Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

• the present invention relates to crystalline compositions of mammalian dipeptidyl peptidase IV (DPP-IV), methods of preparing said compositions, methods of determining the three-dimensional (3-D) X- ray atomic coordinates of said composition, methods of identifying ligands of DPP-IV using structure based drug design, the use of 3-D crystal structures to design, modify and assess the activity of potential inhibitors, and to the use of such inhibitors for the treatment of, for example, diabetes, glucose tolerance obesity, appetite regulation, hpidemia, osteoporosis neuropeptide metabolism and T-cell activation, among others
• DPP-IV se ⁇ ne peptidase dipeptidyl peptidase IV
• DPP-IV is a multifunctional type II cell surface glycoprotein, which is widely expressed in a variety of cell types, particularly on differential epithelial cells of the intestine, liver, prostate tissue, corpus luteum, and kidney proximal tubules (Thoma et al , Structure, 11 , 947-959, 947 (2003) citing Hartel et al , Histochemistry 89, 151 -161 (1988), cCaughan et al , Hepatology 11 , 534-544 (1990) as well as leukocytes subsets (Thoma et al , (2003) citing Gorrell et al , Cell Immunol 134, 205-215 (1991 )) DPP-IV has roles in many biological processes including its ability to modulate the biological activity of several peptide hormones, chemokines and neuropeptides by specifically cleaving
• DPPs such as DPP-IV as a single molecule of an asymmetric unit
• X-ray or NMR studies or from homology models
• analyzing the structures using computational methods facilitates such discovery efforts.
• SUMMARY OF THE INVENTION The present invention provides crystalline compositions of DPP-IV, and specifically of DPP-IV, having one molecule per asymmetric unit.
• the invention further provides methods of preparing said compositions, methods of determining the 3-D X-ray atomic coordinates of said crystalline compositions, methods of using the atomic coordinates in conjunction with computational methods to identify binding site(s), methods to elucidate the 3-D structure of homologues of DPP-IV, and methods to identify ligands which interact with the binding site(s) to agonize or antagonize the biological activity of DPP-IV, methods for identifying inhibitors of DPP-IV, pharmaceutical compositions of inhibitors, and methods of treatment of Type 2 diabetes, Type 1 diabetes, impaired glucose tolerance, hyperglycemia, metabolic syndrome (syndrome X and/or insulin resistance syndrome), glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, acute
• the invention provides crystalline compositions of the extracellular domain of DPP-IV (residues 31 -766 of SEQ ID NO:1 ), whereby the crystal structure is derived from 5 mammal, preferably human.
• One aspect of the present invention provides methods for crystallizing a DPP-IV polypeptide.
• the methods for crystallizing the DPP-IV polypeptide comprising an amino acid sequence spanning the amino acids 31 to 766 listed in SEQ ID NO:1 comprise the steps of: (a) preparing solutions of the polypeptide and precipitant; (b) growing a crystal comprising molecules of the polypeptide from said 10 mixture solution; and (c) separating said crystal from said solution.
• the crystallization growth can be carried out by various techniques known by those skilled in the art, such as for example, batch crystallization, liquid bridge, vapor diffusion, crystallization, or dialysis crystallization. Preferably, the crystallization growth is achieved using vapor diffusion techniques.
• the present invention provides vectors useful in methods for preparing a substantially purified extracellular domain of DPP-IV comprising the polypeptide with an amino acid 20 sequence spanning amino acids Gly31 to Pro766 listed in SEQ ID NO:1.
• Yet another embodiment of the present invention provides a DPP-IV crystal of SEQ ID NO:2, or a homologue, analogue or variant thereof.
• the present invention provides methods for determining the X-ray atomic coordinates of the crystalline compositions at a 2.7A resolution. 25
• the present invention provides a molecule or molecular complex crystal, wherein the crystal has substantially similar atomic coordinates to the atomic coordinates listed in FIG. 2 or portions thereof, or any scalable variations thereof.
• the present invention provides a molecule or molecular complex crystal, wherein the crystal comprises a polypeptide with an amino acid sequence spanning the amino acids Gly31 to Pro766 30 listed in SEQ ID NO:1.
• a further embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 98% or 95% homologous to a polypeptide with an amino acid sequence spanning the amino acids Gly31 to Pro766 listed in SEQ ID NO:1.
• An even further embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 98% or 95% homologous to a polypeptide with an amino acid sequence spanning the amino 35 acids Gly31 to Pro766 listed in SEQ ID NO:1 , and having the atomic coordinates listed in FIG. 2.
• the present invention provides a molecule or molecular complex crystal, wherein the crystal comprises a polypeptide, or a portion thereof, with atomic coordinates having a root mean square deviation from the protein backbone atoms (N, C ⁇ , C, and O) listed in FIG. 2 of less than 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or 2.5 A. 40.
• the present invention provides a scalable, or translatable, three dimensional configuration of points derived from structural coordinates of at least a portion of a DPP-IV molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning the amino acids Gly31 to Pro766 listed in SEQ ID NO:1.
• the invention also comprises the structural coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to a DPP-IV molecule or molecular complex.
• the configuration of points derived from a homologous molecule or molecular complex has a root mean square deviation of less than about 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or 2.5 A from the structural coordinates provided in FIG. 2.
• the invention provides computers for producing a three-dimensional respresentation of aspect eight of the present invention can be used to design and identify potential ligands or inhibitors of DPP-IV by, for example commercially available molecular modeling software in conjunction with structure-based drug design as provided herein.
• the present invention provides computer for producing three-dimensional representations of: a. a molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning amino acids Gly31 to Pro766 listed in SEQ ID NO:1 , or a homologue, or a variant thereof; b.
• a molecule or molecular complex wherein the atoms of the molecule or molecular complex are represented by atomic coordinates that are substantially similar to, or are subsets of, the atomic coordinates listed in FIG. 2; c. a molecule or molecular complex, wherein the molecule or molecular complex comprises atomic coordinates having a root mean square deviation of less than 0.2, 0.5, 0.7, 1.0, 1.2 , 1.5, 2.0 or even 2.5 A from the atomic coordinates for the carbon backbone atoms listed in FIG. 2; or d. a molecule or molecular complex, wherein the molecule or molecular complex comprises a binding pocket or site defined by the structure coordinates that are substantially similar to the atomic coordinates listed in FIG.
