-amino acids, rare amino acids, such as 4-hydroxyproline, and hydroxylysine, or a non-protein amino acid, such as .beta.-alanine, ornithine and homoserine. Also included with the scope of the present invention are amino acids that have been altered by chemical means such as methylation (e.g., .alpha.-methylvaline), deamidation, amidation of the C-terminal amino acid by an alkylamine such as ethylamine, ethanolamine, and ethylene diamine, and acylation or methylation of an amino acid side chain function (e.g., acylation of the epsilon amino group of lysine), deimination of arginine to citrulline, isoaspartylation, or phosphorylation on serine, threonine, tyrosine or histidine residues. Importantly, each of these altered amino acids provide a functional handle, e.g. amine, alcohol, aryl halide, and the like, which can be regioselectively derivatized to further reduce the affinity of these ligands for disease-specific T cell receptors. Peptide analogs may be further derivatized with substitutions, including, without limitation, ethers, amines, esters, amides, carbonates, carbamates, carbazates, ureas and C--C coupled derivatives. Other examples include oxidation of alcohols to ketones, followed by further modifications of the resulting carbonyl group, e.g. via preparation of oximes) or the carbon atom adjacent to the ketone. Such derivatives are encompassed by the term "analog".

The proteolytic stability of gluten oligopeptides can be attributed, at least in part, to the presence of PXP motifs, which are resistant to enzymatic degradation. Preferred analogs of immunogenic gluten oligopeptides will comprise one or more proline residues, and may comprise one or more PXP motifs.

An immunogenic gluten peptide of particular interest is the 33-mer (SEQ ID NO:1) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, which is described in detail in International Patent Application US03/04743, herein specifically incorporated by reference. This peptide is both immunogenic and highly stable to proteases. T cell epitopes present in the 33-mer peptide include, inter alia, (SEQ ID NO:12) PFPQPQLPY, (SEQ ID NO:13) PQPQLPYPQ, (SEQ ID NO:14) PFPQPELPY; (SEQ ID NO:15) PQPELPYPQ; (SEQ ID NO:16) PYPOPELPY and (SEQ ID NO:17) PYPQPQLPY. In one embodiment of the invention, the immunogenic gluten oligopeptide analog is an analog of a peptide that comprises at least one T cell epitope selected from the group consisting of (SEQ ID NO:12) PFPQPQLPY, (SEQ ID NO:13) PQPQLPYPQ, (SEQ ID NO:14) PFPQPELPY; (SEQ ID NO:15) PQPELPYPQ; (SEQ ID NO:16) PYPQPELPY and (SEQ ID NO:17) PYPQPQLPY.

The structure of an immunogenic gluten oligopeptide bound to a presenting molecule, e.g. HLA-DQ2; HLA-DQ8; et6. can be determined, e.g. by crystallography, NMR, etc., and used to identify residues in a peptide that are involved in the binding to the MHC molecule, and that are involved in the binding to a T cell antigen receptor. Residues identified as accessible for interacting with the T cell receptor may be modified to decrease the interaction, e.g. by increasing steric hindrance, altering hydrophilicity or hydrophobicity, etc. Residues identified as involved in interaction with the binding cleft of an MHC molecule may be modified to increase the interaction, e.g. by incorporating amino acids known to interact strongly with the binding cleft.

One inhibitor of interest is an oligopeptide or peptidomimetic that comprises the sequence PXPQPELPY, where X is Gly, Ala, Tyr, Trp, Arg, Lys, p-iodo-Phe, 3-iodo-Tyr, p-amino-Phe, 3-amino-Tyr, hydroxylysine, ornithine, Asp, Glu, or any residue that is substantially bulkier or hydrophilic than Phe. Examples of suitable modifications include ethers, amines, esters, amides, carbonates, carbamates, carbazates, ureas and C--C coupled derivatives. Other examples include oxidation of alcohols to ketones, followed by further modifications of the resulting carbonyl group (e.g. via preparation of oximes) or the carbon atom adjacent to the ketone. The peptide may comprise modifications that increase binding potency to an MHC molecule, by varying residues that facilitate peptide docking into the binding cleft. Examples of such residues include Gln4, Glu-6, Leu-7, and Tyr-9 (numbering based on the epitope PFPQPELPY). Each of these residues interacts closely with several residues in the DQ2 binding pocket. By using structure-based molecular design methods, these interactions can be optimized.

