WO2001011025A2 - Cristallisation et determination de la structure de la thymidylate kinase de staphylococcus aureus - Google Patents

Cristallisation et determination de la structure de la thymidylate kinase de staphylococcus aureus Download PDF

Info

Publication number
WO2001011025A2
WO2001011025A2 PCT/US2000/021425 US0021425W WO0111025A2 WO 2001011025 A2 WO2001011025 A2 WO 2001011025A2 US 0021425 W US0021425 W US 0021425W WO 0111025 A2 WO0111025 A2 WO 0111025A2
Authority
WO
WIPO (PCT)
Prior art keywords
molecule
thymidylate kinase
molecular complex
binding pocket
tmp
Prior art date
Application number
PCT/US2000/021425
Other languages
English (en)
Other versions
WO2001011025A3 (fr
Inventor
Timothy E. Benson
Original Assignee
Pharmacia & Upjohn Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacia & Upjohn Company filed Critical Pharmacia & Upjohn Company
Priority to JP2001515812A priority Critical patent/JP2003517818A/ja
Priority to EP00953850A priority patent/EP1200565A2/fr
Priority to AU66227/00A priority patent/AU781654B2/en
Priority to CA002378010A priority patent/CA2378010A1/fr
Publication of WO2001011025A2 publication Critical patent/WO2001011025A2/fr
Publication of WO2001011025A3 publication Critical patent/WO2001011025A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • This invention relates to the crystallization and structure determination of thymidylate kinase (TMK) from Staphylococcus aureus.
  • TMK thymidylate kinase
  • Thymidylate kinase catalyzes the synthesis of (deoxy)thymidine diphosphate (dTDP) from (deoxy)thymidine monophosphate (dTMP) and ATP along the pathway leading to the synthesis of (deoxy)thymidine triphosphate (dTTP) necessary for DNA synthesis ( Figure 1). Since the phosphorylation of dTDP to dTTP is conducted by a nonspecific diphosphate kinase, TMK is a key player in the regulation of DNA synthesis and is a potential antibacterial target.
  • AZT 3'-azido-3'-deoxythymidine
  • Activation of AZT to azidothymidine triphosphate (AZT-TP) proceeds along cellular phosphorylation pathways to produce the species which is incorporated into growing DNA chains by HIV reverse transcriptase.
  • thymidylate kinase catalyzes the rate limiting phosphorylation of AZT-monophosphate to AZT-diphosphate (AZT-DP).
  • AZT-DP phosphorylation to AZT-TP is then catalyzed by a nonspecific diphosphate kinase.
  • the present invention provides a method for crystallizing an S. aureus thymidylate kinase molecule or molecular complex that includes preparing purified S. aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing S. aureus thymidylate kinase from a solution including about 5 wt. % to about ' 50 wt. % PEG (preferably having a number average molecular weight between about 200 and about 20,000), about 0.05 M to about 0.5 M MgCl 2 , and about 0 wt. % to about 20 wt.
  • a solution including about 5 wt. % to about ' 50 wt. % PEG (preferably having a number average molecular weight between about 200 and about 20,000), about 0.05 M to about 0.5 M MgCl 2 , and about 0 wt. % to about 20 wt.
  • the present invention provides a method for crystallizing an S. aureus thymidylate kinase molecule or molecular complex that includes preparing purified S. aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing S. aureus thymidylate kinase from a solution including about 2 mM to about 20 mM ⁇ , ⁇ -difluoromethylene- bisphosphonate adenosine monophosphate and about 0 wt. % to about 20 wt.
  • the present invention provides crystalline forms of an S. aureus thymidylate kinase molecule.
  • a crystal of S. aureus thymidylate kinase is provided having the trigonal space group symmetry P2 ⁇ .
  • the present invention provides a scalable three dimensional configuration of points derived from structure coordinates of at least a portion of an S. aureus thymidylate kinase molecule or molecular complex.
  • the scalable three dimensional set of points is derived from structure coordinates of at least the backbone atoms of the amino acids representing a TMP and/or TMP/ATP substrate binding pocket of an S. aureus thymidylate kinase molecule or molecular complex.
  • the scalable three dimensional configuration of points is derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an S.
  • aureus thymidylate kinase molecule or molecular complex On a molecular scale, the configuration of points derived from a homologous molecule or molecular complex have a root mean square deviation of less than about 2.1 A from the structure coordinates of the molecule or complex
  • the present invention provides a molecule or molecular complex that includes at least a portion of an S. aureus thymidylate kinase TMP and/or TMP/ATP substrate binding pocket.
  • the S. aureus thymidylate kinase TMP substrate 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 substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 A, preferably less than about 1.5 A, more preferably less than about 1.0 A, and most preferably less than about 0.5 A from points representing the backbone atoms of the amino acids.
  • the S. aureus thymidylate kinase TMP substrate 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 substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 A, preferably less
  • aureus thymidylate kinase TMP/ATP substrate binding pocket includes the amino acids listed in Table 4, preferably the amino acids listed in Table 5, and more preferably the amino acids listed in Table 6, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 A, preferably less than about 1.5 A, more preferably less than about 1.0 A, and most preferably less than about 0.5 A from points representing the backbone atoms of the amino acids.
  • the present invention provides molecules or molecular complexes that are structurally homologous to an S. aureus thymidylate kinase molecule or molecular complex.
  • the present invention provides a machine readable storage medium including the structure coordinates of all or a portion of an S. aureus thymidylate kinase molecule, molecular complex, a structurally homologous molecule or complex, including structurally equivalent structures, as defined herein, particularly a substrate binding pocket thereof, or a similarly shaped homologous substrate binding pocket.
  • a storage medium encoded with these data is capable of displaying on a computer screen, or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises a substrate binding pocket or a similarly shaped homologous substrate binding pocket.
  • the present invention provides a method for identifying inhibitors, ligands, and the like for an S. aureus thymidylate kinase molecule by providing the coordinates of a molecule of 5. aureus thymidylate kinase to a computerized modeling system; identifying chemical entities that are likely to bind to or interfere with the molecule (e.g., screening a small molecule library); and, optionally, procuring or synthesizing and assaying the compounds or analogues derived therefrom for bioactivity.
  • the present invention provides methods for designing inhibitors, ligands, and the like by providing the coordinates of a molecule of S.
  • aureus thymidylate kinase to a computerized modeling system; designing a chemical entity that is likely to bind to or interfere with the molecule; and optionally synthesizing the chemical entity and assaying the chemical entity for bioactivity.
  • the present invention provides inhibitors and ligands designed or identified by the above methods.
  • a composition is provided that includes an inhibitor or ligand designed or identified by the above method.
  • the composition is a pharmaceutical composition.
  • the present invention provides a method involving molecular replacement to obtain structural information about a molecule or molecular complex of unknown structure.
  • the method includes crystallizing the molecule or molecular complex, generating an x-ray diffraction pattern from the crystallized molecule or molecular complex, and applying at least a portion of the structure coordinates set forth in Fig. 2 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex.
  • the present invention provides a method for homology modeling an S. aureus thymidylate kinase homolog.
  • Staphylococcus aureus S. aureus
  • Thymidylate kinase T. kinase or TMK
  • TMP Thymidine 5'-monophosphate
  • Thymidine 5'-diphosphate TDP
  • Thymidine 5 '-triphosphate TTP
  • Phospho(enol)pyruvate Phospho(enol)pyruvate (PEP) Reduced nicotinamide adenine dinucleotide (NADH)
  • LDH Lactate dehydrogenase
