WO2005103241A1 - Structure cristalline de 3'-5'-phosphodiesterase nucleotidique cyclique 9a (pde9a) et ses utilisations - Google Patents

Structure cristalline de 3'-5'-phosphodiesterase nucleotidique cyclique 9a (pde9a) et ses utilisations Download PDF

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WO2005103241A1
WO2005103241A1 PCT/IB2005/001046 IB2005001046W WO2005103241A1 WO 2005103241 A1 WO2005103241 A1 WO 2005103241A1 IB 2005001046 W IB2005001046 W IB 2005001046W WO 2005103241 A1 WO2005103241 A1 WO 2005103241A1
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pde9a
coordinates
computer
ligand
amino acid
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PCT/IB2005/001046
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Shenping Liu
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Pfizer Products Inc.
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    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
    • 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 3', 5'-Cyclic Nucleotide Phosphodiesterase 9A (PDE9A), methods of preparing the compositions, methods of determining the three-dimensional X-ray atomic structure coordinates of the composition, methods of identifying ligands of PDE9A using structure based drug design, the use of the three-dimensional crystal structure to design, modify and assess the activity of potential inhibitors, and to the use of such inhibitors for treating a variety of diseases, particularly diabetes, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome and/or cardiovascular disease.
  • diseases particularly diabetes, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome and/or cardiovascular disease.
  • Cyclic nucleotide second messengers play a central role in signal transduction and regulation of physiologic responses. Their intracellular levels are controlled by the complex superfamily of cyclic nucleotide phosphodiesterase (PDE) enzymes.
  • PDE cyclic nucleotide phosphodiesterase
  • the PDE superfamily is comprised of metallophosphohydrolases (e.g., Mg 2+ , and Zn 2+ ) that specifically cleave the 3',5'-cyclic phosphate moiety of cAMP and/or cGMP to produce the corresponding 5'-nucleotide.
  • Cyclic nucleotide PDEs provide the major pathway for eliminating the cyclic nucleotide signal for the cell.
  • PDEs are critical determinants for modulation of cellular levels of cAMP and/or cGMP by many stimuli.
  • Members of the PDE superfamily differ in their tissue distributions, physicochemical properties, substrate and inhibitor specificities and regulatory mechanisms. Based on differences in primary structure of known PDEs, they have been subdivided into two major classes, class I and class II. To date, no mammalian PDE has been included in class II.
  • Class I contains the largest number of PDEs and includes all known mammalian PDEs. Each class I PDE contains a conserved segment of -250-300 amino acids in the carboxyl- terminal portion of the proteins, and this segment has been demonstrated to include the catalytic site of these enzymes. All known class I PDEs are contained within cells and vary in subcellular distribution, with some being primarily associated with the particulate fraction of the cytoplasmic fraction of the cell, others being evenly distributed in both compartments. PDEs from mammalian tissues have been subdivided into 11 families that are derived from separate gene families. The families are named PDE1 , PDE2, PDE3...to PDE11.
  • PDEs within a given family may differ significantly but the members of each family are functionally related to each other through similarities in amino acid sequences, specificities and affinities for cGMP (PDE5, PDE6, and PDE9) or cAMP (PDE4, PDE7, and PDE8) or accommodation of both (PDE1 , PDE2, PDE3, PDE10, and PDE11), inhibitor specificities, and regulatory mechanisms.
  • PDE5 PDE5, PDE6, and PDE9
  • cAMP PDE4, PDE7, and PDE8
  • Comparison of the amino acid sequences of PDEs suggests that all PDEs may be chimeric multidomain proteins possessing distinct domains that provide for catalysis and a number of regulatory functions.
  • the amino acid sequences of all mammalian PDEs identified to date include a highly conserved region of approximately 270 amino acids located in the carboxy terminal half of the proteins. (Charbonneau, et al., Proc. Natl. Acad., Sci. (USA) 83:9308-9312 (1986)).
  • the conserved domain includes the catalytic site for cAMP and/or cGMP hydrolysis and two putative metal (presumably zinc) binding sites as well as family specific determinants. (Beavo, Physiol. Rev. 75: 725-748 (1995); Francis, et al., J. Biol. Chem. 269:22477-22480 (1994)).
  • the amino terminal region of the various PDEs are highly variable and include other family specific determinants such as : (i) calmodulin binding sites (PDE1 ); (ii) non-catalytic cGMP binding sites (PDE2, PDE5, PDE6); (iii) membrane targeting sites (PDE4); (iv) hydrophobic membrane association sites (PDE3); and (v) phosphorylation sites for either the calmoduline-dependent kinase (II) (PDE1), the cAMP-dependent kinase (PDE1 , PDE3, PDE4), or the cGMP dependent kinase (PDE5) (Beavo, Physiol. Rev.