Another inhibitor of interest is a oligopeptide or peptidomimetic that comprises the sequence PFPQX.sub.1ELX.sub.2Y, where X.sub.1 and X.sub.2 are independently selected from 4-hydroxy-Pro (either isomer at C-4), 4-amino-Pro (either isomer atC-4), or 3-hydroxy-Pro (either isomer atC-3), and proline, with the proviso that at least one of X.sub.1 and X.sub.2 is a residue other than proline.

Peptides and peptide analogs may be synthesized by standard chemistry techniques, including synthesis by automated procedure. In general, peptide analogs are prepared by solid-phase peptide synthesis methodology which involves coupling each protected amino acid residue to a resin support, preferably a 4-methylbenzhydrylamine resin, by activation with dicyclohexylcarbodiimide to yield a peptide with a C-terminal amide. Alternatively, a chloromethyl resin (Merrifield resin) may be used to yield a peptide with a free carboxylic acid at the C-terminus. After the last residue has been attached, the protected peptide-resin is treated with hydrogen fluoride to cleave the peptide from the resin, as well as deprotect the side chain functional groups. Crude product can be further purified by gel filtration, HPLC, partition chromatography, or ion-exchange chromatography.

The present invention provides crystals and structures of HLA-DQ2 bound to antigen, where the antigen is an immunogenic gluten peptide QLQPFPQPELPYP, which may be referred to for brevity as an "HLA-DQ2/peptide complex". The structures and structural coordinates are useful in structural homology deduction, and in developing and screening agents that affect the gluten antigen presentation and immunogenicity. The structure information may be provided in a computer readable form, e.g. as a database of atomic coordinates, or as a three-dimensional model. The structures are useful, for example, in modeling interactions of the HLA molecule with the antigen, effect of inhibitors, etc. The structures are also used to identify molecules that bind to or otherwise interact with structural elements. One aspect of the present invention provides crystals of the HLA-DQ2/peptide complex, which can effectively diffract X-rays for the determination of the atomic coordinates.

The present invention further includes methods of using the structural information provided herein to derive a detailed structure of related peptide binding interactions, particularly other gluten peptides, or analogs and mimetics thereof. Such structural homology determination may utilize modeling, alone or in combination with structure determination.

The present invention provides three-dimensional coordinates for the HLA-DQ2/peptide complex. Such a data set may be provided in computer readable form. Methods of using such coordinates (including in computer readable form) in drug assays and drug screens as exemplified herein, are also part of the present invention. In a particular embodiment of this type, the coordinates contained in the data set can be used to identify potential modulators of the HLA-DQ2/peptide complex, including molecules that mimic the binding of the peptide to the HLA molecule, but which lack, or are substantially diminished in the ability to stimulate a T cell response.

In one embodiment, a potential agent for modulation of HLA-DQ2/peptide complex is selected by performing rational drug design with the three-dimensional coordinates determined for the crystal structures. Preferably the selection is performed in conjunction with computer modeling. Rational design may also be used in the genetic modification of immunogenic peptides by modeling the potential effect of a change in the amino acid sequence.

Computer analysis may be performed with one or more of the computer programs including: GRASP, O (Jones et al. (1991) Acta Cryst. A47:110); QUANTA, CHARMM, INSIGHT, SYBYL, MACROMODEL; ICM, and CNS (Brunger et al. (1998) Acta Cryst. D54:905). In a further embodiment of this aspect of the invention, an initial drug screening assay is performed using the three-dimensional structure so obtained, preferably along with a docking computer program. Such computer modeling can be performed with one or more Docking programs such as DOC, GRAM and AUTO DOCK. See, for example, Dunbrack et al. (1997) Foldinq & Design 2:27 42.