  • NDP-Kinase Nucleoside-5 '-diphosphate kinase
  • dTMP Deoxythymidine monophosphate
  • ADP Adenosine 5'-diphosphate
  • Adenosine 5 '-triphosphate ATP
  • Isopropylthio- ⁇ -D-galactoside IPTG
  • DTT Dithiothreitol
  • DMSO Dimethyl sulfoxide
  • PEG Polyethylene glycol
  • FIGURES Figure 1 shows the biosynthetic pathway for the synthesis of thymidylate. The reaction catalyzed by thymidylate kinase is boxed.
  • Figure 2 lists the atomic structure coordinates for recombinant S. aureus thymidylate kinase (with a His 6 tag) as derived by x-ray diffraction from a crystal of that complex. The following abbreviations are used in Figure 2:
  • “Atom” refers to the element whose coordinates are measured.
  • the second column defines the number of the atom in the structure.
  • the letters in the third column define the element.
  • the fourth and fifth columns define the amino acid and the number of the amino acid in the structure, respectively.
  • "X, Y, Z" crystallographically define the atomic position of the element measured.
  • Occ is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates.
  • a value of " 1 " indicates that each atom has the same conformation, i.e., the same position, in all molecules of the crystal.
  • FIG. 3 depicts S. aureus thymidylate kinase using (a) a ribbon diagram showing the backbone structure of the enzyme and (b) a schematic diagram showing the secondary structure for a TMK monomer Disordered loops are indicated by arrows.
  • Figure 4 depicts a structural comparison of E. coli TMK + AP 5 T and S. aureus TMK. The overall fold of the two proteins is well- conserved, but note that the lid in the E. coli TMK is not present in the S. aureus TMK due to the absence of a ligand.
  • Figure 5 depicts (a) a stereo view of a superposition of S. aureus thymidylate kinase and E. coli thymidylate kinase and (b) the amino acid sequence alignment of S. aureus thymidylate kinase (S ⁇ Q ID NO: 1) (capital letters, upper sequence) and E. coli thymidylate kinase (S ⁇ Q ID NO:2) (lower sequence). Dots in the sequences indicate gaps inserted in order to optimize the alignment. Identical residues are indicated by
  • Figure 6 depicts (a) a stereo view of a superposition of S.
  • aureus thymidylate kinase and S. cerevisiae thymidylate kinase and (b) the sequence alignment of S. aureus thymidylate kinase (S ⁇ Q ID NO:l) (capital letters, upper sequence) and S. cerevisiae thymidylate kinase (S ⁇ Q ID NO:3) (lower sequence). Dots in the sequences indicate gaps inserted in order to optimize the alignment. Identical residues are indicated by
  • Figure 7 depicts a) a substrate-based inhibitor (AP 5 T) for thymidylate kinase with a K d of 20 nM for E. coli TMK (A. Lavie et al., Biochemistry 37:3677-86 (1998); A. Lavie et al., Proc. Natl. Acad. Sci. USA. 95:14045-50 (1998)).
  • b) protein ligand interactions for E. coli TMK (shaded boxes, from A. Lavie et al., Proc. Natl. Acad. Sci. USA, 95:14045- 50 (1998)) with the corresponding residues from S. aureus TMK underlined (conservative mutations are marked with an asterisk). Active site residues from the S. cerevisiae are boxed (where no corresponding residue from E. coli TMK is present, an arrow indicates the point of contact with the substrate).
  • Figure 8 depicts the anomalous difference Patterson maps at
  • Figure 9 depicts electron density maps of residues 76 to 82 from molecule 1 of S. aureus thymidylate kinase (SEQ ED NO:l) at (a) 2.7 A and (b) at 2.3 A resolution.
  • Figure 10 lists the structure factors and multiple anomalous dispersion phases for the crystal structure of S. aureus thymidylate kinase (SEQ ID NO:l).
  • INDE refers to the indices h, k, and 1 (columns 2, 3, and 4 respectively) of the lattice planes.
  • FOBS refers to the structure factor (F) of the observed reflections.
  • SIGMA is the standard deviation for the observations.
  • PHAS refers to the phase used for the observations.
  • FOM refers to the figure of merit.
  • Figure 11 depicts a surface representation of a) E. coli TMK with the inhibitor AP 5 T and b) S. aureus TMK with a hypothetical positioning of AP 5 T based on a structural alignment of C ⁇ atoms from the E. coli TMK + AP 5 T structure.
  • the invention includes a TMK crystal and/or a crystal with TMK co-crystallized with a ligand, such as an inhibitor.
  • the crystal has trigonal space group symmetry P2].
  • the crystallized enzyme is a dimer with a single dimer in the asymmetric unit.
  • Purified S. aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml may be crystallized, for example, by using a streak seeding procedure from a solution including about 5 wt. % to about 50 wt.
  • % PEG (preferably having a number average molecular weight between about 200 and about 20,000), about 0.05 M to about 0.5 M MgCl 2 , and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7.
  • a buffer having a pK a of between about 5 and 8 is preferred.
  • aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml may also be crystallized, for example, from a solution including about 2 mM to about 20 mM ⁇ , ⁇ -difluoromethylene-bisphosphonate adenosine monophosphate and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7.
  • a "molecular complex" means a protein in covalent or non-covalent association with a chemical entity.
  • a buffer having a pK a of between about 5 and 8 is preferred for use in the crystallization method.
  • a particularly preferred buffer is about 0.4M to about 2.0M sodium citrate. Variation in buffer and buffer pH as well as other additives such as PEG is apparent to those skilled in the art and may result in similar crystals.
  • the invention further includes an S. aureus thymidylate kinase crystal or S. aureus thymidylate kinase/ligand crystal that is isomorphous with an S.
  • structure coordinates refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of x-rays by the atoms (scattering centers) of an S. aureus thymidylate kinase complex in crystal form.
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density maps are then used to establish the positions of the individual atoms of the S. aureus thymidylate kinase protein or protein/ligand complex.
  • Slight variations in structure coordinates can be generated by mathematically manipulating the S. aureus thymidylate kinase or S. aureus thymidylate kinase/ligand structure coordinates.
  • the structure coordinates set forth in Figure 2 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
  • modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also yield variations in structure coordinates.
  • Such slight variations in the individual coordinates will have little effect on overall shape. If such variations are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to be structurally equivalent. Structural equivalence is described in more detail below.
  • the phrase "associating with” refers to a condition of proximity between a chemical entity, or portions thereof, and an S. aureus thymidylate kinase molecule or portions thereof.
  • the association may be non-covalent, wherein the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or it may be covalent.
  • a ligand that bound to a substrate binding pocket of S. aureus thymidylate kinase would also be expected to bind to or interfere with another substrate binding pocket whose structure coordinates define a shape that falls within the acceptable error.
  • Applicants' invention has provided, for the first time, information about the shape and structure of the substrate binding pockets of S. aureus thymidylate kinase.
  • the structures of both the TMP and the TMP/ATP substrate binding pockets are elucidated.
  • the secondary structure of the S. aureus thymidylate kinase monomer includes a five stranded parallel ⁇ sheet surrounded by nine ⁇ helices ( Figure 3). This solved crystal structure of S. aureus thymidylate kinase does not contain any ligand which has resulted in a disordered loop between helices ⁇ 7 and ⁇ 8 ( Figure 4).
  • This loop has been called the "lid" in the structures of thymidylate kinase homologs from E. coli and S. cerevisiae.
  • the lid contains Arg 153 which is responsible for phosphate binding of the ATP substrate as shown in the X-ray crystal structure of the E. coli enzyme with the AP 5 T inhibitor, a transition state analog of TMP/ATP (A. Lavie et al., Biochemistry 37:3677-86 (1998)).
  • the analogous arginine in S. cerevisiae comes from the P loop (Arg 15) between ⁇ l and ⁇ l (A. Lavie et al., Proc. Natl. Acad. Sci. USA. 95:14045-50 (1998)).