  • Human PDE9A represented the first human member of the PDE9 family that had been cloned and characterized. (Fisher, et al., Journal of Biological Chemistry, 273:5:15559-15564 (1998)). By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to
  • PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity).
  • PDE9A 21 are also conserved in PDE9A.
  • the one change is a very conservative Tyr to Phe change at amino acid 24 of the catalytic domain. All the features noted above are also conserved in the murine and rattus norvegicus PDE9A sequence, which share about 90% amino acid sequence identity with the human sequence in the catalytic domain. (Fisher, et al., (1998)).
  • PDE9A Selective inhibition of PDE9A has been investigated for the treatment of various conditions including diabetes, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and/or cardiovascular disease.
  • a preferred condition comprises diabetes, metabolic syndrome, and/or cardiovascular disease.
  • Several methods have been used in the past and continue to be used to discover selective inhibitors of biomolecular targets such as PDE9.
  • the various approaches include ligand-directed drug discovery (LDD), quantitative structure activity relationship (QSAR) analyses; and comparative molecular field analysis (CoMFA).
  • LDD ligand-directed drug discovery
  • QSAR quantitative structure activity relationship
  • CoMFA comparative molecular field analysis
  • CoMFA is a particular type of QSAR method that uses statistical correlation techniques for the analysis of the quantitative relationship between the biological activity of a set of compounds with a specified alignment, and their three-dimensional electronic and steric properties.
  • this information can, in turn, be used for predicting ligand-receptor complex formation, and for designing ligands and protein mutations that produce desired ligand receptor interactions.
  • Regulation of PDEs is important for controlling myriad physiological functions, including the visual response, smooth muscle relaxation, platelet aggregation, fluid homeostasis, immune responses, and cardiac contractility.
  • PDEs are critically involved in feedback control of cellular cAMP and cGMP levels.
  • the PDE superfamily continues to be a major target for pharmacological intervention in a number of medically important maladies including cardiovascular diseases, asthma, depression, and male impotence. For example, PDE5, found in varying concentrations in a number of tissues, has been recognized in recent years as an important therapeutic agent.
  • the present invention provides crystalline compositions of PDE, and specifically of the catalytic region of PDE9A.
  • the invention further provides methods of preparing said compositions, methods of determining the three-dimensional X-ray atomic structure coordinates of said crystalline compositions, methods of using the atomic structure coordinates in conjunction with computational methods to identify binding site(s), methods to elucidate the three-dimensional structure of homologues of PDE9A, and methods to identify ligands which interact with the binding site(s) to agonize or antagonize the biological activity of PDE9A, methods for identifying inhibitors of PDE9A, pharmaceutical compositions of inhibitors, and methods of treatment of a variety of conditions including, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and/or cardiovascular disease using said pharmaceutical compositions.
  • the invention provides crystalline compositions of the catalytic region of PDE9A.
  • One aspect of the present invention provides methods for crystallizing a PDE9A polypeptide ligand complex comprising a polypeptide.
  • the methods for crystallizing a PDE9A polypeptide ligand complex comprising an amino acid sequence spanning the amino acids 239 to 562 listed in SEQ ID NO:1 , or a homologue, an analogue or a variant thereof comprising the steps of: comprising the steps: (a) preparing a mixture of an aqueous solution comprising a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , or a homologue, or a variant thereof, with a ligand in a 2:1 molar ration; (b) mixing said aqueous solution with a reservoir solution comprising a precipitant to from a mixed volume; and (c) crystallizing said mixed volume.
  • Crystallization can be carried out by various techniques known by those skilled in the art, such as for example, batch crystallization, liquid bridge crystallization, or dialysis crystallization. Preferably, the crystallization is achieved using vapor diffusion techniques.
  • the present invention provides vectors useful in methods for preparing a substantially purified C-terminal catalytic domain of PDE9A comprising the polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , or a homologue, an analogue or a variant thereof.
  • the present invention provides methods for determining the X-ray atomic structure coordinates of the crystalline compositions at a 2.7 A resolution.
  • the present invention provides a molecule or molecular complex crystal, wherein the crystal has substantially similar atomic structure coordinates to the atomic structure coordinates listed in FIG.4 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 Asp239 to Glu562 listed in SEQ ID NO:1.
  • a further embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 90% homologous to a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1.
  • Yet another embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 95% homologous to a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , and which has the atomic structure coordinates listed in FIG. 4.
  • a further embodiment of the present invention provides a crystal comprising an amino acid sequence that is at least 98% homologous to a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , and which has the atomic structure coordinates listed in FIG. 4
  • the present invention provides a molecule or molecular complex crystal, wherein the crystal comprises a polypeptide, or a portion thereof, with atomic structure coordinates having a root mean square deviation from the protein backbone atoms (N, C ⁇ , C, and O) listed in FIG.1 of less than 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or 2.5 A.