It should be understood that in the drug screening and protein modification assays provided herein, a number of iterative cycles of any or all of the steps may be performed to optimize the selection. For example, assays and drug screens that monitor the activity of the T cells in the presence and/or absence of a potential inhibitor are also included in the present invention and can be employed as an assay or drug screen, usually as a single step in a multi-step protocol.

The structure of the HLA-DQ2/peptide complex is useful in the design of agents that mimic the activity and/or specificity of the binding interaction. The structures encoded by the data may be computationally evaluated for an ability to associate with chemical entities. This provides insight into an element's ability to associate with chemical entities. Chemical entities that are capable of associating with these domains may alter immunogenicity. Such chemical entities are potential drug candidates. Alternatively, the structure encoded by the data may be displayed in a graphical format. This allows visual inspection of the structure, as well as visual inspection of the structure's association with chemical entities.

In one embodiment of the invention, an invention is provided for evaluating the ability of a chemical entity to associate with any of the molecules or molecular complexes set forth above. This method comprises the steps of employing computational means to perform a fitting operation between the chemical entity and the interacting surface of the polypeptide or nucleic acid; and analyzing the results of the fitting operation to quantify the association. The term "chemical entity", as used herein, refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds or complexes. Molecular design techniques are used to design and select chemical entities, including inhibitory compounds, capable of binding to the HLA molecule, or to the gluten peptide. Such chemical entities may interact directly with certain key features of the structure.

It will be understood by those skilled in the art that not all of the atoms present in a significant contact residue need be present in a competitive binding agent. In fact, it is only those few atoms that shape the loops and actually form important contacts that are likely to be important for activity. Those skilled in the art will be able to identify these important atoms based on the structure model of the invention, which can be constructed using the structural data herein.

The design of compounds that bind to HLA-DQ2 according to this invention generally involves consideration of two factors. First, the compound must be capable of either competing for binding with an immunogenic gluten peptide; or physically and structurally associating with the HLA-DQ2 domains. Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions and electrostatic interactions.

The compound must be able to assume a conformation that allows it to interact with the binding pocket. Although certain portions of the compound will not directly participate in these associations, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity in relation to all or a portion of the binding pocket, or the spacing between functional groups of an entity comprising several interacting chemical moieties.

Computer-based methods of analysis fall into two broad classes: database methods and de novo design methods. In database methods the compound of interest is compared to all compounds present in a database of chemical structures and compounds whose structure is in some way similar to the compound of interest are identified. The structures in the database are based on either experimental data, generated by NMR or x-ray crystallography, or modeled three-dimensional structures based on two-dimensional data. In de novo design methods, models of compounds whose structure is in some way similar to the compound of interest are generated by a computer program using information derived from known structures, e.g. data generated by x-ray crystallography and/or theoretical rules. Such design methods can build a compound having a desired structure in either an atom-by-atom manner or by assembling stored small molecular fragments. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the interacting surface of the RNA. Docking may be accomplished using software such as Quanta (Molecular Simulations, San Diego, Calif.) and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include: SmoG, GRID (Goodford (1985) J. Med. Chem., 28, pp. 849 857; Oxford University, Oxford, UK; MCSS (Miranker et al. (1991) Proteins: Structure, Function and Genetics, 11, pp. 29 34; Molecular Simulations, San Diego, Calif.); AUTODOCK (Goodsell et al., (1990) Proteins: Structure, Function, and Genetics, 8, pp. 195 202; Scripps Research Institute, La Jolla, Calif.); and DOCK (Kuntz et al. (1982) J. Mol. Biol., 161:269 288; University of California, San Francisco, Calif.)

Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or complex. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates. Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include: CAVEAT (Bartlett et al., (1989) In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc., 78, pp. 182 196; University of California, Berkeley, Calif.); 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.); and HOOK (available from Molecular Simulations, San Diego, Calif.).

Other molecular modeling techniques may also be employed in accordance with this invention. See, e.g., N. C. Cohen et al., "Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883 894 (1990). See also, M. A. Navia et al., "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202 210 (1992).

Once the binding entity has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency o f fit by the same computer methods described above.

Another approach made possible and enabled by this invention, is the computational screening of small molecule databases. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy. Generally the tighter the fit, the lower the steric hindrances, and the greater the attractive forces, the more potent the potential modulator since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug the more likely that the drug will not interact as well with other proteins. This will minimize potential side effects due to unwanted interactions with other proteins.

Compounds of interest can be systematically modified by computer modeling programs until one or more promising potential analogs are identified. In addition systematic modification of selected analogs can then be systematically modified by computer modeling programs until one or more potential analogs are identified. Alternatively a potential modulator could be obtained by initially screening a random peptide library, for example one produced by recombinant bacteriophage. A peptide selected in this manner would then be systematically modified by computer modeling programs as described above, and then treated analogously to a structural analog.

Once a potential modulator/inhibitor is identified it can be either selected from a library of chemicals as are commercially available from most large chemical companies including Merck, GlaxoWelcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn, or alternatively the potential modulator may be synthesized de novo. The de novo synthesis of one or even a relatively small group of specific compounds is reasonable in the art of drug design.

The success of both database and de novo methods in identifying compounds with activities similar to the compound of interest depends on the identification of the functionally relevant portion of the compound of interest. For drugs, the functionally relevant portion may be referred to as a pharmacophore, i.e. an arrangement of structural features and functional groups important for biological activity. Not all identified compounds having the desired pharmacophore will act as a modulator of inflammation. The actual activity can be finally determined only by measuring the activity of the compound in relevant biological assays. However, the methods of the invention are extremely valuable because they can be used to greatly reduce the number of compounds that must be tested to identify an actual inhibitor.

In order to determine the biological activity of a candidate pharmacophore it is preferable to measure biological activity at several concentrations of candidate compound. The activity at a given concentration of candidate compound can be tested in a number of ways.

For example, an HLA molecule can be attached to a solid support. Methods for placing proteins on a solid support are well known in the art and include such steps as linking biotin to the protein, and linking avidin to the solid support. The solid support can be washed to remove unreacted species. A solution of a labeled candidate agent can be contacted with the solid support. The solid support is washed again to remove the potential modulator not bound to the support. The amount of labeled potential modulator remaining with the solid support and thereby bound to the protein can be determined. Alternatively, or in addition, the dissociation constant between the labeled candidate agent and the protein can be determined.

Crystals of the binding complex of the present invention can be grown by a number of techniques including batch crystallization, vapor diffusion (either by sitting drop or hanging drop) and by microdialysis. Seeding of the crystals in some instances is required to obtain X-ray quality crystals. Standard micro and/or macro seeding of crystals may therefore be used. The crystals may be shrunk by transfer into solutions of different composition, e.g. by the addition of metal ions such as Mn.sup.2+, Pb.sup.2+, etc. Crystals may also be generated that include cofactors, substrates, candidate inhibitors, and the like, that interact with the protein, e.g. by cocrystallization of soaking protein crystals in a solution comprising an inhibitor or other agent.

Alternative methods may also be used. For example, crystals can be characterized by using X-rays produced in a conventional source (such as a sealed tube or a rotating anode) or using a synchrotron source. Methods of characterization include, but are not limited to, precision photography, oscillation photography and diffractometer data collection. Selenium-methionine may be used as described in the examples provided herein, or alternatively a heavy metal derivative data set (e.g., using PCMB) may be used in place of the selenium-methionine derivatization.

Electron density maps may be built from crystals using phase information from multiple isomorphous heavy-atom derivatives, molecular replacement or selenomethionine incorporated multiwavelength anomalous disperson technique. Model building is facilitated by the use of sequence markers, especially selenomethionine residues. Anomalous difference Fourier maps may be calculated with data from selenomethionine-substituted HLA-DQ2/GLUTEN EPITOPE and with experimental multiple isomorphous replacement with anomalous scattering (MIRAS) phases (Hemming and Edwards (2000) J. Biol. Chem. 275:2288). Maps are improved by phase combination, where MIRAS phases are combined by the program SIGMAA (Jones et al., supra.) Phase combination may be followed by solvent flattening with DM (Carson (1997) Methods Enzymol. 277:493). Improved maps may be obtained by combination of the MIRAS phases with improved phases from combined polyalanine and atomic models in an iterative process. The model can be refined by classical positional and B-factor minimization, and with manual rebuilding.

HLA-DQ2/peptide complex structure models and databases of structure information are provided. The structural models find use in determining the structure of related and/or analogous peptide complexes. In some cases, modeling will be based on the provided structure. In other embodiments, modeling will utilize the provided structure in combination with features present in homologous and/or related structures, where relationship may be defined by protein sequence similarity, or structural similarity, e.g. in the presence of specific features as described above.

The structure model may be implemented in hardware or software, or a combination of both. For most purposes, in order to use the structure coordinates generated for the structure, it is necessary to convert them into a three-dimensional shape. This is achieved through the use of free or commercially available software that is capable of generating three-dimensional graphical representations of molecules or portions thereof from a set of structure coordinates.

In one embodiment of the invention, a machine-readable storage medium is provided, the medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of any of the structures of this invention that have been described above. Specifically, the computer-readable storage medium is capable of displaying a graphical three-dimensional representation of the HLA-DQ2/peptide complex.

Thus, in accordance with the present invention, data providing structural coordinates, alone or in combination with software capable of displaying the resulting three dimensional structure of the complex, portions thereof, and their structurally similar analogs, is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery, analysis of interactions between cellular components during translation, modeling of vaccines, and the like.

Preferably, the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer may be, for example, a personal computer, microcomputer, or workstation of conventional design.

Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language.

Each such computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.

The HLA-binding peptide inhibitors are incorporated into a variety of formulations for therapeutic administration. In one aspect, the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration can be achieved in various ways, usually by oral administration. The HLA-binding peptide inhibitors may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the HLA-binding peptide inhibitors may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The agents may be combined, as previously described, to provide a cocktail of activities. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations comprise enteric coatings, so that the active agent is delivered to the intestinal tract. Enteric formulations are often used to protect an active ingredient from the strongly acid contents of the stomach. Such formulations are created by coating a solid dosage form with a film of a polymer that is insoluble in acid environments, and soluble in basic environments. Exemplary films are cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate, methacrylate copolymers, and cellulose acetate phthalate.

Other enteric formulation comprise engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings, can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches. The polymers maintain contact with intestinal epithelium for extended periods of time and actually penetrate it, through and between cells. See, for example, Mathiowitz et al., (1997) Nature 386 (6623): 410 414. Drug delivery systems can also utilize a core of superporous hydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh et al. (2001) J Control Release 71(3):307 18.

Formulations are typically provided in a unit dosage form, where the term "unit dosage form," refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of glutenase calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Methods of Treatment

The subject methods are used to treat individuals suffering from Celiac Sprue and/or dermatitis herpetiformis, by administering an effective dose through a pharmaceutical formulation. Diagnosis of suitable patients may utilize a variety of criteria known to those of skill in the art. A quantitative increase in antibodies specific for gliadin, and/or tissue transglutaminase is indicative of the disease. Family histories and the presence of the HLA alleles HLA-DQ2 [DQ(a1*05, b1*02)] and/or DQ8 [DQ(a1*03, b1*0302)] are indicative of a susceptibility to the disease. Specific peptide analogs may be administered therapeutically to decrease inflammation, and/or to induce antigen-specific tolerance to treat autoimmunity. Methods for the delivery of peptides that are altered from a native peptide are known in the art. Alteration of native peptides with selective changes of crucial residues can induce unresponsiveness or change the responsiveness of antigen-specific autoreactive T cells.

The therapeutic effect may be measured in terms of clinical outcome, or may rely on immunological or biochemical tests. Suppression of the deleterious T-cell activity can be measured by enumeration of reactive Th1 cells, by quantitating the release of cytokines at the sites of lesions, or using other assays for the presence of autoimmune T cells known in the art. Alternatively, one may look for a reduction in symptoms of a disease.

Various methods for administration may be employed. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. Such treatment could either be before meals or on a once-a-day basis or on a once-a-week basis, depending on the half-life of the inhibitor. A typical dose is at least about 1 .mu.g, usually at least about 10 .mu.g, more usually at least about 0.1 mg, and not more than about 10 mg, usually not more than about 1 mg. Enteric coating of these peptides may also enhance their lifetimes in the gut, thereby permitting delivery to the proximal and distal small intestinal tissue. Treatment of other autoimmune disorders such as Type I diabetes with such ligands may involve oral, intravenous or intramuscular administration. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or more often fractionated into smaller doses and administered daily, with meals, semi-weekly, etc. to maintain an effective dosage level.

The HLA-binding peptide inhibitors of the invention may be administered in the treatment of Type 1 diabetes (IDDM). IDDM and celiac disease are both immunologic disorders where specific HLA alleles are associated with disease risk. Transglutaminase autoantibodies can be found in some patients with IDDM. The prevalence of transglutaminase autoantibodies is higher in diabetic patients with HLA DQ2 or DQ8.

Human type I or insulin-dependent diabetes mellitus (IDDM) is characterized by autoimmune destruction of the .beta. cells in the pancreatic islets of Langerhans. The depletion of .beta. cells results in an inability to regulate levels of glucose in the blood. Overt diabetes occurs when the level of glucose in the blood rises above a specific level, usually about 250 mg/dl. In humans a long presymptomatic period precedes the onset of diabetes. During this period there is a gradual loss of pancreatic beta cell function. IDDM is currently treated by monitoring blood glucose levels to guide injection, or pump-based delivery, of recombinant insulin. Diet and exercise regimens contribute to achieving adequate blood glucose control. The inhibitors of the invention may be administered alone, or in combination with other therapies. The route of administration may be oral, as described for treatment of Celiac Sprue, or may be injected, e.g. i.v., i.m., etc. Administration may be performed during the pre-symptomatic phase, or in overt diabetes.
 

Claim 1 of 3 Claims

1. A computer-assisted method for identifying potential modulators of Celiac Sprue and/or dermatitis herpetiformis, using a programmed computer comprising a processor, a data storage system, an input device, and an output device, comprising the steps of: (a) inputting Into the programmed computer through said input device data comprising the three-dimensional coordinates of a subset of the atoms, generated from a complex of DQ2 molecule bound to an immunogenic gluten oligopeptide selected from the group consisting of PFPQPQLPY (SEQ ID NO: 12); PQPQLPYPQ (SEQ ID NO: 13); PFPQPQLPY (SEQ ID NO: 14); PQPQLPYPQ (SEQ ID NO: 15); PYPQPELPY (SEQ ID NO: 16); and PYPQPQLPY (SEQ ID NO: 17) PYPQPQLPY, thereby generating a criteria data set; (b) comparing, using said processor, said criteria data set to a computer database of chemical structures stored in said computer data storage system; (c) selecting from said database, using computer methods, chemical structures having a portion that is structurally similar to said criteria data set; (d) outputting to said output device the selected chemical structures having a portion similar to said criteria data set.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.