• said computer comprises: (i) a computer-readable data storage medium comprising a data storage medium encoded with computer-readable data, wherein said data comprises the structure coordinates of FIG.
• the present invention provides methods involving molecular replacement to obtain structural information about a molecule or molecular complex of unknown structure.
• the method includes crystallizing the molecule or molecular complex, generating an x-ray diffraction pattern from the crystallized molecule or molecular complex, and applying at least a portion of the structure coordinates set forth in FIG. 2 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex.
• the present invention provides methods for generating 3-D atomic coordinates of protein homologues, analogues, or variants of DPP-IV using the x-ray coordinates of DPP- IV described in FIG. 2, comprising: a. identifying one or more homologous polypeptide sequences to DPP-IV; b.
• DPP-IV which comprises a polypeptide with an amino acid sequence spanning amino acids Gly31 to Pro766 listed in SEQ ID NO:1 ; c identifying structurally conserved and structurally variable regions between said homologous sequence(s) and DPP-IV; d. generating 3-D coordinates for structurally conserved residues of the said homologous sequence(s) from those of DPP-IV using the coordinates listed in FIG. 2; e. generating conformations for the loops in the structurally variable regions of said homologous sequence(s); f. building the side-chain conformations for said homologous sequence(s); and g.
• Embodiments of the ninth aspect provide methods which further comprise refining and evaluating the full or partial 3-D coordinates. These methods may thus be used, for example, to generate 3- dimensional structures for proteins for which 3-dimensional atomic coordinates have not been determined. As such, the newly generated structure can help to elucidate enzymatic mechanisms, or be used in conjunction with other molecular modeling techniques in structure based drug design.
• the present invention provides methods for identifying inhibitors, ligands, and the like, of DPP-IV by providing the coordinates of a molecule of DPP-IV to a computerized modeling system; identifying chemical entities that are likely to bind or interact with the molecule (e.g., by screening a small molecule library); and, optionally, procuring or synthesizing and assaying the compounds or analogues derived thereof for bioactivity.
• Further aspects of the present invention relate to methods for identifying potential ligands for DPP-IV or homologues, or analogue or variants thereof comprising: a.
• DPP-IV enzyme or homologue or analogue or variant thereof as defined by atomic coordinates that are substantially similar to the atomic coordinates listed in FIG. 2 on a computer display screen; b. optionally replacing one or more the enzyme amino acid residues listed in SEQ ID NO:1 , or preferably one or more amino acid residues selected from Glu205, Glu206, Tyr547, Ser630, Tyr631 , Tyr662, Tyr666, Asp708, Asn710 and His740, in said three-dimensional structure with a different naturally occurring amino acid or an unnatural amino acid to display a variant structure; c.
• the structural aspects of the ligand may be modified to generate a structural analog of the ligand.
• This analog can then be used in the above methods to identify binding ligands.
• One of ordinary skill in the art will know the various ways a structure may be modified.
• the methods further comprise computationally modifying the structure of the ligand; computationally determining the fit of the modified ligand using the three-dimensional coordinates described in FIG. 2, or portions thereof; contacting said modified ligand with said enzyme, or homologue, or variant thereof in an in vitro or in vivo setting; and measuring the ability of said ligand to modulate the activity of said enzyme.
• the present invention provides compositions, such as, pharmaceutical compositions comprising the inhibitors or ligands designed according to any of the methods of the present invention.
• a composition is provided that includes an inhibitor or ligand designed or identified by any of the above methods.
• the composition is a pharmaceutical composition.
• the twelfth aspect of the present invention are methods for treating Type 2 diabetes, Type 1 diabetes, impaired glucose tolerance, hyperglycemia, metabolic syndrome (syndrome X and/or insulin resistance syndrome), glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, acute coronary syndrome, short stature due to growth hormone deficiency, infertility due to polycystic ovary syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, eating disorders, chronic pain, alcohol addiction, diseases associated with intestinal motility, ulcers, irritable bowel syndrome, inflammatory bowel syndrome; short bowel syndrome; and the prevention of disease progression in Type 2 diabetes, comprising administering pharmaceutical compositions, identified by structure based design using the atomic coordinates, or portions thereof,
• Figure 1 is an orthogonal view of an embodiment of DPP-IV shown in a ribbon representation.
• FIG. 2 is a list of the X-ray coordinates of a DPP-IV crystal as described in the Examples.
• DETAILED DESCRIPTION OF THE INVENTION The present invention relates to crystalline compositions of DPP-IV, 3-D X-ray atomic coordinates of said crystalline compositions, methods of preparing said compositions, methods of determining the 3-D X-ray atomic coordinates of said crystalline compositions, and methods of using said atomic coordinates in conjunction with computational methods to identify binding site(s), or identify ligands which interact with said binding site(s) to agonize or antagonize DPP-IV.
• affinity refers to the tendency of a molecule to associate with another.
• the affinity of a drug is its ability to bind to its biological target (receptor, enzyme, transport system, etc.)
• affinity can be thought of as the frequency with which the drug, when brought into the proximity of a receptor by diffusion, will reside at a position of minimum free energy within the force field of that receptor.
• agonist refers to an endogenous substance or a drug that can interact with a receptor and initiate a physiological or a pharmacological response characteristic of that receptor (contraction, relaxation, secretion, enzyme activation, etc.)
• analog refers to a drug or chemical compound whose structure is related in some way to that of another drug or chemical compound, but whose chemical and biological properties may be quite different.
• antagonist refers to a drug or a compound that opposes the physiological effects of another. At the receptor level, it is a chemical entity that opposes the receptor- associated responses normally induced by another bioactive agent.
• asymmetric unit refers to the basic motif, which is repeated in 3-D space by the symmetry operators of the crystallographic space group, of which the coordinated of the structure are determined. It is the smallest part of the crystal structure from which the complete structure can be built using space group symmetry.
• binding site refers to a specific region (or atom) in a molecular entity that is capable of entering into a stabilizing interaction with another molecular entity. In certain embodiments the term also refers to the reactive parts of a macromolecule that directly participate in its specific combination with another molecule. In other embodiments, a binding site may be comprised or defined by the three dimensional arrangement of one or more amino acid residues within a folded polypeptide.
