| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | November 19, 2002 |
| PATENT TITLE |
Crystal structure |
| PATENT ABSTRACT | A crystal of pantothenate synthetase (PS) has a monoclinic space group P2.sub.1 and unit cell dimensions of a=66.0.+-.0.2 .ANG., b=78.1.+-.0.2 .ANG., c=77.1.+-.0.2 .ANG. and .beta.=103.7.+-.0.2.degree.. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | September 11, 2000 |
| PATENT REFERENCES CITED |
Alexeev et al. The Crystal Structure of 8-Amino-7-oxononanoate Synthase: A Bacterial PLP-dependent, Acyl-CoA-condensing Enzyme. Journal of Molecular Biology,. Nov. 1998, vol. 284, No. 2, pp. 401-419.* Fraser et al. Phosphorylated and Dephosphorylated Structures of Pig Heart, GTP-specific Succinyl-CoA Synthetase. Journal of Molecular Biology. Jun. 2000, vol. 299, No. 5, pp. 1325-1339.* Barker et al., "Conserved cysteine and histidine residues in the structures of the tyrosyl and methionyl-tRNA synthetases", FEBS Letters, vol. 145, No. 2, Aug. 1982, pp. 191-193. Bohacek et al., "The Art and Practice of Structure-Based Drug Design: A Molecular Modeling Perspective", Medicinal Research Reviews, vol. 16, (1996), pp. 3-50. Brick et al., "Structure of Tyrosyl-tRNA Synthetase Refined at 2-3, A Resolution", J. Med. Biol. (1988), 208, pp. 83-98. Greer et al., "Application of the Three-Dimensional Structures of Protein Target Molecules in Structure-Based Drug Design", Journal of Medicinal Chemistry, Apr. 15, 1994, vol. 37, No. 8, pp. 1035-1054. Izard et al., "The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity", The EMBO Journal, vol. 18, No. 8, (1999), pp. 2021-2030. Jones et al., "Docking small-molecule ligands into active sites", Current Opinion in Biotechnology, vol. 6, (1995), pp. 652-656. Mechulam et al., "Crystal Structure of Escherichia coli Methionyl-tRNA synthetase Highlights Species-specific Features", J. Mol. Biol., (1999), 294, pp. 1287-1297. Nureki et al., "Architectures of Class-Defining and Specific Domains of Glutamyl-tRNA Synthetase", Science, vol. 267, Mar. 31, 1995, p. 1958-1965. Perona et al., "Structural Basis for Transfer RNA Aminoacylation of Escherichia coli Glutaminyl-tRNA Synthetase", Biochemistry, 1993, 32, pp. 8758-8771. Shuker et al., "Discovering High-Affinity Ligands for Proteins: SAR by NMR", Science, vol. 274, Nov. 29, 1996, pp. 1531-1534. Stout et al., "The additivity of substrate fragments in enzyme-ligand binding", Structure, 6, (1998), pp. 839-848 (Research Article). Van Duyne et al., "Atomic Structures of the Human Immunophilin FKBP-12 Complexes with FK506 and Rapamycin", J. Mol. Biol. (1993), 229, pp. 105-124. Verlinde et al., "In search of new lead compounds for trypanosomiasis drug design: A protein structure-based linked-fragment approach", Journal of Computer Aided Molecular Design, 6, (1992), pp. 131-147. Weber et al., "A prototypical cytidylyltransferase: CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis", Structure with Folding and Design, 7, (1999), pp. 1113-1124. Delft et al, "The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylytransferase superfamily," Structure, vol. 9, May 2001, pp. 439-450; XP002187263. Qoronfleh et al, "Production of selenomethionine-labeled recombinant . . . ", Journal of Biotechnology Elsevier Science Publishers, Amsterdam, NL, vol. 39, No. 2, Apr. 15, 1995, pp. 119-128; XP004036977. Shao et al, "Accessibility of selenomethionine . . . ", FEBS Letters, Elsevier Science Publishers, Amsterdam, NL, vol. 441, No. 1, Dec. 11, 1998, pp. 77-82; XP004258875. Jhoti, "High-throughput structural . . . ", Trends in Biology, Elsevier Publications, Cambridge, GB, vol. 19, No. 10, Oct. 1, 2001, pp. S67-S71; XP004310381. |
| PATENT CLAIMS |
What is claimed is: 1. A crystal of pantothenate synthetase (PS) having a monoclinic space group P2.sub.1, and unit cell dimensions of a=66.0.+-.0.2 .ANG., b=78.1.+-.0.2 .ANG., c=77.1.+-.0.2 .ANG. and f=103.7.+-.0.2.degree.. 2. A crystal of PS having the three dimensional atomic coordinates of Table 1. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to the enzyme pantothenate synthetase, and in particular its crystal structure and the use of this structure in drug discovery. BACKGROUND OF THE INVENTION Pantothenic acid (vitamin B.sub.5) is found in coenzyme A (CoA) and the acyl carrier protein (ACP), both of which are involved in fatty acid metabolism. Pantothenic acid can be synthesised by plants and microorganisms but animals are apparently unable to make the vitamin, and require it in their diet. However, all organisms are able to convert pantothenic acid to its metabolically active form, coenzyme A. The pathway for the synthesis of pantothenic acid is shown in FIG. 1. It provides a potential target for the treatment of infectious disease, since inhibitors of the pathway should be damaging to bacteria and fungi but not to human or animal subjects infected by bacteria. Of specific interest is pantothenate synthetase (D-pantoate: .beta.-alanine ligase (AMP-forming); EC 6.3.2.1) This enzyme catalyses the condensation between .beta.