  • the present invention provides a scalable, or translatable, three dimensional configuration of points derived from structural coordinates of at least a portion of a PDE9A molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning the amino acids Asp239 to Glu562 listed in SEQ ID NO:1.
  • the invention also comprises the structural coordinates of at least a portion of a molecule or ⁇ molecular complex that is structurally homologous to a PD ⁇ 9A molecule or molecular complex.
  • the configuration of points derived from a homologous molecule or molecular complex have 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. 4.
  • the present invention provides a computer for producing a three- dimensional representation of: a. a molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 , or a homologue, an analogue, or a variant thereof; b.
  • a molecule or molecular complex wherein the atoms of the molecule or molecular complex are represented by atomic structure coordinates that are substantially similar to, or are subsets of the atomic structure coordinates listed in FIG. 4; c. a molecule or molecular complex, wherein the molecule or molecular complex comprises atomic structure 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 structure coordinates for the carbon backbone atoms listed in FIG.1 ; 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 structure 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 computer configured according to this aspect of the invention can be used to design and identify potential modulators of PDE9A by, for example commercially available molecular modeling software in conjunction with structure-based drug design as provided herein.
  • the potential modulators designed and identified using the computer configurations according to the present invention can be for example ligands or inhibitors.
  • the present invention provides methods for designing a compound that binds to PDE9A using the molecular or molecular complex, comprising selecting a compound by performing structure- based drug design with the atomic structure coordinates determined for the crystal, wherein said selecting is performed in conjunction with computer modeling.
  • 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.
  • the present invention provides methods for generating three-dimensional atomic structure coordinates of a protein homologue or a variant of PDE9A using the X-ray coordinates of PDE9A described in FIG. 4, comprising, a. identifying one or more homologous polypeptide sequences to PDE9A; b. aligning said sequences with the sequence of PDE9A which comprises a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 ; c.
  • Embodiments of the ninth aspect provide methods, which further comprise refining and evaluating the full or partial three-dimensional coordinates. These methods may thus be used to generate three-dimensional structures for proteins for which heretofore three- dimensional atomic structure coordinates have not been determined. Depending on the extent of sequence homology, 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 modulators, such as for example inhibitors, ligands, and the like of PDE9A by providing the coordinates of a molecule of PDE9A to a computerized modeling system; identifying chemical entities that are likely to bind to or interfere with the molecule (e.g., by screening a small molecule library); and, optionally, procuring or synthesizing and assaying the compounds or analogues derived for bioactivity.
  • the present invention relate to methods for identifying potential modulators for PDE9A or homologues, an analogue or variants thereof comprising: a.
  • the structural aspects of the modulator may be modified to generate a structural analog of the modulator. This analog can then be used in the above method to identify binding modulators.
  • preferred modulators are ligands and include selective inhibitors of PDE9A.
  • 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.
  • the present invention provides compositions and pharmaceutical preparations comprising the modulator designed according to any of the above methods.
  • 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 present invention provides methods for treating conditions, diseases, or symptoms selected from the group consisting of type 1 diabetes, type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and cardiovascular disease, comprising administering to a patient in need of such treatment the pharmaceutical composition of ligands identified by structure-based drug design using the atomic structure coordinates substantially similar to, or portions of, the coordinated listed in FIG. 4.
  • Figure 1 is an orthogonal view of the embodiment of PDE9A in ribbon representation.
  • the compound of Formula 1 is shown in ball-and-stick representation.
  • Figure 2 is another orthogonal view of the embodiment of the compound of Formula 1 with PDE9A.
  • Figure 3 is schematic diagram showing the interactions of the compound of Formula
  • FIG. 1 with PDE9A is a list of the X-ray coordinates of the PDE9A C-terminal catalytic domain crystal as described in the Examples.
  • FIG. 4 is a list of the X-ray coordinates of the PDE9A C-terminal catalytic domain crystal as described in the Examples.
  • DETAILED DESCRIPTION OF THE INVENTION The present invention relates to crystalline compositions of PDE9A, three- dimensional X-ray atomic structure coordinates of said crystalline composition, methods of preparing said compositions, methods of determining the three-dimensional X-ray atomic structure coordinates of said crystalline compositions, and methods of using said atomic structure 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 PDE9A.
  • 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.
  • “Atomic coordinates” or “atomic structural coordinates” are the Cartesian coordinates corresponding to an atom's spatial relationship to other atoms in a molecule or molecular complex.
  • Structural coordinates may be obtained using x-ray crystallography techniques or NMR techniques, or may be derived using molecular replacement analysis or homology modeling.
  • Various software programs allow for the graphical representation of a set of structural coordinates to obtain a three dimensional representation of a molecule or molecular complex.
  • the structural coordinates of the present invention may be modified from the original sets provided in FIG. 4 by mathematical manipulation, such as by inversion or integer additions or subtractions.
  • 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. In further embodiments, 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 PDE9A activity that exhibits a biological property conventionally associated with a PDE9A agonist or antagonist, such as a property that would allow treatment of one or more of the various diseases of the central nervous system.
  • catalytic domain refers to the catalytic domain of the PDE9A 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.
  • To clone 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.
  • the term "functional equivalence” refers to the assembly of one or more amino acid residues that form a binding site in an enzyme. These residues may have one or more intervening residues which are distant from the binding site, and therefore may minimally interact with a ligand in the binding sites.
  • the binding site may be defined for the purpose of structure based drug design as comprising only a handful of amino acid residues.
  • the ligand binding site can alternatively comprise at least about 80% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516 of SEQ ID No:1.
  • the ligand binding site comprises at least about 90% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512 Gln513 and Phe516 of SEQ ID No:1.
  • any molecular assembly that has a root mean square deviation from the atomic structure coordinates of the protein backbone atoms (N, C ⁇ , C, and O), or the side chain atoms, of one or more of Met425,
  • the functional equivalents may be different from the peptides described herein at one, two, three, four, or more amino acid positions. In the most common instances, such differences will involve conservative amino acid substitutions.
  • the terms “gene”, “recombinant gene” and “gene construct” refer to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • the term “intron” refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.
  • 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. In a preferred case, 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, oliogpeptide, or portion thereof, having preferably at least 90% amino acid sequence identity with PDE9A enzyme as described in SEQ ID No:1 or SEQ ID No:2 or any catalytic domain described herein, or any functional or structural domain of lipid binding protein.
  • SEQ ID No:1 is the full- length amino acid sequence of the wild-type human PDE9A.
  • SEQ ID No:2 is the amino acid sequence of the wild type carboxy-terminal catalytic domain of human PDE9A that was crystallized in the Examples.
  • SEQ ID No:3 is the wild-type mus musculus (mouse) PDE9A amino acid sequence, (Soderling, et al., J. Biol. Chem., 273: 15553-15558, 1998) and SEQ ID No:4 is the wild-type rattus norvegicus (rat) PDE9A amino acid sequence, (Andreeua, S.G., Rosenberg, P.A., "Characterization of PDE9A in the rat brain", Submitted (Apr.
  • the term "substantially similar atomic structure coordinates" or atomic structure coordinates that are “substantially similar” refers to any set of structure coordinates of PDE9A or PDE9A homologues, or PDE9A variants, polypeptide fragments, described by atomic coordinates that have a root mean square deviation for the atomic structure 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. 4.
  • 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 structure coordinates have a root mean square deviation for the atomic structure 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 structure coordinates of similar or the same amino acids from the coordinates listed in FIG. 4.
  • an example of an assembly of amino acids may be the amino acid residues that form a binding site in an enzyme. These residues may have one or more intervening residues which are distant from the binding site, and therefore may minimally interact with a ligand in the binding sites.
  • the binding site may be defined for the purpose of structure based drug design as comprising only a handful of amino acid residues.
  • the ligand binding site can alternatively comprise at least about 80% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516 of SEQ ID No:1.
  • the ligand binding site comprises at least about 90% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512 Gln513 and Phe516 of SEQ ID No:1.
  • any molecular assembly that has a root mean square deviation from the atomic structure coordinates of the protein backbone atoms (N, C ⁇ , C, and O), or the side chain atoms, of one or more of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501, Ala512, Gln513 and Phe516 of SEQ ID NO:1 , any conservative substitutions thereof, or any functional equivalence 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 will be considered substantially similar to the coordinates listed in FIG.4.
  • substantially similar atomic structure coordinates are considered identical to the coordinates, or portions thereof, listed in FIG. 4.
  • the coordinates listed in FIG. 4 or portions thereof may be transformed into a different set of coordinates using various mathematical algorithms without departing from the present invention.
  • the coordinates listed in Fig. 4, or portions thereof may be transformed by algorithms which translate or rotate the atomic structure coordinates.
  • molecular mechanics, molecular dynamics or ab intio algorithms may modify the atomic structure coordinates.
  • Atomic coordinates generated from the coordinates listed in FIG. 4, or portions thereof, using any of the aforementioned algorithms shall be considered identical to the coordinates listed in FIG. 4.
  • 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-dime ⁇ sional 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 is designed or developed with reference to the crystal structure of PDE9A as represented by the atomic structure coordinates listed in FIG. 4.
  • the ligand is an agonist, whereby the molecule upregulates (i.e., activate or stimulate, 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., inhibit or suppress, 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 an activity.
  • modulator refers to any molecule, e.g., protein, peptide, peptidomimetics, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., which can either 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 an activity.
  • upregulation i.e., activation or stimulation, e.g., by agonizing or potentiating
  • down-regulation i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting
  • pharmacophore refers to the ensemble of steric and electronic features of a particular structure that is necessary to ensure ⁇ ns optimal supramolecular interactions with a specific biological target structure and to trigger (or to block) its biological response.
  • a pharmacophore may or may not represent a real molecule or a real association of functional groups.
  • 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. Accordingly, in the context of enzyme ligands, such as for example agonists or antagonists, a pharmacophore may represent an ensemble of steric and electronic factors which are necessary to insure supramolecular interactions with a specific biological target structure. As such, 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.
  • precipitant as used herein is includes any substance that, when added to a solution, causes a precipitate to form or crystals to grow.
  • examples of precipitants within the scope of this invention 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 (e.g., Mn, or Zn) metal salts.
  • alkali e.g., Li, Na, or K
  • alkaline earth metal e.g., Mg, or Ca
  • transition e.g., Mn, or Zn
  • Common counterions to the metal ions include, but are not limited to, halides, phosphates, citrates and sulfates.
  • prodrug as used herein refers to drugs that, once administered, are chemically modified by metabolic processes in order to become pharmaceutically active.
  • 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 undesireable, 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 said 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 PDE9A inhibitor refers to a substance, for example an organic molecule that effectively inhibits an enzyme from the PDE9A family to a greater extent than any other PDE enzyme, particularly any enzyme from the PDE 1-9 families or any PDE10 or PDE11 enzyme.
  • a selective PDE9A inhibitor is a substance, for example, a small organic molecule having a K; for inhibition of PDE9A that is less than about one-half, one-fifth, or one-tenth the Kj that the substance has for inhibition of any other PDE enzyme.
  • the substance inhibits PDE9A 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 PDE enzyme.
  • a substance is considered to effectively inhibit PDE9A if it has an IC 50 or Ki of less than or about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM or even 10 nM.
  • small molecules refers to preferred drugs as they are orally available (unlike proteins which must be administered by injection or topically). Size of small molecules is generally under 1000 Daltons, but many estimates seem to range between 300 to 700 Daltons.
  • therapeutically effective amount is meant that amount which is capable of at least partially reversing 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 art employing such factors and using no more than routine experimentation.
  • 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 or one or more amino acids from or to the sequence providing a resultant polypeptide sequence for an enzyme having PDE9A activity.
  • the variant, homologue, fragment or portion, of SEQ ID NO:1 or SEQ ID NO:2 comprise 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.
  • 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.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • expression vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".
  • 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 other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • nucleotide sequence coding for a PDE9A polypeptide, or functional fragment, including the C-terminal peptide fragment of the catalytic domain of PDE9A protein, derivatives or analogs thereof, including a chimeric protein, thereof can be inserted into an appropriate 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 PDE9A polypeptide of the invention or a functional fragment comprising the C-terminal peptide fragment of the catalytic domain of PDE9A protein, derivatives or analogs 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 Asp239 to Glu562 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 PDE9A 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 PDE9A 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. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant PDE9A protein of the invention may be expressed chromosomally
  • 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 PDE9A protein is cultured in an appropriate cell culture medium under conditions that provide for expression of PDE9A 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.
  • PDE9A protein may be controlled by any promoter/enhancer element known in the art, and these regulatory elements must be functional in the host selected for expression.
  • Vectors containing a nucleic acid encoding a PDE9A 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 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 vectors containing the PDE9A protein insert can be identified by the absence of the PDE9A protein gene function.
  • 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.
  • 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.
  • 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. Cham. 267:953-967; 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 structure 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 preferably a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1.
  • the present invention discloses a crystalline PDE9A molecule comprising a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 listed in SEQ ID NO:1 complexed with one or more ligands.
  • the crystallized complex is characterized by the structural coordinates listed in FIG. 4, or portions thereof.
  • the atoms of the ligand are within about 4, 7, or 10 angstroms of one or more PDE9A amino acids in SEQ ID NO: 1 preferably selected from Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516, or a conservative replacements, or functional equivalence thereof.
  • PDE9A amino acids in SEQ ID NO: 1 preferably selected from Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516, or a conservative replacements, or functional equivalence thereof.
  • the ligand may be a small molecule which binds to a PDE9A catalytic domain defined by SEQ ID NO: 2, or portions thereof, with a K, of less than about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM, or even 10 nM.
  • the ligand is the compound of Formula I, [2-(3-lsopropyl-7- oxo-6, 7-dihydro-1 H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid.
  • the ligand is a substrate or substrate analog of PDE9A.
  • the ligand(s) may be a competitive or uncompetitive inhibitor of PDE9A.
  • the ligand is a covalent inhibitor of PDE9A.
  • 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 PDE9A 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); all remaining structures are working structures (i.e., moving structures).
  • atom equivalency within molecular similarity applications is defined by user input, for the purpose of this invention 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.
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the molecular similarity application.
  • 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 PDE9A or a binding pocket portion thereof, as defined by the structural coordinates of PDE9A described herein.
  • the structure is composed of a single domain of fourteen ⁇ helices and two 3 10 helices arranged in a compact fold (FIG. 1).
  • the numbering of the helix is shown below. We have the followed the numbering conversion established by Xu et al., Science, 288:1822-25 (2000), and the start and end points of the helices are determined according to Kabsch and Sander, Biopolymers, 22(12): 2577-637 (1983).
  • Two metal ions are in the catalytic site. The first is determined to be Zn 2+ , by analogy with PDE4b, and from an analysis of its coordination geometry.
  • the metal is coordinated by His352 (N ⁇ 2-Zn 2.2A), His316 (N ⁇ 2-Zn 2.2A), As ⁇ 462 (O ⁇ 1-Zn 2.0A), and Asp353 (O ⁇ 2-Zn 2. ⁇ A). These residues are completely conserved across the PDE gene family.
  • the second metal ion is coordinated to Asp353 (O ⁇ 1-Mg 2.1A) and to a water network that stabilizes the metal environment. Due to the coordination geometry and the relative observed electron density, this second metal ion has been refined as a Mg 2+ in accordance with a similar observation in the PDE4 structure. (Xu et al., Science, 288:1822-25 (2000)).
  • One molecule of the inhibitor ([2-(3-lsopropyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3- d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid) is seen bound within the active site.
  • the inhibitor binding site is bounded by H12 and H13 on one side, and by the N-terminus of H12 and the 3 10 helix, A1.
  • Protein-inhibitor interactions are shown schematically in the FIG. 3. The majority of the interactions between the inhibitor and the protein are hydrophobic in nature; with only two hydrogen bonds observed (FIG. 2).
  • the present invention provides a molecule or molecular complex that includes at least a portion of a PDE9A and/or substrate binding pocket.
  • the PDE9A binding pocket includes the amino acids listed in Table 1 , preferably the amino acids listed in Table 2, and more preferably the amino acids listed in Table 3, 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 Tables 1-3.
  • the PDE9A substrate binding pocket includes the amino acids selected from Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516 from SEQ ID NO:1
  • the ligand binding site can alternatively comprise at least about 80% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516.
  • the ligand binding site comprises at least about 90% of the amino acid residues selected from the group consisting of Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512, Gln513 and Phe516.
  • Table 1 Residues near the binding pocket in PDE9A catalytic domain. Identified residues are 10 A away from the compound of Formula 1
  • One embodiment of the invention describes an isolated polypeptide consisting of a portion of PDE9A which functions as the binding site when folded in the proper 3-D orientation.
  • One embodiment is an isolated polypeptide comprising a portion of PDE9A, wherein the portion starts at about amino acid residue Asp239, and ends at about amino acid residue Glu562 as described in SEQ ID NO:1 , or a sequence that is at least 95%, or 98% homologous to a polypeptide with an amino acid sequence spanning amino acids Asp239 to Glu562 as listed in SEQ ID NO:1 , such as, for example the polypeptide of the wild-type mus musculus (mouse) PDE9A enzyme, disclosed in SEQ ID No:3, or the wild- type rattus norvegicus (rat) PDE9A.
  • variants of PDE9A comprises crystalline compositions comprising variants of PDE9A.
  • 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 contemplated.
  • the variant may 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.
  • 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:
  • Homology is a measure of the identity of nucleotide sequences or amino acid sequences. In order 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 (DNAStar Inc. Madison, WI); the GCG® program package (Devereux, J., et al.
  • 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. Ninety percent of homology is therefore determined, for example, using the
  • BESTFIT® program with parameters set such that the percentage of identity is calculated over the full length of the reference sequence, e.g., SEQ ID NO:1 , and wherein up to 10% of the amino acids in the reference sequence may be substituted with another amino acid.
  • Percent homologies are likewise determined, for example, to identify preferred species, within the scope of the claims appended hereto, which reside within the range of about 90% to 100% homology to SEQ ID NO: 1 as well as the binding site thereof.
  • N- or C-terminal extensions shall not be construed as affecting homology.
  • the reference sequence is generally the shorter of the two sequences.
  • 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 4. 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.4, 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. 4, wherein the amino acid sequence is at least 90%, 95%, or 98% homologous to the amino acid sequence represented by SEQ ID NO:1.
  • Various methods for obtaining atomic structure coordinates of structurally homologous molecules and molecular complexes using homology modeling are disclosed in US Patent 6,356,845, which is hereby incorporated by reference in its entirety.
  • a potential 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 PDE9A 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 PDE9A.
  • One embodiment of the present invention relates to a method of identifying an agent that binds to a binding site on PDE9A catalytic domain wherein the binding site comprises amino acid residues Met425, Glu466, Leu480, Leu481 , Tyr484, Phe501 , Ala512 Gln513 and Phe516 of SEQ ID NO: 1 comprising contacting PDE9A with a test ligand under conditions suitable for binding of the test ligand 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,
  • a potential ligand may be obtained by screening a random oep de library produced by ⁇ r ⁇ combin ⁇ nt bacteriophag ⁇ for example, (Scott ⁇ nd Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad.
  • 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 PDE9A activity.
  • a supplemental crystal can be grown comprising a protein-ligand complex formed between a PDE9A protein and the drug.
  • the crystal effectively diffracts X-rays allowing the determination of the atomic structure 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 involves using 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 A ORE (J.
  • the present invention discloses ligands which interact with a binding site of the PDE9A catalytic domain defined by a set of points having a root mean square deviation of less than about 2.5 A from points representing the backbone atoms of the amino acids represented by the structure coordinates listed in FIG.4.
  • a further embodiment of the present invention comprises binding agents which interact with a binding site of PDE9A 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. 4.
  • Such embodiments represent variants of the PDE9A crystal.
  • the present invention describes ligands which bind to a correctly folded polypeptide comprising an amino acid sequence spanning amino acids 239 to 562 listed in SEQ ID NO:1 , or a homologue or variant thereof.
  • the ligand is a competitive or uncompetitive inhibitor of PDE9A.
  • the ligand inhibits PDE9A with an IC 50 or Ki of less than about 10 mM, 1 mM, 500 nM, 100 nM, 50 nM or 10 nM.
  • the ligand inhibits PDE9A 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 PDE enzyme.
  • the substance inhibits PDE9A 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 PDE enzyme.
  • ligands such as proteins, peptides, peptidomimetics, small organic molecules, etc., designed or developed with reference to the crystal structure of PDE9A as represented by the coordinates presented herein in FIG. 4, and portions thereof.
  • binding agents interact with the binding site of the PDE9A represented by one or more amino acid residues selected from Met425, Glu466, Leu480, Lsu481 , Tyr484, Pr.3501 , Ala512 Gln513 and Phe51 ⁇ .
  • F. Machine Readable Storage Media Transformation of the structure coordinates for all or a portion of PDE9A, or the PDE9A/ligand complex 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 PDE9A C-terminal catalytic domain or 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 2.5 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.
  • 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.
  • necessary hardware e.g., drives, controllers, power supplies, etc.
  • any necessary media e.g., disks, flash cards, etc.
  • compositions contemplates methods for treating certain diseases in a mammal, preferably a human, 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.
  • Pharmaceutical compositions containing the ligand can be used for the treatment of a variety of conditions including diabetes, including type 1 and type 2 diabetes, hyperglycemia, dyslipidemia, impaired glucose tolerance, metabolic syndrome, and/or cardiovascular disease.
  • a preferred condition comprises diabetes, metabolic syndrome, and/or cardiovascular disease.
  • 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, issued 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. Such techniques are explained fully in the literature.
  • the resulting construct was subcloned into a into Ncol/BamHI into pFastBac-1 (Invitrogen, Fredrick, MD) in order to generate recombinant baculovirus using the Bac-to-Bac system (Gibco Carlsbad, CA), corresponding to the amino acids in SEQ ID NO:2.
  • the protein was expressed in SF9.
  • the insect cells were infected with the recombinant baculovirus at a MOI (multiplicity of infection) of 0.5 and harvested 72 hrs. post infection. Pellets of infected cells were frozen at -80°C for transfer to purification.
  • Example 2 Purification of PDE9A wild type catalytic domain Baculovirus cell paste (80 g) containing the over expressed PDE9A N3C2 recombinant protein was resuspended in 3 volumes buffer A, containing 20mM Tris (trimethoxylmethaneamine) pH 8.0, 150mM NaCl (sodium chloride), 5% (v/v) glycerol, 2mM TCEP (Tri(2-carboxyethyl) phosphine hydrochloride) and 1 CompleteTM protease inhibitor cocktail tablet/50mL (EDTA free) (Roche, Applied Science, Indianapolis, IN).
  • the cells were sonicated using handheld sonicator (VirTis Virsonic 100) for 3 x 30 sonications (level 7), the cell debris was removed by centrifugation at 4°C for 45 minutes at 35,000xg. The resulting clarified supernatant is collected and batch-bound to Ni-NTA Superflow resin (Qiagen) for 30 min at 4°C on rocker table. Ni-NTA resin is first washed with dH 2 0 and equilibrated in Ni-NTA buffer A (20 mM Tris 8.0, 150 mM NaCl, 2 mM TCEP, 5% glycerol, 20 mM Imadizole).
  • Ni-NTA Superflow resin Qiagen
  • Ni-NTA purified protein is cleaved with Thrombin (High Activity Thrombin - Calbiochem) at 5 Units / mg.
  • Cleaved protein is then repurified over Ni-NTA resin (adding 20 mM Imadizole to the sample before loading) using same Ni-NTA buffers.
  • Flow- thru containing cleaved protein is collected and concentrated to approx 10 mg / mL (using 10K MWCO concentrator).
  • Example 3 Crystallization of PDE10A wild-type catalytic domain with compound of Formula 1 [2-(3-lsopropyl-7-oxo-6,7-dihydro-1 H-pyrazolo[4,3-d]pyrimidin-5-ylmethyl)- phenoxyj-acetic acid) Compound of Formula I in 100% DMSO at 30mM was added to SX200 pure protein at to a final concentration of 300 uM and incubated on ice for 30 min.
  • Protein complex was then concentrated (using 10K MWCO concentrator) to 8mg / mL for crystallization trials. Concentration was assessed by Protein Determination using Coomassie Plus Protein Reagent (Pierce, Biotechnology Inc., Rockford, IL). PDE9 co-crystals were grown using the vapor diffusion method in 24 well VDX plates (Hampton Research, Aliso Viejo, CA). Crystals were grown in 19 - 21% Polyacrylic Acid, 0.1 M MES pH 6.5, 0.2M MgCI 2 (magnesium chloride) using 1 :1 drop ratios of protein : precipitant. Drop size was 3 uL and precipitant well volume was 800 uL. Plates were incubated at 22°C.
  • Example 4 X-ray data collection, structure determination and refinement of PDE9A: compound of Formula 1 ([2-(3-lsopropyl-7-oxo-6,7-dihydro-1 H-pyrazolo[4,3- d]pyrimidin-5-ylmethyl)-phenoxy]-acetic acid) complex
  • the crystals prepared in Example 3 were transferred to a cryoprotectant solution, made up of the reservoir solution, with 30% glycerol, and then flash-frozen in a stream of cold nitrogen gas at 100K or in liquid nitrogen. A full data set was collected from one crystal frozen in this manner at synchrotron beam line COMCAT at APS in Chicago. Data were processed using the HKL2000 suite of software (Otwinowski, Z. & Minor, W.

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Abstract

Une structure cristalline d'une phosphodiestérase 9A (PDE9A)/complexe ligand PDE9A et leurs utilisations dans l'identification de modulateurs PDE9A, y compris des composés ligands PDE9A. Des procédés d'identification de tels modulateurs PDE9A utilisés pour traiter une grande variété de pathologies, y compris le diabète, dont le diabète de type I et de type II, l'hyperglycémie, la dyslipidémie, la tolérance déficiente au glucose, le syndrome métabolique et/ou les maladies cardio-vasculaires.
PCT/IB2005/001046 2004-04-26 2005-04-14 Structure cristalline de 3'-5'-phosphodiesterase nucleotidique cyclique 9a (pde9a) et ses utilisations WO2005103241A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053495A1 (fr) * 2002-12-09 2004-06-24 Bayer Healthcare Ag Diagnostic et traitement de maladies associees a la phosphodiesterase 9a1(pde9a1)humaine
WO2004069989A2 (fr) * 2003-02-07 2004-08-19 Affinium Pharmaceuticals, Inc. Nouveaux polypeptides de phosphodiesterase purifies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053495A1 (fr) * 2002-12-09 2004-06-24 Bayer Healthcare Ag Diagnostic et traitement de maladies associees a la phosphodiesterase 9a1(pde9a1)humaine
WO2004069989A2 (fr) * 2003-02-07 2004-08-19 Affinium Pharmaceuticals, Inc. Nouveaux polypeptides de phosphodiesterase purifies

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FISHER D A ET AL: "Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 273, no. 25, 19 June 1998 (1998-06-19), pages 15559 - 15564, XP002091363, ISSN: 0021-9258 *
HUAI QING ET AL: "Crystal structure of phosphodiesterase 9 shows orientation variation of inhibitor 3-isobutyl-1-methylxanthine binding.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 29 JUN 2004, vol. 101, no. 26, 29 June 2004 (2004-06-29), pages 9624 - 9629, XP002334764, ISSN: 0027-8424 *
HUAI QING ET AL: "Crystal structures of phosphodiesterases 4 and 5 in complex with inhibitor 3-isobutyl-1-methylxanthine suggest a conformation determinant of inhibitor selectivity.", THE JOURNAL OF BIOLOGICAL CHEMISTRY. 26 MAR 2004, vol. 279, no. 13, 26 March 2004 (2004-03-26), pages 13095 - 13101, XP002334765, ISSN: 0021-9258 *

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