• the binding site further comprises prosthetic groups, water molecules or metal ions which may interact with one or more amino acid residues.
• Prosthetic groups, water molecules, or metal ions may be apparent from X-ray crystallographic data, or may be added to an apo protein or enzyme using in silico methods.
• bioactivity refers to DPP-IV activity that exhibits a biological property conventionally associated with a DPP-IV agonist or antagonist, such as a property that would allow treatment of one or more of the various diseases such as, for example, diabetes, glucose tolerance, obesity, appetite regulation, lipidemia, osteoporosis, neuropeptide metabolism and T-cell activation, among others.
• catalytic domain refers to the catalytic domain of the DPP-IV class of enzymes, which features a conserved segment of amino acids in the carboxy-terminal portion of the proteins, wherein this segment has been demonstrated to include the catalytic site of these enzymes. This conserved catalytic domain extends approximately from residue 552 to 766 of the full-length enzyme of DPP-IV (SEQ ID NO:1 ). "To clone” as used herein, means obtaining exact copies of a given polynucleotide molecule using recombinant DNA technology.
• to clone into may be meant as inserting a given first polynucleotide sequence into a second polynucleotide sequence, preferably such that a functional unit combining the functions of the first and the second polynucleotides results.
• a polynucleotide from which a fusion protein may be translationally provided which fusion protein comprises amino acid sequences encoded by the first and the second polynucleotide sequences.
• co-crystallization is taken to mean crystallization of a preformed protein/I igand complex.
• complex or “co-complex” are used interchangeably and refer to a DPP-IV molecule, or a variant, or homologue of DPP-IV in covalent or non-covalent association with a substrate, ligand, inhibitor.
• contacting as used herein applies to in silico, in vitro, and/or in vivo experiments.
• Diseases and particularly “diseases that are associated with proteins that are subject to processing by DPP-IV”, include, but are not limited to, for example, Type 2 diabetes, Type 1 diabetes, impaired glucose tolerance, hyperglycemia, metabolic syndrome (syndrome X and/or insulin resistance syndrome), glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, acute coronary syndrome, short stature due to growth hormone deficiency, infertility due to polycystic ovary syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, eating disorders, chronic pain, alcohol addiction, diseases associated with intestinal motility, ulcers, irritable bowel syndrome, inflammatory bowel syndrome; short bowel syndrome; and the prevention of disease progression in Type 2 diabetes.
• extracellular domain refers to the extracellular domain of DPP-IV, which features a conserved segment of amino acids, whereby this segment has been demonstrated to include glycosylation sites, a cysteine-rich region and the catalytic active site. This conserved extracellular domain extends approximately from residue Gly31 to Pro766 of the full length enzyme (SEQ ID NO:1 ).
• gene refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
• hDPP-IV Human DPP-IV
• ADA adenosine deaminase binding protein
• hDPP-IV is a single polypeptide chain of 766 amino acids, which consists if five regions: a cytoplasmic region (residues about 1 -6), a transmembrane region (residues about 7-28), a highly gycosylated region (residues about 29-323), a cysteine-rich region (residues about 324-551 ), and a catalytic region (residues about 552-766) (Hiramatsu et al., (2003)).
• the term "high affinity” as used herein means strong binding affinity between molecules with a dissociation constant K D of no greater than 1 ⁇ M.
• the K D is less than 100 nM, 10 nM, 1 nM, 100 pM, or even 10 pM or less.
• the two molecules can be covalently linked (K D is essentially 0).
• the term "homologue” as used herein means a protein, polypeptide, oligopeptide, or portion thereof, having preferably at least 95% amino acid sequence identity with DPP-IV enzyme as described in SEQ ID NO: 1 or SEQ ID NO:2 or with any extracellular domain described herein, or with any functional or structural domain of lipid binding protein.
• SEQ ID NO:1 is the full-length amino acid sequence of a wild- type human DPP-IV
• SEQ ID NO:2 is the hDPP-IV construct including residues 31 -766 which, as described in the Examples, was purified, expressed and crystallized.
• residues 31 -766 which, as described in the Examples, was purified, expressed and crystallized.
• the term "substantially similar atomic coordinates" or atomic coordinates that are “substantially similar” refers to any set of structure coordinates of DPP-IV or DPP-IV homologues, or DPP-IV variants, polypeptide fragments, described by atomic coordinates that have a root mean square deviation for the atomic coordinates of protein backbone atoms (N, C ⁇ , C, and O) of less than about 2.5, 2.0 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed using backbone atoms of structure coordinates listed in FIG. 2.
• structures that have substantially similar coordinates as those listed in FIG. 2 shall be considered identical to the coordinates listed in FIG. 2.
• substantially similar also applies to an assembly of amino acid residues that may or may not form a contiguous polypeptide chain, but whose three dimensional arrangement of atomic coordinates have a root mean square deviation for the atomic coordinates of protein backbone atoms (N, C ⁇ , C, and O), or the side chain atoms, of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed-using backbone atoms, or the side chain atoms- of the atomic coordinates of similar or the same amino acids from the coordinates listed in FIG. 2.
• Those skilled in the art further understand that the coordinates listed in FIG.
• the coordinates listed in Fig. 2, or portions thereof may be transformed by algorithms which translate or rotate the atomic coordinates.
• molecular mechanics, molecular dynamics or ab intio algorithms may modify the atomic coordinates.
• Atomic coordinates generated from the coordinates listed in FIG. 2, or portions thereof, using any of the aforementioned algorithms shall be considered identical to the coordinates listed in FIG. 2.
• the term "in silico" as used herein refers to experiments carried out using computer simulations.
• the in silico methods are molecular modeling methods wherein 3-dimensional models of macromolecules or ligands are generated.
• the in silico methods comprise computationally assessing ligand binding interactions.
• ligand describes any molecule, e.g., protein, peptide, peptidomimetics, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., which binds or interacts, generally but not necessarily specifically to or with another molecule.
• the ligand is an agonist, whereby the molecule upregulates (i.e., activates or stimulates, e.g., by agonizing or potentiating) activity, while in another aspect of the invention the ligand is an inhibitor or antagonist, whereby the molecule down regulates (i.e., inhibits or suppresses, e.g. by antagonizing, decreasing or inhibiting) the activity.
• modulate refers to both upregulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down-regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting) of a bioactivity.
• pharmacophore refers to the " ensemble of steric and electronic features of a particular structure that is necessary to ensure the optimal supramolecular interactions with a specific biological target structure and to trigger (or to block) its biological response.
• a pharmacophore is an abstract concept that accounts for the common molecular interaction capacities of a group of compounds towards their target structure.
• the term can be considered as the largest common denominator shared by a set of active molecules.
• Pharmacophoric descriptors are used to define a pharmacophore, including H-bonding, hydrophobic and electrostatic interaction sites, defined by atoms, ring centers and virtual points.
• a pharmacophore may represent an ensemble of steric and electronic factors which are necessary to insure supramolecular interactions with a specific biological target structure.
• a pharmacophore may represent a template of chemical properties for an active site of a protein/enzyme representing these properties' spatial relationship to one another that theoretically defines a ligand that would bind to that site.
• the term "precipitant" as used herein includes any substance that, when added to a solution, causes a precipitate to form or crystals to grow.
• Suitable precipitants include, but are not limited to, alkali (e.g., Li, Na, or K), or alkaline earth metal (e.g., Mg, or Ca) salts, and transition metal (e.g., Mn, or Zn) salts.
• Common counter ions to the metal ions include, but are not limited to, halides, phosphates, citrates and sulfates.
• prodrug refers to drugs that, once administered, are chemically modified by metabolic processes to become pharmaceutically active. In certain embodiments the term also refers to any compound that undergoes biotransformation before exhibiting its pharmacological effects.
• Prodrugs can thus be viewed as drugs containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate properties, usually undesirable, in the parent molecule.
• the term "receptor” as used here in refers to a protein or a protein complex in or on a cell that specifically recognizes and binds to a compound acting as a molecular messenger (neurotransmitter, hormone, lymphokine, lectin, drug, etc.). In a broader sense, the term receptor is used interchangeably with any specific (as opposed to non-specific, such as binding to plasma proteins) drug binding site, also including nucleic acids such as DNA.
• recombinant protein refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding a polypeptide is inserted into a suitable expression vector which is, in turn, used to transform a host cell to produce the polypeptide encoded by the DNA.
• This polypeptide may be one that is naturally expressed by the host cell, or it may be heterologous to the host cell, or the host cell may have been engineered to have lost the capability to express the polypeptide which is otherwise expressed in wild type forms of the host cell.
• the polypeptide may also be, for example, a fusion polypeptide.
• the phrase "derived from”, with respect to a recombinant gene, is meant to include within the meaning of "recombinant protein” those proteins having an amino acid sequence of a native polypeptide, or an amino acid sequence similar thereto which is generated by mutations, including substitutions, deletions and truncation, of a naturally occurring form of the polypeptide.
• selective DPP-IV inhibitor refers to a substance, such as for example, an organic molecule, that effectively inhibits an enzyme from the DDP-IV family to a greater extent than any other DPP enzyme.
• a selective DPP-IV inhibitor is a substance, having a K, for inhibition of DPP-IV that is less than about one-half, one-fifth, or one-tenth the K, that the substance has for inhibition of any other DPP enzyme.
• the substance inhibits DPP-IV activity to the same degree at a concentration of about one-half, one-fifth, one-tenth or less than the concentration required for any other DPP enzyme.
• a substance is considered to effectively inhibit DPP-IV if it has an IC 50 or K, of less than or about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM or 10 nM.
• small molecules refers to drugs as they are orally available (unlike proteins which must be administered by injection, topically or inhalation).
• the size of the small molecules is generally under 1000 Daltons, but many estimates seem to range between 300 to 700 Daltons.
• space group refers to the lattice and symmetry of the crystal. In a space group designation the capital letter indicates the lattice type and the other symbols represent symmetry operations that can be carried out on the contents of the asymmetric unit without changing its appearance.
• therapeutically effective amount is meant that amount which is capable of at least partially reversing and/or treat the symptoms of the disease.
• a therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on a consideration of the species of the mammal, the size of the mammal, the type of delivery system used, and the type of administration relative to the progression of the disease.
• a therapeutically effective amount can be determined by one of ordinary skill in the arts.
• the term "transfection” means the introduction of a nucleic acid, e.g., via an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
• Transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA and, for example, the transformed cell expresses a recombinant form of a polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the polypeptide is disrupted.
• variants in relation to the polypeptide sequence in SEQ ID NO:1 or SEQ ID NO:2 include any substitution of, variation of, modification of, replacement of, deletion of, or addition of one or more amino acids from or to the sequence providing a resultant polypeptide sequence for an enzyme having DPP-IV activity.
• the variant, homologue, fragment or portion, of SEQ ID NO:1 or SEQ ID NO:2 comprises a polypeptide sequence of at least 5 contiguous amino acids, preferably at least 10 contiguous amino acids, preferably at least 15 contiguous amino acids, preferably at least 20 contiguous amino acids, preferably at least 25 contiguous amino acids, or preferably at least 30 contiguous amino acids.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• One type of preferred vector is an episome, i.e., a nucleic acid capable of extra- chromosomal replication.
• Suitable host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
• virus e.g., vaccinia virus, adenovirus, etc.
• insect cell systems infected with virus e.g., baculovirus
• microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
• the expression elements of vectors vary in their strengths and specificities. As those depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
• Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which
• expression vectors In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
• plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.
• the invention is intended to include such othefr forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
• nucleotide sequence coding for a DPP-IV polypeptide, or a functional fragment, including the C-terminal peptide fragment of the catalytic domain of DPP-IV protein, and/or derivatives or analogs thereof, including a chimeric protein, thereof can be inserted into a suitable expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
• the nucleic acid encoding a DPP-IV polypeptide of the invention or a functional fragment comprising the extracellular domain of of the DPP-IV protein, or a homologue, an analog, or variant thereof is operationally associated with a promoter in an expression vector of the invention.
• the expression vector contains the nucleotide sequence coding for the polypeptide comprising the amino acid sequence spanning amino acids Gly31 to Pro766 listed in SEQ ID NO:1. Both cDNA and genomic sequences can be cloned and expressed under the control of such regulatory sequences.
• An expression vector also preferably includes a replication origin.
• the necessary transcriptional and translational signals can be provided on a recombinant expression vector.
• all genetic manipulations described for the DPP-IV gene in this section may also be employed for genes encoding a functional fragment, including the C-terminal peptide fragment of the catalytic domain of the DPP-IV protein, derivatives or analogs thereof, including a chimeric protein thereof.
• Suitable host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
• virus e.g., vaccinia virus, adenovirus, etc.
• insect cell systems infected with virus e.g., baculovirus
• microorganisms such as yeast containing yeast vectors
• bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
• the expression elements of vectors vary in their strengths and specificities. As those depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
• a recombinant DPP-IV protein of the invention may be expressed chromosom
• any of a number of amplification systems may be used to achieve high levels of stable gene expression.
• a suitable cell for purposes of this invention is one into which the recombinant vector comprising the nucleic acid encoding DPP-IV protein is cultured in an appropriate cell culture medium under conditions that provide for expression of DPP-IV protein by the cell.
• Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences.
• DPP-IV protein expression may be controlled by any promoter/enhancer element known in the art, provided that these regulatory elements must be functional in the host selected for expression, as would be appreciated by those of skill in the art.
• Vectors containing a nucleic acid encoding a DPP-IV protein of the invention can be identified, for example, by four general approaches: (1 ) PCR amplification of the desired plasmid DNA or specific mRNA; (2) nucleic acid hybridization; (3) presence or absence of selection marker gene functions; and (4) expression of inserted sequences.
• the invention is further intended to include other forms of identification of vectors, containing a nucleic acid encoding a DPP-IV protein of the present invention, which serve equivalent functions and which become known in the art subsequently hereto.
• the nucleic acids can be amplified by PCR to provide for detection of the amplified product.
• the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene.
• the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., beta-galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector.
• selection marker e.g., beta-galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
• recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant vector, provided that the expressed protein assumes a functionally active conformation.
• a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention as known by those of skill in the art. Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity.
• the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda); and plasmid and cosmid DNA vectors, to name but a few.
• Vectors can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621 -14624; Hartmut et al., Canadian Patent Application No. 2,012,311 , filed Mar. 15, 1990).
• X-ray structure coordinates define a unique configuration of points in space.
• a set of structure coordinates for a protein or a protein/ligand complex, or a portion thereof define a relative set of points that, in turn, define a configuration in three dimensions.
• a similar or identical configuration can be defined by an entirely different set of coordinates, provided the distances and angles between atomic coordinates remain essentially the same.
• a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same.
• One aspect of the present invention relates to a crystalline composition
• a crystalline composition comprising a polypeptide with an amino acid sequence spanning amino acids Gly31 to Pro766 listed in SEQ ID NO:1.
• the crystallized complex is characterized by the structural coordinates listed in FIG. 2, or portions thereof.
• the atoms of the ligand are within about 5 angstroms of one or more DPP-IV amino acids in SEQ ID NO: 1 preferably selected from Glu205, Glu206, Tyr547, Ser630, Tyr631 , Tyr662, Tyr666, Asp708, Asn710 and His740.
• One embodiment of the crystallized complex is characterized as belonging to the space group P4 3 2 !
• the ligand may be a small molecule which binds to DPP-IV extracelluar domain defined by SEQ ID NO:2, or portions thereof, with a Kj of less than about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM, or 10 nM.
• Various computational methods can be used to determine whether a molecule or a binding pocket portion thereof is "structurally equivalent,” defined in terms of its three-dimensional structure, to all or part of DPP-IV or its binding pocket(s).
• Such methods may be carried out in current software applications, such as the molecular similarity application of QUANTA (Accelrys Inc., San Diego, Calif.).
• the molecular similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
• the procedure used in molecular similarity to compare structures is divided into four steps: (1 ) load the structures to be compared; (2) optionally define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results.
• Each structure is identified by a name.
• One structure is identified as the target (i.e., the fixed structure), while all remaining structures are working structures (i.e., moving structures).
• equivalent atoms are defined as protein backbone atoms (N, C ⁇ , C, and O) for all conserved residues between the two structures being compared.
• a conserved residue is defined as a residue that is structurally or functionally equivalent (See Table 4 infra).
• rigid fitting operations are considered.
• flexible fitting operations may be considered.
• Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structural coordinates listed in FIG.
• root mean square deviation means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object.
• root mean square deviation defines the variation in the backbone of a protein from the backbone of DPP-IV or a binding pocket portion thereof, as defined by the structural coordinates of DPP-IV described herein.
• the refined x-ray coordinates of the extracellular domain of DDP-IV (amino acids 38 to 766 as listed in SEQ ID NO:2), Zn 2+ , Mg 2+ , and 32 water molecules are as listed in FIG. 2.
• One orthogonal view of the molecule is shown in FIG. 1.
• the crystal structure of the extracellular domain (amino acids 31 -766 of SEQ ID NO:1 ) was solved to a resolution 2.7 A.
• the asymmetric unit is composed of one dimer.
• the structure includes two domains, the ⁇ -propeller domain (residues 55-497) and the catalytic domain (residues 508-766), together with a couple of linker regions (1 -54 and 498-507).
• the propeller domain packs against the hydrolase domain, and the catalytic triad of DPP-IV composed of residues Ser630, His740 and Asp708, which are located which the last 140 residues of the C-terminal region is at the interface of the two domains.
• the present invention provides a molecule or molecular complex that includes at least a portion of a DDP-IV and/or substrate binding pocket.
• the DDP-IV binding pocket includes the amino acids listed in Table 1 , the binding pocket being defined by a set of points having a root mean square deviation of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A, from points representing the backbone atoms of the amino acids in Table 1.
• the DPP-IV substrate binding pocket includes the amino acids selected from Glu205, Glu206, Tyr547, Ser630, Tyr631 , Tyr662, Tyr666, Asp708, Asn710 and His740 from SEQ ID NO:1.
• Table 1 Identified residues 5 A away from the binding pocket of the DPP-IV crystal structure.
• One embodiment of the invention describes an isolated polypeptide consisting of a portion of DPP-IV which functions as the binding site when folded in a 3-D orientation.
• One embodiment is an isolated polypeptide comprising a portion of DPP-IV, wherein the portion starts at about amino acid residue Gly31 , and ends at about amino acid residue Pro766 as described in SEQ ID NO: , or a sequence that is at least 95% or 98% homologous to a polypeptide with an amino acid sequence spanning amino acids Gly31 to Pro766 as listed in SEQ ID NO:1.
• Another embodiment comprises crystalline compositions comprising variants of DPP-IV.
• Variants of the present invention may have an amino acid sequence that is different by one or more amino acid substitutions to the sequence disclosed in SEQ ID NO:1 or SED ID NO:2.
• Embodiments which comprise amino acid deletions and/or additions are also provided.
• the variant may, for example, have conservative changes (amino acid similarity), wherein a substituted amino acid has similar structural or chemical properties, for example, the replacement of leucine with isoleucine.
• determining which and how many amino acid residues may be substituted, inserted, or deleted without adversely affecting biological or pharmacological activity may be reasonably inferred in view of this disclosure, and may further be found using computer programs well known in the art, for example, DNAStar® software (DNAStar Inc.
• amino acid substitutions may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues provided that a biological and/or pharmacological activity of the native molecule is retained.
• Negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids, with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, and valine; amino acids with aliphatic head groups include glycine, alanine; asparagine, glutamine, serine; and amino acids with aromatic side chains include threonine, phenylalanine, and tyrosine. Examples of conservative substitutions are set forth in Table 4 as follows: Table 4:
• “Homology” is a measure of the identity of nucleotide sequences or amino acid sequences. To characterize the homology, subject sequences are aligned so that the highest percentage homology (match) is obtained, after introducing gaps, if necessary, to achieve maximum percent homology. N- or C- terminal extensions shall not be construed as affecting homology. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. Computer program methods to determine identity between two sequences, for example, include DNAStar® software; the GCG® program package (Devereux, J., et al. Nucleic Acids Research (1984) 12(1 ): 387); BLASTP, BLASTN, FASTA (Atschul, S.F.
• the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence or amino acid sequence and that gaps in homology of up to about 90% of the total number of nucleotides in the reference sequence are allowed.
• Similarity between two sequences includes direct matches as well a conserved amino acid substitutes which possess similar structural or chemical properties, e.g., similar charge as described in Table 2. Percentage similarity (conservative substitutions) between two polypeptides may also be scored by comparing the amino acid sequences of the two polypeptides by using programs well known in the art, including the BESTFIT program, by employing default settings for determining similarity.
• a further embodiment of the invention is a crystal comprising the coordinates of FIG. 2, wherein the amino acid sequence is represented by SEQ ID NO:1.
• a further embodiment of the invention is a crystal comprising the coordinates of FIG.2, wherein the amino acid sequence is at least 95% or 98% homologous to the amino acid sequence represented by SEQ ID NO:1.
• Various methods for obtaining atomic coordinates of structurally homologous molecules and molecular complexes using homology modeling are disclosed in, for example, US Patent No: 6,356,845.
• a ligand (antagonist or agonist) may be examined through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (See for example, Morris et al., J. Computational Chemistry, 19:1639-1662 (1998)). This procedure can include in silico fitting of potential ligands to the DPP-IV crystal structure to ascertain how well the shape and the chemical structure of the potential ligand will complement or interfere with the catalytic domain of DPP-IV.
• One embodiment of the present invention relates to methods of identifying agents that bind to a binding site on DPP-IV extracellular domain wherein the binding site comprises amino acid residues Glu205, Glu206, Tyr547, Ser630, Tyr631 , Tyr662, Tyr666, Asp708, Asn710 and His740 of SEQ ID NO:1 , comprising: contacting DPP-IV with a test ligand under conditions suitable for binding of the test compound to the binding site, and determining whether the test ligand binds in the binding site, wherein if binding occurs, the test ligand is an agent that binds in the binding site.
• the testing may be carried out in silico using a variety of molecular modeling software algorithms including, but not limited to, DOCK, ALADDIN, CHARMM simulations, AFFINITY, C2-LIGAND FIT, Catalyst, LUDI, CAVEAT, and CONCORD.
• molecular modeling software algorithms including, but not limited to, DOCK, ALADDIN, CHARMM simulations, AFFINITY, C2-LIGAND FIT, Catalyst, LUDI, CAVEAT, and CONCORD.
• a potential ligand may be obtained by screening a random peptide library produced by a recombinant bacteriophage (Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)) or a chemical library, or the like.
• a ligand selected in this manner can be then be systematically modified by computer modeling programs until one or more promising potential ligands are identified.
• Such analysis for example, has been shown to be effective in the development of HIV protease inhibitors.
• a potential ligand (agonist or antagonist)
• it can be either selected from a library of chemicals as are commercially available from most large chemical companies or, alternatively, the potential ligand may be synthesized de novo. As mentioned above, the de novo synthesis of one or even a relatively small group of specific compounds is reasonable in the art of drug design.
• the potential ligand can be placed into any standard binding assay as well known to those skilled in the art to test its effect on DPP-IV activity.
• a supplemental crystal can be grown comprising a protein- ligand complex formed between a DPP-IV protein and the drug.
• the crystal diffracts X-rays allowing the determination of the atomic coordinates of the protein-ligand complex to a resolution of less than 5.0 Angstroms, more preferably less than 3.0 Angstroms, and even more preferably less than 2.0 Angstroms.
• the three-dimensional structure of the supplemental crystal can be determined by Molecular Replacement Analysis. Molecular replacement uses a known three-dimensional structure as a search model to determine the structure of a closely related molecule or protein-ligand complex in a new crystal form.
• the measured X-ray diffraction properties of the new crystal are compared with the search model structure to compute the position and orientation of the protein in the new crystal.
• Computer programs that can be used include: X-PLOR and AMORE (J. Navaza, Acta Crystallographies ASO, 157-163 (1994)).
• X-PLOR and AMORE J. Navaza, Acta Crystallographies ASO, 157-163 (1994)
• an electron density map can be calculated using the search model to provide X-ray phases. Thereafter, the electron density is inspected for structural differences, and the search model is modified to conform to the new structure.
• the claimed structure of DPP-IV can be used to solve the three- dimensional structures of any such DPP-IV complexed with a ligand.
• STAT crystals include: QUANTA; CHARMM; INSIGHT; SYBYL; MACROMODEL; and ICM.
• Suitable in silico methods for screening, designing or selecting ligands are disclosed in, for example, U.S. Patent No. 6,356,845.
• the present invention discloses binding agents which interact with a binding site of DPP-IV defined by a set of points having a root mean square deviation of less than about 2.5, 2.0, 1.7, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A from points representing the backbone atoms of the amino acids represented by the structure coordinates listed in FIG. 2. Such embodiments represent variants of the DPP-IV crystal.
• the present invention provides ligands which bind to a folded polypeptide comprising an amino acid sequence spanning amino acids 31 to 766 listed in SEQ ID NO:1 , or a homologue or variant thereof.
• the ligand is a competitive or uncompetitive inhibitor of DPP-IV.
• the ligand inhibits DPP-IV with an IC 50 or K, of less than about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM or 10 nM. In yet further embodiments, the ligand inhibits DPP-IV with a K, that is less than about one-half, one-fifth, or one-tenth the K, that the substance has for inhibition of any other DPP-IV enzyme. In other words, the substance inhibits DPP-IV activity to the same degree at a concentration of about one-half, one-fifth, one-tenth or less than the concentration required for any other DPP enzyme.
• One embodiment of the present invention relates to ligands, such as proteins, peptides, peptidomimetics, small organic molecules, etc., designed or developed with reference to the crystal structure of DPP-IV as represented by the coordinates presented herein in FIG. 2, and portions thereof.
• Such binding agents interact with the binding site of the DPP-IV represented by one or more amino acid residues selected from Glu205, Glu206, Tyr547, Ser630, Tyr631 , Tyr662, Tyr666, Asp708, Asn710 and His740.
• Machine Readable Storage Media Transformation of the structure coordinates for all or a portion of DPP-IV or one of its binding pockets, for structurally homologous molecules as defined below, or for the structural equivalents of any of these molecules or molecular complexes as defined above, into three-dimensional graphical representations of the molecule or complex can be conveniently achieved through the use of commercially-available software.
• the invention thus further provides a machine-readable storage 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 molecule or molecular complexes of this invention that have been described above.
• the machine-readable data storage medium comprises 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 a molecule or molecular complex comprising all or any parts of a DPP-IV binding pocket, as defined above.
• the machine-readable data storage medium is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex defined by the structure coordinates of the amino acids listed in FIG. 4, plus or minus a root mean square deviation from the backbone atoms of said amino acids of not more than 2.0 .A.
• the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structural coordinates set forth in FIG. 2, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structural coordinates corresponding to the second set of machine readable data.
• a system for reading a data storage medium may include a computer comprising a central processing unit (“CPU"), a working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube (“CRT”) displays, light emitting diode (“LED”) displays, liquid crystal displays (“LCDs”), electroluminescent displays, vacuum fluorescent displays, field emission displays (“FEDs”), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, touch screens, etc.), one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.
• CPU central processing unit
• working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e
• the system may be a stand-alone computer, or may be networked (e.g., through local area networks, wide area networks, intranets, extranets, or the internet) to other systems (e.g., computers, hosts, servers, etc.).
• the system may also include additional computer controlled devices such as consumer electronics and appliances.
• Input hardware may be coupled to the computer by input lines and may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device.
• Output hardware may be coupled to the computer by output lines and may similarly be implemented by conventional devices.
• the output hardware may include a display device for displaying a graphical representation of a binding pocket of this invention using a program such as QUANTA as described herein.
• Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
• a CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage devices, accesses to and from working memory, and determines the sequence of data processing steps.
• a number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein.
• Machine-readable storage devices useful in the present invention include, but are not limited to, magnetic devices, electrical devices, optical devices, and combinations thereof.
• Examples of such data storage devices include, but are not limited to, hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device.
• these storage devices include necessary hardware (e.g., drives, controllers, power supplies, etc.) as well as any necessary media (e.g., disks, flash cards, etc.) to enable the storage of data.
• compositions The present invention provides methods for treating certain diseases in a mammal, preferably a human being, in need of such treatment using the ligands, and preferably the inhibitors, as described herein.
• the ligand can be advantageously formulated into pharmaceutical compositions comprising a therapeutically effective amount of the ligand, a pharmaceutically acceptable carrier and other compatible ingredients, such as adjuvants, Freund's complete or incomplete adjuvant, suitable for formulating such pharmaceutical compositions as is known to those skilled in the art.
• compositions containing the ligand can be used for treatment of diseases that are associated with proteins that are subject to processing by DPP-IV, such as Type 2 diabetes, Type 1 diabetes, impaired glucose tolerance, hyperglycemia, metabolic syndrome (syndrome X and/or insulin resistance syndrome), glucosuria, metabolic acidosis, arthritis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, obesity, conditions exacerbated by obesity, hypertension, hyperlipidemia, atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone fracture, acute coronary syndrome, short stature due to growth hormone deficiency, infertility due to polycystic ovary syndrome, anxiety, depression, insomnia, chronic fatigue, epilepsy, eating disorders, chronic pain, alcohol addiction, diseases associated with intestinal motility, ulcers, irritable bowel syndrome, inflammatory bowel syndrome; short bowel syndrome; and the prevention of disease progression in Type 2 diabetes.
• the pharmaceutical composition is administered to the mammal in a therapeutically effective amount such that treatment of the disease occurs.
• the present invention is further illustrated by the following examples, which should not be construed as limiting in any way.
• the contents of all cited references (including literature references, patents, published patent applications as cited throughout this application) are hereby expressly incorporated by reference in their entireties.
• the practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology and recombinant DNA, X-ray crystallography, and molecular modeling which are within the skill of the art. As those of skill in the art will understand, such techniques are explained fully in the literature.
• hDPP-IV Construction of hDPP-IV: Residues 31 to 766 of homo sapien (human) wild type DPP-IV (SEQ ID NO:1) were amplified by PCR using the following primers DPPIV-Fc31 - BamF (5'-
• TTAAGGATCCTGGCACAGATGATGCTACAGCTGAC-3' (SEQ ID NO: 3)), which introduced a Bam HI site at the N-terminus
• DPPIV-ChisT-XhoR (5'-AATTCTCGAGTTACTAGTGAT GATGGTGGTGATGGCTGCCGCGCGGCACCAGAGGTAAAGAGAAACATTGTTTTATGAAGTGGC-3' (SEQ ID NO: 4)), which introduced His 6 -tag, thrombin cleavage site and Xho I site at the C-terminus.
• the vector contained a polyhedrin promoter and the honeybee melittin secretion signal for efficient, high-level secretion of the recombinant protein.
• the transfection mixture was removed after 5 h of incubation (27°C) and the cells were overlaid with 25 ml of Sf-900 II SFM.
• the recombinant virus were harvested at 72h post-transfection and further amplification of the virus was achieved by infecting 100 ml of Sf-9 cells (1.2 X 10 6 cells/ml) with 2 ml of the recombinant virus for 65-72 h.
• BIIC Baculo Infected Insect Cells
• E coli clones with recombinant bacmid were obtained after transformation of E. coli DH1 OBac cells (Invitrogen Corp., Frederick, MD) with 5 ng of DPPIV-HBM31 -HT plasmid DNA and blue/white-screening according to manufacturer's protocol (Invitrogen).
• Monolayers of Sf9 cells (20 X 10 6 cells in a 162 cm 2 culture flask) were transfected by overlaying 20 ml of transfection mixture containing 100 ⁇ l of mini-prep bacmid DNA and 100 ⁇ l of CellFECTIN reagent (Gibco BRL) in Sf-900 II SFM.
• the transfection mixture was removed after 5 h of incubation (27 a C) and the cells were overlaid with 25 ml of Sf-900 II SFM.
• the recombinant virus were harvested at 72h post-transfection and further amplification of the virus was achieved by infecting 100 ml of Sf-9 cells (1.2 X 10 6 cells/ml) with 2 ml of the recombinant virus for 65-72 h.
• BIIC Baculo Infected Insect Cells
• Example 2 Purification of His6-tagged DPP-IV wild type extracellular domain After clarification by centrifugation and filtration, 10 liters of culture media containing secreted human DPP-IV-31-766-C-his 6 was concentrated 10-20 fold using a hollow fiber filter unit which had been washed with exchange Buffer A (50 mM Tris, 0.3 M NaCI, 1 mM TCEP, pH 8). The concentrated media was exchanged with 5 volumes of Buffer A. After clarification by filtration, imidazole was added to 10 mM by addition of Buffer B (50 mM TrisCI, 0.3 M NaCI, 0.25 M imidazole, 1 mM TCEP, pH 8).
• Buffer A 50 mM Tris, 0.3 M NaCI, 1 mM TCEP, pH 8.
• the sample was applied to a 40 mL immobilized metal affinity column (Ni-NTA Superflow, Qiagen), which had been equilibrated in Buffer A (50 mM TrisCI, 0.3 M NaCI, 1 mM TCEP, pH 8) at 6-8 mL/min.
• Buffer A 50 mM TrisCI, 0.3 M NaCI, 1 mM TCEP, pH 8
• the column was washed with Buffer A to achieve a stable baseline at 280 nm.
• Bound protein was eluted at a lower flow rate in a linear gradient from 0 - 20%B in 4 column volumes (cv) (5%B / cv) followed by a step to 100% B, held isocratic ally for 4 cv.
• Fractions were analyze by SDS-PAGE on 4-12% bis-tris in MOPS buffer using the NuPAGE system (Invitrogen). Fractions containing DPP-IV were pooled and dialyzed at 4 degrees C against 2 changes of dialysis buffer (50 mM TrisCI, 0.1 M NaCI, 1 mM TCEP, pH 8. After dialysis, the sample was concentrated to 6-10 mg/mL and fractionated by size exclusion chromatography on Superdex 200 prep grade HiLoad 16/60 (Amersham Biosciences). DPP-IV eluted as an apparent dimer. Fractions were analyze by SDS-PAGE on 4-12% bis-tris in MOPS buffer using the NuPAGE system (Invitrogen).
• Example 3 Crystallization of DPP-IV DPP-IV of Example 2 was concentrated into buffer containing 50 mM TrisCI, 25 mM NaCI, 1 mM TCEP, pH 8, to 8-10 mg/mL. Leads were obtained through sparse matrix screening at 22°. Optimized crystals grew in drops made from 1.5 ⁇ L of protein + 1.5 ⁇ L of reservoir solution (0.1 M TrisCI, pH 8.5, 0.2 M sodium acetate, 10-16% PEG 4000) equilibrating over the same reservoir solution.
• Crystals were transferred to a solution containing 0.1 M TrisCI, pH 8.5, 0.2 M sodium acetate, 14-16% PEG 4000, and 20% ethylene glycol. Crystals were flash frozen in gaseous or liquid nitrogen for data collection.
• Example 4 X-ray data collection, structure determination and refinement of DPP-IV The crystals prepared in Example 3 were transferred to a cryoprotectant solution, made up of the reservoir solution, with 15-25% ethylene glycol, and then flash-frozen in a stream of cold nitrogen gas at 100K. A full data set was collected from one crystal frozen in this manner at the Advanced Photon Sources of Argonne National Laboratory on a on a ADSC Quantum 210 CCD detector.
• the data is 96.5% complete to 2.7 A resolution with an R m ⁇ rge ⁇ f 0.062 and an average redundancy of 4.4.
• the final model was built with manual rebuilding on the graphics screen, using the program XtalView (McRee, .D. E., Practical Protein Crystallography, Academic Press, San Diego, 1993). Refinement in Refmac was carried out using all data in the resolution range 50.0-2.7A.
• the R-factor for the current model is 0.257 (free R-factor, 5% of the data, 0.340).
• the refinement statistics are summarized in Table 5b.
• the current model contains residues 38-766 (others are disordered in the crystal), 12 sugar and 32 water molecules. Table 5a -Data statistics

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