-alanine and pantoic acid, the final steps in pantothenic acid biosynthesis. Inhibitors (whether competitive, non-competitive, uncompetitive or irreversible) of pantothenate synthetase would be of significant technical and commercial interest. Purification of pantothenate synthetase (PS) to homogeneity was achieved by Miyatake et. al, (J. Biochem., 79, (1976), 673-678). The enzyme was reported to require stoichiometric amounts of ATP as an energy source which is hydrolysed to AMP and inorganic pyrophosphate. The mechanism of the enzymic reaction involves pantoate adenylate as an intermediate. However, until now no one has successfully determined the structure of PS. This has prevented PS inhibitors being developed via structure-based drug design methodologies. Knowledge of the structure of PS would significantly assist the rational design of novel therapeutics based on PS inhibitors. SUMMARY OF THE INVENTION The present invention is at least partly based on overcoming several technical hurdles: we have (i) produced PS crystals of suitable quality, including crystals of selenium atom PS derivatives, for performing X-ray diffraction analyses, (ii) collected X-ray diffraction data from the crystals, (iii) determined the three-dimensional structure of PS, and (iv) identified sites on the enzyme which are likely to be involved in the enzymic reaction. In a first aspect, the present invention provides a crystal of PS having a monoclinic space group P2.sub.1, and unit cell dimensions of a=66.0.+-.0.2 .ANG., b=78.1.+-.0.2 .ANG., c=77.1.+-.0.2 .ANG. and .beta.=103.7.+-.0.2.degree.. Preferably the PS is a dimer. In a second aspect, the invention also provides a crystal of PS having the three dimensional atomic coordinates of Table 1. In a third aspect, the invention provides a method for crystallizing a selenium atom PS derivative which comprises producing PS by recombinant production in a bacterial host (e.g. E. coli) in the presence of selenomethionine, recovering a selenium atom PS derivative from the host and growing crystals from the recovered selenium atom PS derivative. Thus, the selenium atom PS derivative and PS produced by crystallising native PS (see the detailed description below) are provided as crystallised proteins suitable for X-ray diffraction analysis. The crystals may be grown by any suitable method, e.g. the hanging drop method. In a fourth aspect, the present invention provides a method for identifying a potential inhibitor of PS comprising the steps of: a. employing a three-dimensional structure of PS, or at least one sub-domain thereof, to characterise at least one PS active site, the three-dimensional structure being defined by atomic coordinate data according to Table 1; and b. identifying the potential inhibitor by designing or selecting a compound for interaction with the active site. By "sub-domain" is meant at least one complete element of secondary structure, i.e. an alpha helix or a beta sheet, as described in the detailed description below. If more than one PS active site is characterised and a plurality of respective compounds are designed or selected, the potential inhibitor may formed by linking the respective compounds into a larger compound which maintains the relative positions and orientations of the respective compounds at the active sites. The larger compound may be formed as a real molecule or by computer modelling. In any event, the determination of the three-dimensional structure of PS provides a basis for the design of new and specific ligands for PS. For example, knowing the three-dimensional structure of PS, computer modelling programs may be used to design different molecules expected to interact with possible or confirmed active sites, such as binding sites or other structural or functional features of PS. More specifically, a potential modulator of PS activity can be examined through the use of computer modelling using a docking program such as GRAM, DOCK, or AUTODOCK (see Walters et al., Drug Discovery Today, Vol.3, No.4, (1998), 160-178, and Dunbrack et al., Folding and Design, 2, (1997), 27-42) to identify potential inhibitors of PS. This procedure can include computer fitting of potential inhibitors to PS to ascertain how well the shape and the chemical structure of the potential inhibitor will bind to the enzyme. Also computer-assisted, manual examination of the active site structure of PS may be performed. The use of programs such as GRID (Goodford, J. Med. Chem., 28, (1985), 849-857)n--a program that determines probable interaction sites between molecules with various functional groups and the enzyme surface--may also be used to analyse the active site to predict partial structures of inhibiting compounds. Computer programs can be employed to estimate the attraction, repulsion, and steric hindrance of the two binding partners (e.g. the PS and a potential inhibitor). Generally the tighter the fit, the fewer the steric hindrances, and the greater the attractive forces, the more potent the potential modulator since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug, the more likely it is that the drug will not interact with other proteins as well. This will tend to minimise potential side-effects due to unwanted interactions with other proteins. Alternatively, step b. may involve selecting the compound by computationally screening a database of compounds for interaction with the active site. For example, a 3-D descriptor for the potential inhibitor may be derived, the descriptor including geometric and functional constraints derived from the architecture and chemical nature of the active