  • S. cerevisiae enzyme as a class I thymidylate kinase (which also includes human thymidylate kinase) and the E. coli enzyme as a class ⁇ thymidylate kinase (A. Lavie, Proc. Natl. Acad. Sci. USA. 95:14045-50 (1998)).
  • S. aureus SEQ ID NO:l
  • SEQ ID NO:2 has greater sequence similarity to the E. coli enzyme (SEQ ID NO:2, 38% identical, 59% similar) than the S.
  • aureus TMK sequence reveals strong conservation of these active site residues with the E.coli active site ( Figure 7b); 15 of the 17 residues involved in the protein-inhibitor complex are identical while the two remaining residues are strongly conserved.
  • An analogous comparison for the S. cerevisiae TMK ( Figure 7b) shows only four of 18 residues conserved within the active site suggesting that specificity between the S. aureus and eukaryotic thymidylate kinases might be attainable.
  • Binding pockets are of significant utility in fields such as drug discovery.
  • the association of natural ligands or substrates with the binding pockets of their corresponding receptors or enzymes is the basis of many biological mechanisms of action.
  • many drugs exert their biological effects through association with the binding pockets of receptors and enzymes.
  • Such associations may occur with all or any parts of the binding pocket.
  • An understanding of such associations helps lead to the design of drugs having more favorable associations with their target, and thus improved biological effects. Therefore, this information is valuable in designing potential inhibitors of S. aureus thymidylate kinase-like substrate binding pockets, as discussed in more detail below.
  • binding pocket refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity.
  • a binding pocket may include or consist of features such as cavities, surfaces, or interfaces between domains.
  • Chemical entities that may associate with a binding pocket include, but are not limited to, cofactors, substrates, inhibitors, agonists, and antagonists.
  • the amino acid constituents of an S. aureus thymidylate kinase substrate binding pocket as defined herein are positioned in three dimensions in accordance with the structure coordinates listed in Figure 2.
  • the structure coordinates defining a substrate binding pocket of S. aureus thymidylate kinase include structure coordinates of all atoms in the constituent amino acids; in another aspect, the structure coordinates of a substrate binding pocket include structure coordinates of just the backbone atoms of the constituent atoms.
  • the TMP substrate binding pocket of S. aureus thymidylate kinase preferably includes the amino acids listed in Table 1, more preferably the amino acids listed in Table 2, and most preferably the amino acids listed in Table 3, as represented by the structure coordinates listed in Figure 2.
  • the TMP substrate binding pocket of S. aureus thymidylate kinase may be defined by those amino acids whose backbone atoms are situated within about 3.5 A, more preferably within about 5 A, most preferably within about 7 A, of one or more constituent atoms of a bound substrate or inhibitor.
  • the TMP substrate binding pocket may be defined by those amino acids whose backbone atoms are situated within a sphere centered on the coordinates representing the alpha carbon atom of residue Ser98, the sphere having a radius of about 10 A, preferably about 15 A, and more preferably about 20 A.
  • the TMP/ATP substrate binding pocket of S. aureus thymidylate kinase preferably includes the amino acids listed in Table 4, more preferably the amino acids listed in Table 5, and most preferably the amino acids listed in Table 6, as represented by the structure coordinates listed in Figure 2.
  • the TMP/ATP substrate binding pocket of S. aureus thymidylate kinase may be defined by those amino acids whose backbone atoms are situated within about 3.5 A, more preferably within about 5 A, most preferably within about 7 A, of one or more constituent atoms of a bound substrate or inhibitor.
  • the TMP/ATP substrate binding pocket may be defined by those amino acids whose backbone atoms are situated within a sphere centered on the coordinates representing the alpha carbon atom of residue Arg93, the sphere having a radius of about 10 A, preferably about 15 A, and more preferably about 20 A.
  • the term "S. aureus thymidylate kinase-like substrate binding pocket” refers to a portion of a molecule or molecular complex whose shape is sufficiently similar to at least a portion of a substrate binding pocket of S. aureus thymidylate kinase as to be expected to bind related TMP and/or ATP structural analogues.
  • a structurally equivalent substrate binding pocket is defined by a root mean square deviation from the structure coordinates of the backbone atoms of the amino acids that make up substrate binding pockets in S. aureus thymidylate kinase (as set forth in Figure 2) of at most about 2.1 A. How this calculation is obtained is described below.
  • the invention provides molecules or molecular complexes comprising an S. aureus thymidylate kinase substrate binding pocket or S. aureus thymidylate kinase-like substrate binding pocket, as defined by the sets of structure coordinates described above.
  • X-ray structure coordinates define a unique configuration of points in space.
  • a set of structure coordinates for protein or an 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 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.
  • the present invention thus includes the scalable three- dimensional configuration of points derived from the structure coordinates of at least a portion of an S. aureus thymidylate kinase molecule or molecular complex, as listed in Figure 2, as well as structurally equivalent configurations, as described below.
  • the scalable three- dimensional configuration includes points derived from structure coordinates representing the locations of a plurality of the amino acids defining an S. aureus thymidylate kinase substrate binding pocket.
  • the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations the backbone atoms of a plurality of amino acids defining the S.
  • the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of the side chain and the backbone atoms (other than hydrogens) of a plurality of the amino acids defining the S. aureus thymidylate kinase TMP substrate binding pocket, preferably the amino acids listed in Table 1, more preferably the amino acids listed in Table 2, and most preferably the amino acids listed in Table 3.
  • the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations the backbone atoms of a plurality of amino acids defining the S. aureus thymidylate kinase TMP/ATP substrate binding pocket, preferably the amino acids listed in Table 4, more preferably the amino acids listed in Table 5, and most preferably the amino acids listed in Table 6.
  • the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of the side chain and the backbone atoms (other than hydrogens) of a plurality of the amino acids defining the S. aureus thymidylate kinase TMP/ATP substrate binding pocket, preferably the amino acids listed in Table 4, more preferably the amino acids listed in Table 5, and most preferably the amino acids listed in Table 6.
  • the invention also includes the scalable three- dimensional configuration of points derived from structure coordinates of molecules or molecular complexes that are structurally homologous to S. aureus thymidylate kinase, as well as structurally equivalent configurations.
  • Structurally homologous molecules or molecular complexes are defined below.
  • structurally homologous molecules can be identified using the structure coordinates of S. aureus thymidylate kinase according to a method of the invention.
  • the configurations of points in space derived from structure coordinates according to the invention can be visualized as, for example, a holographic image, a stereodiagram, a model or a computer-displayed image, and the invention thus includes such images, diagrams or models.
  • Various computational analyses can be used to determine whether a molecule or a substrate binding pocket portion thereof is "structurally equivalent,” defined in terms of its three-dimensional structure, to all or part of S. aureus thymidylate kinase or its substrate binding pockets.
  • Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, CA) version 4.1, and as described in the accompanying User's Guide.
  • 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) 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).
  • 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 which is structurally or functionally equivalent. Only rigid fitting operations are 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 atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
  • Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structure coordinates listed in Figure 2 ⁇ a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 2.1 A.
  • the root mean square deviation is less than about 1.0 A.
  • Another embodiment of this invention is a molecular complex defined by the structure coordinates listed in Figure 2 for those amino acids listed in Table 1, ⁇ a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 2.1 A, preferably less than about 1.0 A .
  • Still another embodiment of this invention is a molecular complex defined by the structure coordinates listed in Figure 2 for those amino acids listed in Table 4, ⁇ a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 2.1 A, preferably less than about 1.0 A.
  • 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.
  • the "root mean square deviation” defines the variation in the backbone of a protein from the backbone of S. aureus thymidylate kinase or a substrate binding pocket portion thereof, as defined by the structure coordinates of S. aureus thymidylate kinase described herein. Machine Readable Storage Media Transformation of the structure coordinates for all or a portion of S. aureus thymidylate kinase or the S.
  • aureus thymidylate kinase/ligand complex or one of its substrate 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 an S. aureus thymidylate kinase substrate binding pocket or an S.
  • 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 defined by the structure coordinates of all of the amino acids listed in Figure 2, ⁇ a root mean square deviation from the backbone atoms of said amino acids of not more than 2.1 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 structure coordinates set forth in Figure 2, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the x-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.
  • a system for reading a data storage medium may include a computer comprising a central processing unit (“CPU"), a working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube (“CRT”) displays, light emitting diode (“LED”) displays, liquid crystal displays (“LCDs”), elecfroluminescent displays, vacuum fluorescent displays, field emission displays (“FEDs”), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, track balls, touch pads, 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
  • 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.
  • the input hardware may comprise CD-ROM drives or disk drives.
  • 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. References to components of the hardware system are included as appropriate throughout the following description of the data storage medium.
  • 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.
  • the structure coordinates set forth in Figure 2 can be used to aid in obtaining structural information about another crystallized molecule or molecular complex.
  • the method of the invention allows determination of at least a portion of the three-dimensional structure of molecules or molecular complexes which contain one or more structural features that are similar to structural features of S. aureus thymidylate kinase, These molecules are referred to herein as "structurally homologous" to S. aureus thymidylate kinase, Similar structural features can include, for example, regions of amino acid identity, conserved active site or binding site motifs, and similarly arranged secondary structural elements (e.g., ⁇ helices and ⁇ sheets).
  • structural homology is determined by aligning the residues of the two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • two amino acid sequences are compared using the Blastp program, version 2.0.9, of the BLAST 2 search algorithm, as described by Tatiana et al., FEMS Microbiol Lett 174, 247-50 (1999), and available at http://www.ncbi.nlm.nih.gov/gorf bl2.html.
  • a structurally homologous molecule is a protein that has an amino acid sequence sharing at least 65% identity with a native or recombinant amino acid sequence of S. aureus thymidylate kinase (for example, SEQ ID NO:l). More preferably, a protein that is structurally homologous to S.
  • aureus thymidylate kinase includes at least one contiguous stretch of at least 50 amino acids that shares at least 80% amino acid sequence identity with the analogous portion of the native or recombinant S. aureus thymidylate kinase (for example, SEQ ID NO: 1).
  • Methods for generating structural information about the structurally homologous molecule or molecular complex are well-known and include, for example, molecular replacement techniques. Therefore, in another embodiment this invention provides a method of utilizing molecular replacement to obtain structural information about a molecule or molecular complex whose structure is unknown comprising the steps of:
  • all or part of the structure coordinates of S. aureus thymidylate kinase or the S. aureus thymidylate kinase/ligand complex as provided by this invention can be used to determine the structure of a crystallized molecule or molecular complex whose structure is unknown more quickly and efficiently than attempting to determine such information ab initio.
  • Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that cannot be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures.
  • this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of S. aureus thymidylate kinase or the S. aureus thymidylate kinase/ligand complex according to Figure 2 within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed x-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown.
  • Phases can then be calculated from this model and combined with the observed x-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown.
  • This can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex (E. Lattman, "Use of the Rotation and Translation Functions," in Meth. Enzymol.. 115, pp. 55-77 (1985); M.G. Rossman, ed., "The Molecular Replacement Method,” Int. Sci. Rev. Ser.. No. 13, Gordon & Breach, New York (1972)).
  • Structural information about a portion of any crystallized molecule or molecular complex that is sufficiently structurally homologous to a portion of S. aureus thymidylate kinase can be resolved by this method.
  • a molecule that shares one or more structural features with S. aureus thymidylate kinase as described above a molecule that has similar bioactivity, such as the same catalytic activity, substrate specificity or ligand binding activity as S. aureus thymidylate kinase, may also be sufficiently structurally homologous to S. aureus thymidylate kinase to permit use of the structure coordinates of S. aureus thymidylate kinase to solve its crystal structure.
  • the method of molecular replacement is utilized to obtain structural information about a molecule or molecular complex, wherein the molecule or molecular complex comprises at least one S. aureus thymidylate kinase subunit or homolog.
  • a "subunit" of S. aureus thymidylate kinase is an S. aureus thymidylate kinase molecule that has been truncated at the N-terminus or the C-terminus, or both.
  • aureus thymidylate kinase is a protein that contains one or more amino acid substitutions, deletions, additions, or rearrangements with respect to the amino acid sequence of S. aureus thymidylate kinase (SEQ ID NOT), but that, when folded into its native conformation, exhibits or is reasonably expected to exhibit at least a portion of the tertiary (three-dimensional) structure of S. aureus thymidylate kinase,
  • structurally homologous molecules can contain deletions or additions of one or more contiguous or noncontiguous amino acids, such as a loop or a domain.
  • Structurally homologous molecules also include "modified" S.
  • aureus thymidylate kinase molecules that have been chemically or enzymatically derivatized at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
  • a heavy atom derivative of S. aureus thymidylate kinase is also included as an S. aureus thymidylate kinase homolog.
  • the term "heavy atom derivative” refers to derivatives of 5. aureus thymidylate kinase produced by chemically modifying a crystal of S., aureus thymidylate kinase, In practice, a crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, e.g., lead chloride, gold thiomalate, thiomersal or uranyl acetate, which can diffuse through the crystal and bind to the surface of the protein.
  • the location(s) of the bound heavy metal atom(s) can be determined by x-ray diffraction analysis of the soaked crystal. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the protein (T.L. Blundell and N.L. Johnson, Protein Crystallography. Academic Press (1976)). Because S. aureus thymidylate kinase can crystallize in more than one crystal form, the structure coordinates of S. aureus thymidylate kinase as provided by this invention are particularly useful in solving the structure of other crystal forms of S. aureus thymidylate kinase or S. aureus thymidylate kinase complexes.
  • the structure coordinates of S. aureus thymidylate kinase as provided by this invention are particularly useful in solving the structure of S. aureus thymidylate kinase mutants.
  • Mutants may be prepared, for example, by expression of S. aureus thymidylate kinase cDNA previously altered in its coding sequence by oligonucleotide-directed mutagenesis. Mutants may also be generated by site-specific incorporation of unnatural amino acids into thymidylate kinase proteins using the general biosynthetic method of C.J. Noren et al., Science. 244:182-188 (1989). In this method, the codon encoding the amino acid of interest in wild-type S.
  • aureus thymidylate kinase is replaced by a "blank" nonsense codon, TAG, using oligonucleotide-directed mutagenesis.
  • a suppressor tRNA directed against this codon is then chemically aminoacylated in vitro with the desired unnatural amino acid.
  • the aminoacylated tRNA is then added to an in vitro translation system to yield a mutant S. aureus thymidylate kinase with the site-specific incorporated unnatural amino acid.
  • Selenocysteine or selenomethionine may be incorporated into wild- type or mutant S. aureus thymidylate kinase by expression of S. aureus thymidylate kinase-encoding cDNAs in auxotrophic E. coli strains (W.A. Hendrickson et al., ⁇ MBO J.. 9(5): 1665-1672 (1990)). In this method, the wild-type or mutagenized S.
  • aureus thymidylate kinase cDNA may be expressed in a host organism on a growth medium depleted of either natural cysteine or methionine (or both) but enriched in selenocysteine or selenomethionine (or both).
  • selenomethionine analogues may be prepared by down regulation methionine biosynthesis.
  • the structure coordinates of S. aureus thymidylate kinase listed in Figure 2 are also particularly useful to solve the structure of crystals of S. aureus thymidylate kinase, S. aureus thymidylate kinase mutants or S. aureus thymidylate kinase homologs co-complexed with a variety of chemical entities.
  • This approach enables the determination of the optimal sites for interaction between chemical entities, including candidate S. aureus thymidylate kinase inhibitors and S. aureus thymidylate kinase. Potential sites for modification within the various binding site of the molecule can also be identified.
  • This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between S. aureus thymidylate kinase and a chemical entity. For example, high resolution x-ray diffraction data collected from crystals exposed to different types of solvent allows the determination of where each type of solvent molecule resides. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their S. aureus thymidylate kinase inhibition activity.
  • the invention also includes the unique three-dimensional configuration defined by a set of points defined by the structure coordinates for a molecule or molecular complex structurally homologous to S. aureus thymidylate kinase as determined using the method of the present invention, structurally equivalent configurations, and magnetic storage media comprising such set of structure coordinates.
  • the invention includes structurally homologous molecules as identified using the method of the invention.
  • a computer model of an S. aureus thymidylate kinase homolog can be built or refined without crystallizing the homolog.
  • a preliminary model of the S. aureus thymidylate kinase , homolog is created by sequence alignment with S. aureus thymidylate kinase, secondary structure prediction, the screening of structural libraries, or any combination of those techniques.
  • Computational software may be used to carry out the sequence alignments and the secondary structure predictions.
  • Structural incoherences e.g., structural fragments around insertions and deletions, can be modeled by screening a structural library for peptides of the desired length and with a suitable conformation.
  • a side chain rotamer library may be employed. If the S. aureus thymidylate kinase homolog has been crystallized, the final homology model can be used to solve the crystal structure of the homolog by molecular replacement, as described above. Next, the preliminary model is subjected to energy n ⁇ nization to yield an energy minimized model.
  • the energy minimized model may contain regions where stereochemistry restraints are violated, in which case such regions are remodeled to obtain a final homology model.
  • the homology model is positioned according to the results of molecular replacement, and subjected to further refinement comprising molecular dynamics calculations.
  • Computational techniques can be used to screen, identify, select and/or design chemical entities capable of associating with S. aureus thymidylate kinase or structurally homologous molecules. Knowledge of the structure coordinates for S. aureus thymidylate kinase permits the design and/or identification of synthetic compounds and/or other molecules which have a shape complementary to the conformation of the S. aureus thymidylate kinase binding site.
  • computational techniques can be used to identify or design chemical entities, such as inhibitors, agonists and antagonists, that associate with an S. aureus thymidylate kinase substrate binding pocket or an S. aureus thymidylate kinase-like substrate binding pocket.
  • Inhibitors may bind to or interfere with all or a portion of an active site of S. aureus thymidylate kinase, and can be competitive, non- competitive, or uncompetitive inhibitors; or interfere with dimerization by binding at the interface between the two monomers. Once identified and screened for biological activity, these inhibitors/agonists/antagonists may be used therapeutically or prophylactically to block S. aureus thymidylate kinase activity and, thus, inhibit the growth of the bacteria or cause its death. Structure-activity data for analogues of ligands that bind to or interfere with S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pockets can also be obtained computationally.
  • chemical entity refers to chemical compounds, complexes of two or more chemical compounds, and fragments of such compounds or complexes. Chemical entities that are determined to associate with S. aureus thymidylate kinase are potential drug candidates.
  • Data stored in a machine-readable storage medium that is capable of displaying a graphical three-dimensional representation of the structure of S. aureus thymidylate kinase or a structurally homologous molecule, as identified herein, or portions thereof may thus be advantageously used for drug discovery.
  • the structure coordinates of the chemical entity are used to generate a three-dimensional image that can be computationally fit to the three-dimensional image of S. aureus thymidylate kinase or a structurally homologous molecule.
  • the three-dimensional molecular structure encoded by the data in the data storage medium can then be computationally evaluated for its ability to associate with chemical entities.
  • the protein structure can also be visually inspected for potential association with chemical entities.
  • One embodiment of the method of drug design involves evaluating the potential association of a known chemical entity with S. aureus thymidylate kinase or a structurally homologous molecule, particularly with an S. aureus thymidylate kinase substrate binding pocket or S. aureus thymidylate kinase-like substrate binding pocket.
  • the method of drug design thus includes computationally evaluating the potential of a selected chemical entity to associate with any of the molecules or molecular complexes set forth above. This method comprises the steps of:
  • the method of drug design involves computer-assisted design of chemical entities that associate with S. aureus thymidylate kinase, its homologs, or portions thereof. Chemical entities can be designed in a step-wise fashion, one fragment at a time, or may be designed as a whole or "de novo.”
  • the chemical entity identified or designed according to the method must be capable of structurally associating with at least part of an S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pockets, and must be able, sterically and energetically, to assume a conformation that allows it to associate with the S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pocket.
  • Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions.
  • Conformational considerations include the overall three-dimensional structure and orientation of the chemical entity in relation to the substrate binding pocket, and the spacing between various functional groups of an entity that directly interact with the S. aureus thymidylate kinase-like substrate binding pocket or homologs thereof.
  • the potential binding of a chemical entity to an S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pocket is analyzed using computer modeling techniques prior to the actual synthesis and testing of the chemical entity. If these computational experiments suggest insufficient interaction and association between it and the S. aureus thymidylate kinase or S.
  • aureus thymidylate kinase-like substrate binding pocket testing of the entity is obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to or interfere with an S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pocket. Binding assays to determine if a compound actually interferes with S. aureus thymidylate kinase can also be performed and are well known in the art.
  • Binding assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof.
  • One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with an S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pocket. This process may begin by visual inspection of, for example, an S. aureus thymidylate kinase or S.
  • aureus thymidylate kinase- like substrate binding pocket on the computer screen based on the S. aureus thymidylate kinase structure coordinates listed in Figure 2 or other coordinates which define a similar shape generated from the machine- readable storage medium.
  • Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the substrate binding pocket. Docking may be accomplished using software such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
  • Specialized computer programs may also assist in the process of selecting fragments or chemical entities. Examples include GRID (P. J. Goodford, J. Med. Chem. 28:849-857 (1985); available from Oxford University, Oxford, UK); MCSS (A. Miranker et al., Proteins: Struct. Funct. Gen.,11 :29-34 (1991); available from Molecular Simulations, San Diego, CA); AUTODOCK (D.S. Goodsell et al., Proteins: Struct. Funct. Genet. 8:195-202 (1990); available from Scripps Research Institute, La JoUa, CA); and DOCK (I.D. Kuntz et al., J. Mol. Biol. 161:269-288 (1982); available from University of California, San Francisco, CA).
  • GRID P. J. Goodford, J. Med. Chem. 28:849-857 (1985); available from Oxford University, Oxford, UK
  • MCSS A. Miranker et al.
  • Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include, without limitation, CAVEAT (P.A. Bartlett et al., in Molecular Recognition in Chemical and Biological Problems.” Special Publ., Royal Chem. Soc, 78:182-196 (1989); G. Lauri et al., J. Comput. Aided Mol. Des. 8:51-66 (1994); available from the University of California, Berkeley, CA); 3D database systems such as ISIS (available from MDL Information Systems, San Leandro, CA; reviewed in Y.C. Martin, J. Med. Chem. 35:2145-2154 (1992)); and HOOK (M.B. Eisen et al., Proteins: Struc. Funct.. Genet. 19:199-221 (1994); available from Molecular Simulations, San Diego, CA).
  • CAVEAT P.A. Bartlett et al., in Molecular Recognition in Chemical and Biological Problems.” Special Publ., Royal Chem
  • S. aureus thymidylate kinase binding compounds may be designed "de novo" using either an empty binding site or optionally including some portion(s) of a known inhibitor(s).
  • de novo ligand design methods including, without limitation, LUDI (H.-J. Bohm, J. Comp. Aid. Molec. Design. 6:61-78 (1992); available from Molecular Simulations Inc., San Diego, CA); LEGEND (Y. Nishibata et al., Tetrahedron. 47:8985 (1991); available from Molecular Simulations Inc., San Diego, CA); LeapFrog (available from Tripos Associates, St. Louis, MO); and SPROUT (V. Gillet et al., J. Comput. Aided Mol. Design 7:127- 153 (1993); available from the University of Leeds, UK).
  • an effective S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pocket inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding).
  • a relatively small difference in energy between its bound and free states i.e., a small deformation energy of binding.
  • aureus thymidylate kinase-like substrate binding pocket inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole; more preferably, not greater than 7 kcal/mole.
  • S. aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pocket inhibitors may interact with the substrate binding pocket in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein.
  • aureus thymidylate kinase or S. aureus thymidylate kinase-like substrate binding pocket may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme and with the surrounding water molecules.
  • Such non-complementary electrostatic interactions include repulsive charge- charge, dipole-dipole, and charge-dipole interactions.
  • This invention also enables the development of chemical entities that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that interferes with or with S. aureus thymidylate kinase.
  • Time-dependent analysis of structural changes in S. aureus thymidylate kinase during its interaction with other molecules is carried out.
  • the reaction intermediates of 5. aureus thymidylate kinase can also be deduced from the reaction product in co- complex with S. aureus thymidylate kinase. Such information is useful to design improved analogues of known S.
  • aureus thymidylate kinase inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the S. aureus thymidylate kinase and inhibitor co-complex. This provides a novel route for designing S. aureus thymidylate kinase inhibitors with both high specificity and stability.
  • Yet another approach to rational drug design involves probing the S. aureus thymidylate kinase crystal of the invention with molecules comprising a variety of different functional groups to determine optimal sites for interaction between candidate S. aureus thymidylate kinase inhibitors and the protein. For example, high resolution x-ray diffraction data collected from crystals soaked in or co-crystallized with other molecules allows the determination of where each type of solvent molecule sticks. Molecules that bind tightly to those sites can then be further modified and synthesized and tested for their thymidylate kinase inhibitor activity (J. Travis, Science. 262:1374 (1993)).
  • iterative drug design is used to identify inhibitors of S. aureus thymidylate kinase. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein compound complexes. In iterative drug design, crystals of a series of protein/compound complexes are obtained and then the three- dimensional structures of each complex is solved. Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three dimensional structure of the complex, and comparing the associations between the new protein/compound complex and previously solved protein/compound complexes.
  • a compound that is identified or designed as a result of any of these methods can be obtained (or synthesized) and tested for its biological activity, e.g., inhibition of thymidylate kinase activity.
  • compositions of this invention comprise an inhibitor of S. aureus TMK activity identified according to the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • pharmaceutically acceptable carrier refers to a carrier(s) that is “acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof.
  • the pH of the formulation is adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form. Methods of making and using such pharmaceutical compositions are also included in the invention.
  • compositions of the invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Oral administration or administration by injection is preferred.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, infra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between about 0.5 and about 75 mg/kg body weight per day of the S. aureus TMK inhibitory compounds described herein are useful for the prevention and treatment of S. aureus TMK mediated disease.
  • the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w).
  • such preparations contain from about 20% to about 80% active compound.
  • the M15-1C Escherichia coli construct expressing S. aureus thymidylate kinase was obtained as a strain in which the Qiagen pREP4 vector was replaced with pREP4UX.
  • Genes and polypeptides derived from S. aureus, including S. aureus and thymidylate kinase, are published in EP 786519 A2 and WO 0012678, both assigned to Human Genome Sciences.
  • This plasmid contains the argU gene which codes for the AGA tRNA and prevents the lysine for arginine substitution which occurred in the original construct from Human Genome Sciences.
  • the construct was grown in a minimal salts medium, M9, which contained glucose and N ⁇ t t Cl as the sources of carbon and nitrogen. Endogenous methionine biosynthesis was then inhibited while adding an excess of selenomethionine to the growth medium just prior to IPTG induction of thymidylate kinase synthesis (T.E. Benson et al., Nat. Struct. Biol.. 2:644-53 (1995); G.D. Van Duyne et al., J. Mol. Biol. 229:105-24 (1993)).
  • basal M9 was Na 2 HPO 4 , 6 g; KH 2 PO 4 , 3 g; NH 4 CI, 1.0 g; and NaCl, 0.5 g per L of deionized water.
  • the pH was adjusted to 7.4 with concentrated KOH and the medium was sterilized by autoclaving.
  • the following filter sterilized solutions were added per L of basal medium: 1M MgSO , 1.0 mL; 1M CaCl 2 , 0.1 mL; trace metal salts solution, 0.1 mL, 10 mM thiamin, 1.0 mL; and 20% glucose, 20 mL.
  • the trace metal salts solution contained per L of deionized water: MgCl 2 -6H 2 O, 39.44 g; MnSO 4 H 2 O, 5.58 g; FeSO 4 -7H 2 O, 1.11 g; Na 2 MoO 4 -2H 2 O, 0.48 g; CaCl 2 , 0.33 g; NaCl, 0.12 g; and ascorbic acid, 1.0 g.
  • Filter sterilized ampicillin and kanamycin were added to the medium at final concentrations of 100 mg/mL and 30mg/mL, respectively. Fermentations were prepared in 100 mL volumes of M9 medium contained in 500 mL wide mouth flasks.
  • a 0.1 mL aliquot of the stock culture was inoculated into the medium and allowed to grow at 30°C for 18 - 20 hours with a shaking rate of 200 rpm.
  • the seed culture was harvested by centrifugation and then resuspended in an equal volume of M9 medium.
  • the resuspended seed was used to inoculate expression fermentations at a rate of 3%.
  • the culture was grown under the same conditions to an A600 of ⁇ 0.6.
  • methionine biosynthesis was down regulated by the addition of L-lysine, L-threonine, and L-phenylalanine at a final concentration for each of 100 mg/mL and L-leucine, L-isoleucine, and L-valine at 50 mg/mL each.
  • D,L-selenomethionine was added simultaneously to a final concentration of 100 mg/mL.
  • expression of thymidylate kinase was induced by addition of EPTG (isopropyl thio- ⁇ -D-galactosidase, Gibco BRL) to 1 mM. Growth of the culture was continued for an additional 3.5 hours until an A600 of 1.5 - 1.6. Cells were then harvested by centrifugation and frozen at -80°C. Under these conditions, the average yield of cell paste was 3.0 to 3.5 g/L.
  • EQ buffer 25 mM Tris (pH 7.8), 500 mM NaCl, 10% glycerol, 25 mM imidazole, 5 mM 2-mercaptoethanol.
  • the column was washed with 7.7 CV of EQ buffer, 12.5 CV of wash buffer (25 mM Tris (pH 7.8), 500 mM NaCl, 10% glycerol, 50 mM imidazole, 5 mM 2-mercaptoethanol) and eluted with 1.4 CV of elution buffer (25 mM Tris (pH 7.8), 500 mM NaCl, 10% glycerol, 300 mM imidazole, 5 mM 2-mercaptoethanol). During the elution the linear velocity was decreased to 42 cm hr.
  • the eluted fraction was treated with DTT to achieve a final concentration of 10 mM and dialyzed extensively against nitrogen sparged dialysis buffer (25 mM Tris (pH 7.8), 500 mM NaCl, 10% glycerol, 10 mM DTT, pH 7.8).
  • the Mono Q analytical run was performed using 50 mL native TMK (14 mg/mL) diluted to 200 mL with 20 mM Tris (pH 8.0). The sample was loaded onto a Mono Q (Amersham Pharmacia Biotech) column equilibrated with 20 mM Tris (pH 8.0) and run through a 20-40% (20 mM Tris (pH 8.0) + 1.0M NaCl) gradient in 40 mL with a flow rate of 1.0 mL/min. The Mono P column run was performed using 50 mL TMK (14mg/mL) diluted to 200 mL with 25 mM bis-Tris (pH 6.71).
  • the sample was injected onto a Mono P column (Amersham Pharmacia Biotech) equilibrated with 25 mM bis-Tris (pH 6.71) and run through a step gradient of 0-100-0 % Polybuffer Mix 96/74 (20:1), pH 5.80.
  • Gel filtration studies were carried out on a Superose 200 column with a 500 mL sample of thymidylate kinase at a concentration of 4.2 mg/mL using 50 mM Tris (pH 8.5), 500 mM NaCl, 5 mM 2-mercaptoethanol, and 0.5% glycerol at a flow rate of 1 mL/min.
  • samples were mixed in 1.5ml eppendorf tubes, then sterile filtered through a 0.22 mm ceramic membrane (Whatman). 20 mL of solution is read in a quartz cuvette in a Dyna Pro Molecular Sizing Instrument (Protein Solutions, Inc., Charlottesville, VA).
  • the native protein was exchanged into 50 mM Tris (pH 7.8), 5 mM 2-mercaptoethanol to a concentration of 15 mg/mL and screened for crystallization conditions using Crystal Screen I, Crystal Screen ⁇ , and MembFac Screen (Hampton Research, Madison Niguel, CA). The most encouraging lead was from Hampton Crystal Screen I condition 23: 30% PEG 400, 0.1M Na EEEPES pH 7.5, 0.2M MgCl 2 .
  • the initial crystals of the thymidylate kinase were stacks of small plates that were inseparable and unusable for diffraction studies.
  • Biochemical analysis of the protein revealed that the sample was substantially pure by sodium dodecylsulfate polyacrylamide-gel electrophoresis (SDS-PAGE) analysis, but isoelectric focusing (EEF) gels revealed at least two distinct isoelectric species. It is likely, although yet unproven, that these isoelectric species were the cause of the morphology of the thymidylate kinase crystals. Further efforts at purification with a Mono Q column indicated that separation of these species would be difficult and it was not clear that large scale isoelectrofocusing using a Mono P column or preparative isoelectric focusing would improve the separation because of the small differences in pi.
  • the stacked plates were eventually transformed into single crystals through iterative streak seeding and crystallization on hanging or sitting drops with thymidylate kinase in 0.1 M PIPES (pH 6.6), 14-19% PEG 400, 0.2 M MgC12.
  • This technique involved taking the multinucleated crystals, crushing them into microcrystals, and using a dilution series of this suspension of microcrystals for seeding. It was observed that this second round of crystals were usually less multinucleated than when crystal formation was allowed to proceed via spontaneous nucleation. A second round of streak seeding was usually necessary in order to obtain multiple single crystals. Refinement of the streak seeding technique resulted in native and selenomethionine TMK crystals on the order of about 100 ⁇ m x about 100 ⁇ m x about 20 ⁇ m.
  • Thymidylate kinase crystals were generally too small for useful data collection using standard x-ray diffraction equipment. Therefore, all data collection was carried out at the Advanced Photon Source (Argonne, IL).
  • Binding assays to determine if a compound actually interferes with S. aureus thymidylate kinase can also be performed.
  • thymidylate kinase activity can be measured by coupling the formation of ADP and TDP to the reactions catalyzed by PD, LDH, and ⁇ DP-Kinase, as shown below. Oxidation of ⁇ ADH is accompanied by a decrease in absorbance at 340 run, which is measured spectrophotometrically. 1) ATP + TMP Thymidylate Kinase ADP + TDP
  • the standard reaction conditions employed during the kinetic characterization of the enzyme were: 50 mM HEPES, pH 8.0, 50 mM KC1, 2 mM MgCl 2 , 4 U/ml PK, 5 U/ml LDH, 2 mM PEP, 1.5 mM ATP, 5 U/ml NDP-Kinase, 1.0 mM TMP, 0.22 mM NADH, and 0.8 ⁇ g/ml T. kinase. All of the reagents except the T. Kinase were added to a cuvette and mixed, and the mixture was incubated at 24.5°C for 2 minutes. To start the reaction, the T. Kinase was added, the contents of the cuvette were mixed, and the decrease in absorbance at 340 run was monitored for 4-5 minutes.
  • SEQ ID NO: 1 recombinant S. aureus thymidylate kinase (with polyhistidine [His 6 ] sequence tag)
  • SEQ ID NO: 3 S. cerevisiae thymidylate kinase

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Analysing Materials By The Use Of Radiation (AREA)

Abstract

L'invention concerne une forme sans ligand de thymidylate kinase (TMK) de Staphylococcus aureus (S. aureus), qui a été cristallisée, la structure cristalline à rayons X tridimensionnelle ayant une résolution de 2,3 Å. La structure cristalline à rayons X s'utilise pour résoudre la structure d'autres molécules ou complexes moléculaires, et désigner des inhibiteurs de l'activité de la thymidylate kinase (TMK) de S. aureus.
PCT/US2000/021425 1999-08-04 2000-08-04 Cristallisation et determination de la structure de la thymidylate kinase de staphylococcus aureus WO2001011025A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2001515812A JP2003517818A (ja) 1999-08-04 2000-08-04 スタフィロコッカス・アウレウスのチミジル酸キナーゼの結晶化および構造決定
EP00953850A EP1200565A2 (fr) 1999-08-04 2000-08-04 Cristallisation et determination de la structure de la thymidylate kinase de staphylococcus aureus
AU66227/00A AU781654B2 (en) 1999-08-04 2000-08-04 Crystallization and structure determination of staphylococcus aureus thymidylate kinase
CA002378010A CA2378010A1 (fr) 1999-08-04 2000-08-04 Cristallisation et determination de la structure de la thymidylate kinase de staphylococcus aureus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14711799P 1999-08-04 1999-08-04
US60/147,117 1999-08-04

Publications (2)

Publication Number Publication Date
WO2001011025A2 true WO2001011025A2 (fr) 2001-02-15
WO2001011025A3 WO2001011025A3 (fr) 2001-08-23

Family

ID=22520348

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/021425 WO2001011025A2 (fr) 1999-08-04 2000-08-04 Cristallisation et determination de la structure de la thymidylate kinase de staphylococcus aureus

Country Status (5)

Country Link
EP (1) EP1200565A2 (fr)
JP (1) JP2003517818A (fr)
AU (1) AU781654B2 (fr)
CA (1) CA2378010A1 (fr)
WO (1) WO2001011025A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007525947A (ja) * 2003-05-07 2007-09-13 スミスクライン・ビーチャム・コーポレイション メチオニンアミノペプチダーゼおよびその使用方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6026425B2 (ja) * 2010-11-02 2016-11-16 プロビオドルグ エージー イソグルタミニルシクラーゼの結晶構造

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus
WO2000012678A2 (fr) * 1998-09-01 2000-03-09 Human Genome Sciences, Inc. Genes de staphylococcus aureus et polypeptides associes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus
WO2000012678A2 (fr) * 1998-09-01 2000-03-09 Human Genome Sciences, Inc. Genes de staphylococcus aureus et polypeptides associes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A LAVIE ET AL: "Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor TP5A at 2.0 A resolution: implications for catalysis and AZT activation" BIOCHEMISTRY,AMERICAN CHEMICAL SOCIETY. EASTON, PA,US, vol. 37, 1998, pages 3677-3686, XP002111244 ISSN: 0006-2960 cited in the application *
A LAVIE ET AL: "Structural basis for the efficient phosphorylation of 3'-azidothymidine monophosphate by E. coli thymidylate kinase" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA,NATIONAL ACADEMY OF SCIENCE. WASHINGTON,US, vol. 95, November 1998 (1998-11), pages 14045-14050, XP002111243 ISSN: 0027-8424 cited in the application *
DE LA SIERRA I LI ET AL: "Crystallization and preliminary X-ray analysis of the thymidylate kinase from Mycobacterium tuberculosis." ACTA CRYSTALLOGRAPHICA SECTION D BIOLOGICAL CRYSTALLOGRAPHY, vol. 56, no. 2, February 2000 (2000-02), pages 226-228, XP000982605 ISSN: 0907-4449 *
OSTERMANN NILS ET AL: "Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate." STRUCTURE (LONDON), vol. 8, no. 6, 2000, pages 629-642, XP000982582 ISSN: 0969-2126 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007525947A (ja) * 2003-05-07 2007-09-13 スミスクライン・ビーチャム・コーポレイション メチオニンアミノペプチダーゼおよびその使用方法

Also Published As

Publication number Publication date
CA2378010A1 (fr) 2001-02-15
WO2001011025A3 (fr) 2001-08-23
AU781654B2 (en) 2005-06-02
AU6622700A (en) 2001-03-05
EP1200565A2 (fr) 2002-05-02
JP2003517818A (ja) 2003-06-03

Similar Documents

Publication Publication Date Title
US20090062286A1 (en) Crystal Structure of SMYD3 Protein
US6387641B1 (en) Crystallized P38 complexes
US8002891B2 (en) Crystallization of C-Jun N-Terminal Kinase 3 (JNK3)
US6356845B1 (en) Crystallization and structure determination of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (S. aureus MurB)
US6988041B2 (en) Crystallization and structure determination of Staphylococcus aureus NAD synthetase
EP1200468A1 (fr) Cristallisation et determination de structure du facteur p d'elongation du staphylococcus aureus
WO1999057253A2 (fr) Complexes de jnk cristallisables
AU781654B2 (en) Crystallization and structure determination of staphylococcus aureus thymidylate kinase
US6689595B1 (en) Crystallization and structure determination of Staphylococcus aureus thymidylate kinase
US20030165984A1 (en) Hepatitis C virus helicase crystals, crystallographic structure and methods
US20030166843A1 (en) Crystallization and structure determination of staphylococcus aureus thioredoxin reductase
EP1911838A1 (fr) Formes crystallines de la protéine PKC alpha kinase, procédé de manufacture desdits cristaux et leur utilisation
US6484103B1 (en) Crystal structure
US20040191271A1 (en) Crystal structures of streptococcus undecaprenyl pyrophosphate synthase and uses thereof
US20050208639A1 (en) Crystal structure of staphylococcus undecaprenyl pyrophosphate synthase and uses thereof
WO2002055550A2 (fr) Cristallisation et determination de la structure de proteines fema et/ou de type fema
JP2007522792A (ja) ｃ−Ａｂ１チロシン・キナーゼ・ドメインの結晶及び構造
WO2003060102A2 (fr) Structures cristallines de complexes d'inhibition de la jnk et poches de liaison de ceux-ci
WO2002002758A2 (fr) Cristallisation et structure de la peptide deformylase du staphylocoque dore

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 66227/00

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2378010

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2000953850

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000953850

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2000953850

Country of ref document: EP