site. The descriptor may then be used to interrogate the compound database, a potential inhibitor being a compound that has a good match to the features of the descriptor. In effect, the descriptor is a type of virtual pharmacophore. Having designed or selected possible binding partners, these can then be screened for activity. Consequently, the method preferably further comprises the further steps of: c. obtaining or synthesising the potential inhibitor; and d. contacting the potential inhibitor with PS to determine the ability of the potential inhibitor to interact with PS. More preferably, in step d. the potential inhibitor is contacted with PS in the presence of a substrate, and typically a buffer, to determine the ability of said potential inhibitor to inhibit PS. The substrate may be e.g. pantoic acid (or a salt thereof), .beta.-alanine (or a salt thereof), or ATP. So, for example, an assay mixture for PS may be produced which comprises the potential inhibitor, substrate and buffer. Instead of, or in addition to, performing e.g. a chemical assay, the method may comprise the further steps of: c. obtaining or synthesising said potential inhibitor; d. forming a complex of PS and said potential inhibitor; and e. analysing said complex by X-ray crystallography to determine the ability of said potential inhibitor to interact with PS. Detailed structural information can then be obtained about the binding of the potential inhibitor to PS, and in the light of this information adjustments can be made to the structure or functionality of the potential inhibitor, e.g. to improve binding to the active site. Steps c. to e. may be repeated and re-repeated as necessary. In a fifth aspect, the invention includes a compound which is identified as an inhibitor of PS by the method of the fourth aspect. In a sixth aspect, the invention provides a method of analysing a PS-ligand complex comprising the step of employing (i) X-ray crystallographic diffraction data from the PS-ligand complex and (ii) a three-dimensional structure of PS, or at least one subdomain thereof, to generate a difference Fourier electron density map of the complex, the three-dimensional structure being defined by atomic coordinate data according to Table 1. Therefore, PS-ligand complexes can be crystallised and analysed using X-ray diffraction methods, e.g. according to the approach described by Greer et al., J. of Medicinal Chemistry, Vol. 37, (1994), 1035-1054, and difference Fourier electron density maps can be calculated based on X-ray diffraction patterns of soaked or co-crystallised PS and the solved structure of uncomplexed PS. These maps can then be used to determine whether and where a particular ligand binds to PS and/or changes the conformation of PS. Electron density maps can be calculated using programs such as those from the CCP4 computing package (Collaborative Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographica, D50, (1994), 760-763.). For map visualisation and model building programs such as "O" (Jones et al., Acta Crystallograhy, A47, (1991), 110-119) can be used. In a seventh aspect, the present invention provides computer readable media with either (a) atomic coordinate data according to Table 1 recorded thereon, said data defining the three-dimensional structure of PS or at least one sub-domain thereof, or (b) structure factor data for PS recorded thereon, the structure factor data being derivable from the atomic coordinate data of Table 1. As used herein, "computer readable media" refers to any media which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. By providing such computer readable media, the atomic coordinate data can be routinely accessed to model PS or a sub-domain thereof. For example, RASMOL (Sayle et al., TIBS, Vol. 20, (1995), 374) is a publicly available computer software package which allows access and analysis of atomic coordinate data for structure determination and/or rational drug design. On the other hand, structure factor data, which are derivable from atomic coordinate data (see e.g. Blundell et al., in Protein Crystallography, Academic Press, New York, London and San Francisco, (1976)), are particularly useful for calculating e.g. difference Fourier electron density maps. In an eighth aspect, the present invention provides systems, particularly a computer systems, intended to generate structures and/or perform rational drug design for PS or PS ligand complexes, the systems containing either (a) atomic coordinate data according to Table 1, said data defining the three-dimensional structure of PS or at least one sub-domain thereof, or (b) structure factor data for PS, said structure factor data being derivable from the atomic coordinate data of Table 1. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Windows NT or IBM OS/2 operating systems. As used herein, "a computer system" refers to the hardware means, software means and data storage means used to analyse the atomic coordinate data of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a monitor is provided to visualise structure data. The data storage means may be RAM or means for accessing computer readable media of the sixth aspect of the invention. |
| PATENT EXAMPLES | Available on request |
| PATENT PHOTOCOPY | Available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |