WO2001090301A2 - Procedes de preparation de modeles, procedes d'utilisation des modeles de la proteine murg, composes liant, inhibant ou stimulant des proteines murg et des compositions therapeutiques associees - Google Patents

Procedes de preparation de modeles, procedes d'utilisation des modeles de la proteine murg, composes liant, inhibant ou stimulant des proteines murg et des compositions therapeutiques associees Download PDF

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WO2001090301A2
WO2001090301A2 PCT/US2001/011500 US0111500W WO0190301A2 WO 2001090301 A2 WO2001090301 A2 WO 2001090301A2 US 0111500 W US0111500 W US 0111500W WO 0190301 A2 WO0190301 A2 WO 0190301A2
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murg
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Suzanne Walker
Sha Ha
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Princeton University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the present invention relates to crystals of the Escherichia coli MurG, a membrane- associated UDP-glycosyltransferase involved in peptidoglycan biosynthesis.
  • the present invention also relates to three-dimensional atomic coordinates of the MurG protein, three-dimensional structures of the protein, and images thereof.
  • the present invention also relates to the atomic coordinates and three-dimensional structures of the ⁇ -carbon backbone of the MurG protein and images thereof.
  • the present invention further relates to the atomic coordinates and three-dimensional structures of the ⁇ -carbon backbone and conserved amino acid residue sidechains of the MurG protein and images thereof.
  • the present invention further relates to three-dimensional atomic coordinates of the donor nucleotide binding site, the acceptor binding site, and the membrane association site of the MurG protein, three-dimensional structures of the binding domains; and images thereof.
  • the present invention also relates to computer readable media encoded with sets of the three dimensional coordinates of the E. coli MurG protein, the ⁇ -carbon backbone of the MurG protein, the ⁇ -carbon backbone and the conserved amino acid residue sidechains of the MurG protein, the donor nucleotide binding site, the acceptor binding site, and the membrane association site.
  • the present invention relates to methods of crystallizing MurG proteins.
  • the present invention relates to models of three dimensional structures of UDP- glycosyltransferases and, in particular, MurG proteins, based on the three dimensional structure of crystals of the Escherichia coli MurG.
  • the present invention also relates to models of the three dimensional structures of the ⁇ -carbon backbone of UDP- glycosyltransferases and MurG proteins.
  • the present invention further relates to models of the three dimensional structure of the ⁇ -carbon backbone and conserved amino acid residue sidechains of gUDP-glycosyltransferases, in particular, MurG proteins.
  • the present invention further relates to models of the three-dimensional structures of donor nucleotide binding sites, acceptor binding sites, and membrane association sites of UDP- glycosyltransferases, in particular, MurG proteins.
  • the present invention also relates to methods of drug design using models of this invention.
  • the present invention further relates to compounds identified using models of the present invention that bind, inhibit or stimulate UDP-glycosyltransferases or MurG proteins.
  • the present invention relates to compositions comprising compounds identified using the models of this invention for therapeutic or diagnositic uses. Also, the present invention relates to methods of making models of the present invention.
  • MurG is the last enzyme involved in the intracellular phase of peptidoglycan synthesis (Bugg & Walsh, 1993). It catalyzes the transfer of N-acetyl glucosamine (NAG) from UDP to the C4 hydroxyl of a lipid-linked N-acetylmuramoyl pentapeptide
  • NAM NAG-NAM disaccharide
  • Fig. 1 ?-linked NAG-NAM disaccharide that is transported across the cell membrane where it is polymerized and cross-linked
  • One three dimensional structure of a MurG protein can be used to construct models of other MurG proteins and to facilitate the structure determination of crystalline forms of other MurG proteins.
  • Structures and models of MurG proteins can also be used to design proteins containing only the donor binding site or the acceptor binding site. These proteins can be used in assays, including NMR-based assays, to identify — or characterize the mode of binding of — ligands that bind in or near the vicinity of the substrates. These ligands or compounds can then be used as leads for the design of inhibitors that have therapeutic activity.
  • Structures and models of MurG proteins can also be used in computer-based drug design.
  • the present invention relates to crystalline Escherichia coli MurG protein. Obtaining such crystals is an unexpected result. It is well known in the protein crystallographic art that obtaining crystals of quality sufficient for determining the structure of a protein is unpredictable. In particular, obtaining crystals of quality sufficient for determining the three-dimensional (3-D) structure of MurG has not been achievable until the crystallization of MurG as disclosed in the present application. As such, determination of the three-dimensional structure of MurG has not been possible until the discovery of the present invention. Additionally, until the discovery of the present invention, derivation of the three-dimensional structure and models of other MurG proteins has not been possible. The present inventors are also the first to define the three-dimensional structure and provide three-dimensional models for drug design for MurG proteins.
  • one object of the present invention is to provide crystals of sufficient quality to obtain a determination of the three-dimensional atomic coordinates and structures of MurG to high resolution, preferably to the resolution of less than 2.0 angstroms (A).
  • the present invention also provides methods for producing crystalline MurG protein.
  • the value of the crystals of E. coli MurG protein extends beyond merely being able to obtain such crystals.
  • the knowledge obtained concerning the MurG crystal structure has been used by the present inventors to define the heretofore unknown tertiary structure of the MurG protein and to identify the location of the glycosyl donor and glycosyl acceptor binding domains, as well as the location of the amino acid residues that are invariant in all MurG proteins. This information can be used to design inhibitors of MurG that have therapeutic utility.
  • the atomic coordinates of E. coli MurG also are used to model the heretofore unknown tertiary structures of other MurG proteins having substantially related linear amino acid sequences, such as for MurG proteins from other microorganisms.
  • Comparison of nucleic acid and amino acid sequences of MurG proteins indicates that the linear amino acid sequences can vary significantly. Homology between MurG proteins from different microorganisms varies from less than 30% to greater than 90%, reflecting the evolutionary relationship between the organisms. The low homology between distantly related MurG homologues is not believed to reflect significantly different folded structures. It is well known that many amino acid sequences are capable of adopting the same general fold. E. coli MurG contains an alpha/beta folding pattern, one of the most common folds known in proteins. It is likely that all MurG homologues contain a similar alpha/beta fold despite the differences in the linear amino acid sequences.
  • a object of the present invention is to provide information regarding the atomic coordinates and three-dimensional structures of (1) the MurG protein, (2) the ⁇ -carbon backbone of the MurG protein, (3) the ⁇ -carbon backbone and conserved amino acid residues of the MurG protein, (4) the donor nucleotide binding site, (5) the acceptor binding site, and (6) the membrane association site MurG proteins.
  • the present invention relates to models of three dimensional structures of UDP- glycosyltransferases, in particular MurG proteins, based on the atomic coordinates of crystalline E. coli MurG protein.
  • Another object of the present invention is to provide computer readable mediums encoded with a set of three-dimensional coordinates of the E. coli MurG protein, the ⁇ - carbon backbone of the MurG protein, the ⁇ -carbon backbone and conserved amino acid residues of the MurG protein, and the nucleotide donor binding site, the acceptor binding site, the membrane association site of the MurG protein.
  • Another embodiment of the present invention provides three-dimensional and two-dimensional computer images of the three dimensional structure of MurG protein, the ⁇ -carbon backbone of the MurG protein, the ⁇ -carbon backbone and conserved amino acid residues of the MurG protein, and the nucleotide donor binding site, the acceptor binding site, the membrane association site of the MurG protein.
  • the knowledge of the three dimensional structure of MurG also provides a means for designing proteins that have altered beneficial functions by analyzing the structure and interactions between individual amino acids of the protein.
  • ⁇ . coli MurG consists of two domains separated by a cleft. Noncovalent interactions between the two domains are not extensive.
  • the present inventors have shown that the domains fold independently and can, therefore, be expressed independently either alone or as part of a recombinant protein containing the acceptor binding site from one MurG homologue and the donor binding site from another MurG homologue. It would be expected that the domains of other MurG proteins could also be expressed independently, either alone or as chimaeras with other MurG domains. Independently expressed domains of the protein are useful for discovering ligands that bind to the individual domains.
  • the knowledge of the three-dimensional structure of E. coli MurG protein and models of other MurG proteins also provides a means for designing and producing compounds that regulate, inhibit or antagonize functions of the MurG protein (i.e., structure based drug design).
  • chemical compounds can be designed to block binding of UDP-GlcNAc to a MurG protein using various computer programs and models.
  • Another embodiment of the present invention is a composition comprising MurG protein in a crystalline form.
  • Yet another embodiment of the present invention is a method for producing crystals of MurG, comprising combining MurG protein in a suitable buffer with a suitable amount of a reservoir buffer containing a detergent, and inducing crystal formation to produce said MurG crystals.
  • FIG. 2 Overall architecture of MurG.
  • A Stereo view of the MurG structure. The N domain is shown in purple; the C domain is shown in green. The figure was generated with the programs MOLSCRIPT (Klaulis, 1991) and RASTER3D (Merrit & Murphy, 1994).
  • B Topology diagram of MurG. Fig. 3. Identification of critical residues in MurG and related glycosyltransferases.
  • A Sequence alignment of E. coli MurG with homologs from seven other bacterial strains, deliberately chosen to represent a disparate group of organisms. The secondary structure of E. coli MurG is shown above the sequences. Gaps mapping to the loop regions of E. coli MurG suggest that some sequences include other structural elements.
  • Residues highlighted in blue are invariant among the eighteen MurG sequences available. Residues highlighted in yellow are identical in 85% of the eighteen homologs, while in the remaining 15%, only closely related amino acid substitutions are found. Highly conserved residues that do not meet the stringent criteria established for highlighting are shown in the consensus sequence. A consensus motif for UDP-glucuronosyltransferases is also shown. Numbering is with respect to the overexpressed E. coli MurG construct, which contains an additional N-terminal methionine. B. Mapping of the G loops and other highlighted residues from Fig. 3a in red on the MurG structure. Side chains for highly conserved residues are also shown. C.
  • Fig 4. Structural analysis of the substrate binding pockets in MurG.
  • the carbonyl oxygen of residue 1245 is shown in red, and its backbone nitrogen is shown in blue.
  • C The surface of E. coli MurG. The G loops and other conserved residues in MurG are colored magenta. The proposed membrane binding interface is also highlighted with hydrophobic residues in yellow and positively charged residues in blue.
  • a or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound.
  • a compound refers to one or more compounds or at least one compound.
  • the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
  • a compound “selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds.
  • an isolated, or pure, protein is a protein that has been removed form its natural milieu.
  • isolated and biologically pure do not necessarily reflect the extent to which the protein has been purified.
  • An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology or can be produced by chemical synthesis.
  • MurG protein can also be recited as “MurG” and such terms can be used to refer to the complete MurG protein, a portion of the MurG protein, such as a polypeptide.
  • naturally occurring amino acids means the L-isomers of the naturally occurring amino acids.
  • the naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, .gamma.- carboxyglutamic acid, arginine, ornithine and lysine. Unless specifically indicated, all amino acids referred to in this application are in the L-form.
  • the term "unnatural amino acids” means amino acids that are not naturally found in proteins.
  • unnatural amino acids examples include racemic mixtures of selenocysteine and selenomethionine.
  • unnatural amino acids include the D or L forms of nor-leucine, para-nitrophenylalanine, homophenylalanine, para- fluorophenylalanine, 3-amino-p2-benzylpropionic acid, homoarginine, and D- phenylalanine.
  • positively charged amino acid includes any naturally occurring or unnatural amino acid having a positively charged side chain under normal physiological conditions.
  • positively charged naturally occurring amino acids are arginine, lysine and histidine.
  • negatively charged amino acid includes any naturally occurring or unnatural amino acid having a negatively charged side chain under normal physiological conditions.
  • negatively charged naturally occurring amino acids are aspartic acid and glutamic acid.
  • hydrophobic amino acid means any amino acid having an uncharged, nonpolar side chain that is relatively insoluble in water.
  • examples of naturally occurring hydrophobic amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • hydrophilic amino acid means any amino acid having an uncharged, polar side chain that is relatively soluble in water.
  • hydrophilic amino acids are serine, threonine, tyrosine, asparagine, glutamine, and cysteine.
  • MurG refers to a UDP-glycosyltransferase that has a two domain strucuture, where each domain contains a set of invariant residues as shown in Figure 3a, including any mutant, homologue or co-complex or any similar enzyme that catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP to the C4 hydroxyl of the lipid- linked MurNAc pentapeptide.
  • GlcNAc N-acetylglucosamine
  • mutant refers to a MurG polypeptide, i.e., a polypeptide displaying the biological activity of a wild-type MurG, characterized by the replacement of at least one amino acid from the wild-type, E. coli MURG sequence according to Ikeda, et al., Nucleic Acids Res. 1990, and Mengin-LeCreuix et al., Nucleic Acids Res. 1990.
  • Such a mutant may be prepared, for example, by expression of MURG cDNA previously altered in its coding sequence by PCR-based mutagenesis method.
  • MurG mutants may also be generated by site-specific incorporation of unnatural amino acids into MURG proteins using the general biosynthetic method of Noren, C. J., et al., Science, 244, pp. 182-188 (1989).
  • the codon encoding the amino acid of interest in wild-type MURG is replaced by a "blank" nonsense codon, TAG, using oligonucleotide-directed mutagenesis (described in detail, infra).
  • 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 MURG enzyme with the site-specific incorporated unnatural amino acid.
  • Selenocysteine or selenomethionine may be incorporated into wild-type or mutant MURG by expression of MURG-encoding cDNAs in auxotrophic E. coli strains. Hendrickson, W. A. et al., EMBO J., 9(5), pp. 1665-1672 (1990).
  • the wild-type or mutagenized MURG 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).
  • altered surface charge means a change in one or more of the charge units of a mutant polypeptide, at physiological pH, as compared to wild-type MURG. This is preferably achieved by mutation of at least one amino acid of wild-type MURG to an amino acid comprising a side chain with a different charge at physiological pH than the original wild-type side chain.
  • the change in surface charge is determined by measuring the isoelectric point (pi) of the polypeptide molecule containing the substituted amino acid and comparing it to the isoelectric point of the wild-type MURG molecule.
  • altered substrate specificity refers to a change in the ability of a mutant MURG to cleave a substrate as compared to wild-type MURG.
  • the "kinetic form" of MURG refers to the condition of the enzyme in its free or unbound fonn or bound to a chemical entity at either its active site or accessory binding site.
  • a “competitive” inhibitor is one that inhibits MURG activity by binding to the same kinetic form, of MURG, as its substrate binds— thus directly competing with the substrate for the active site of MURG.
  • Competitive inhibition can be reversed completely by increasing the substrate concentration.
  • an “uncompetitive” inhibitor is one that inhibits MURG by binding to a different kinetic form of the enzyme than does the substrate. Such inhibitors bind to MURG already bound with the substrate and not to the free enzyme. Uncompetitive inhibition cannot be reversed completely by increasing the substrate concentration.
  • a “non-competitive" inhibitor is one that can bind to either the free or substrate bound form of MURG.
  • inhibitors may be identified as competitive, uncompetitive or non-competitive, by computer fitting enzyme kinetic data using standard equations according to Segel, I. H., Enzvme Kinetics, J. Wiley & Sons, (1975). It should also be understood that uncompetitive or non-competitive inhibitors according to this invention may bind to the accessory binding site.
  • homolog means a protein having at least 25% amino acid sequence identity with MURG or any functional part of MURG, and including certain invariant amino acid residues corresponding to G14, G15, G18, H19, G104, H124, E125, G190, G191, S192, G194, A195, R261, G263, A264, E269, P281, Q289, N292 and A293 (as numbered in the E.coli MurG sequence set forth in Figure 3a) and also including three glycine rich loops.
  • a homolog may contain some or all of the invariant residues.
  • co-complex means MURG or a mutant or homologue of MURG in covalent or non-covalent association with a chemical entity or compound.
  • association refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a MurG molecule or portions thereof.
  • the association may be non-covalent-wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions-or it may be ⁇ ovalent.
  • .beta.-sheet refers to the conformation of a polypeptide chain stretched into an extended zig-zig conformation. Portions of polypeptide chains that run “parallel” all run in the same direction. Polypeptide chains that are "antiparallel” run in the opposite direction from the parallel chains.
  • atomic coordinates or “structure coordinates” refer to mathematical 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 a MurG molecule 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 used to establish the positions of the individual atoms within the unit cell of the crystal.
  • the term "heavy atom derivatization” refers to the method of producing a chemically modified form of a crystal of MURG.
  • a MurG crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, e.g., lead chloride, gold thiomalate, thimerosal, uranyl acetate or mercuric chloride, 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 enzyme. Blundel, T. L. and N. L. Johnson, Protein Crystallography, Academic Press (1976).
  • unit cell refers to a basic parallelepiped shaped block.
  • the entire volume of a crystal may be constructed by regular assembly of such blocks.
  • Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal.
  • space group refers to the arrangement of symmetry elements of a crystal.
  • molecular replacement refers to a method that involves generating a preliminary model of a MurG crystal whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known (e.g., MURG coordinates from Table 1, 2, or 3) within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal.
  • a molecule whose structure coordinates are known e.g., MURG coordinates from Table 1, 2, or 3
  • the present invention relates to the discovery of the three-dimensional structure of the crystalline form of the E. coli MurG protein, models of such three-dimensional structures, a method of structure based drug design using such structures, methods to identify ligands or compounds that interact or bind with such structures, the compounds identified by such methods, and the use of such compounds in therapeutic compositions.
  • the present invention relates to novel crystals of E. coli MurG protein, methods of production of such crystals, three dimensional coordinates of MurG protein, MurG structures and models derived from the E. coli MurG structure, and uses of such structures and models to derive other MurG structures and in ligand discovery and drug design strategies.
  • the present invention also relates to three-dimensional structures and coordinates of the donor nucleotide binding site, the acceptor binding site, and the membrane association site of the MurG protein, structures and models of the binding sites, and uses of such structures and models to derive the binding sites of other MurG proteins and in drug design strategies.
  • the description of the invention is divided into the following sections: (1) crystals of MurG protein; (2) methods of crystallization; (3) three- dimensional crystal coordinates and structure of E. coli MurG; (4) three-dimensional coordinates and structure of the donor nucleotide binding site of MurG; (5) coordinates and structure of the acceptor binding site of MurG; (5) three dimensional coordinates and structure of the membrane association site; (6) two dimensional and three dimensional images of the protein, ⁇ -carbon backbone, ⁇ -carbon backbone with conserved amino acid residues, and binding sites; and (7) computer readable mediums comprising the three dimensional coordinates of the MurG protein, ⁇ -carbon backbone, ⁇ -carbon backbone with conserved amino acid residues, and binding sites; (8) images of structures of MurG protiensand binding sites; (9) models of MurG proteins and binding sites thereof and methods of using the structure of MurG to determine the structures of other MurG proteins and binding sites; (10) structure based drug design using models of MurG protein and
  • One embodiment of the present invention includes a composition comprising a MurG protein in a crystalline form (i.e., MurG crystals).
  • a composition comprising a MurG protein in a crystalline form (i.e., MurG crystals).
  • crystalline MurG and “MurG crystal” both refer to crystallized MurG protein and are intended to be used interchangeably.
  • an embodiment of the present invention includes a composition comprising an E. coli MurG protein in a crystalline form.
  • a crystalline MurG is produced using the crystal formation method described herein, in particular according to the method disclosed in Example 1.
  • a MurG crystal of the present invention comprises any crystal structure and preferably precipitates as a triclinic crystal.
  • a preferred crystal of the present invention provides X-ray diffraction data for determination of atomic coordinates to a resolution of about 3.0 A, preferably to about 2.4 A ' , and more preferably to about 1.8 A.
  • a donor nucleotide is UDP or UDP-GlcNAc (UDP-N-acetylglucosamine) or an analog thereof.
  • the substrate or substrate analog is preferably Lipid I or Lipid II, or analogs of Lipid I or Lipid II. More specifically, Lipid I and II analogs are as described in PCT/US99/02187, published as WO99/38958 and US Provisional Application Nos. 60/122,966 filed March 3, 1999 and 60/137,696 filed June 4, 1999, and International Application No. PCT/USOO/05554 entitled "Bacterial transglycosylases: Assays for monitoring the activity using lipid II substrate analogs and methods for discovering antibiotics," all incorporated herein by reference in their entirety.
  • MurG proteins from numerous organisms can be used to prepare MurG crystals, including but not limited to, microorganisms such as bacteria, higher-order bacteria, thermal stable bacteria, spirochetes, small pathogenic organisms, fungi, protozoa, cyanobacteria, and trypanosomes.
  • microorganisms such as bacteria, higher-order bacteria, thermal stable bacteria, spirochetes, small pathogenic organisms, fungi, protozoa, cyanobacteria, and trypanosomes.
  • bacteria such as but not limited to, Escherichia coli, Bacillus subtilis, Aquefex aeolicus, Borrelia burgdorferi, Chlamydia pneumoniae, Chlamydia trachoma ⁇ s, Enterococcus faecais, Enterococcus hirae, Haemophilus in ⁇ uenzae, Helicobacter pylori J99, Helicobacter pylori, Mycobacterium tuberculosis, Porphyromonas gingivalis, Rickettsia prowazekii.Streptomyces coelicolor, Streptomyces collinus, Streptococcus pneumoniae, Synechocystis sp. (strain PCC6803), Thermotoga maritime, and Treponema pallidum.
  • the MurG proteins or fragments thereof, mutants or homologs are expressed in, for example, an E. coli host cell for use expressing sufficient quantities of sufficiently purified protein to form crystals.
  • the present inventors have demonstrated that it is possible to express Enterococcus. faecalis MurG in E. coli cells - so the MurG proteins from many organisms can be cloned into expression vectors suitable for expression in E. coli cells. This would facilitate obtaining sufficient quanitites of isolated or purified MurG proteins.
  • the expression of E. faecalis MurG protein in E. coli host cells is performed, for example, by expressing the E.
  • the MurG protein is over-expressed with a C-terminal his tag (LEHHHHHH) which allows the protein to be purified using a His-tag affinity column.
  • the protein is then crystallized and the atomic coordinates are determined using X-ray diffraction and methods known to those skilled in the art.
  • the present invention provides information that makes it possible to make chimaeric proteins containing the donor or acceptor binding site from E. coli MurG and the corresponding acceptor or donor binding site from another organism. Chimaeric proteins could be easier to express, handle, or crystallize. For example, we have found that ⁇ . faecalis MurG is more difficult to solubilize that E. coli MurG (requiring more detergent).
  • crystalline MurG can be used to determine the ability of a chemical compound to bind to a MurG protein in a manner predicted by a structure based drug design method of the present invention.
  • a MurG crystal is soaked in a solution containing a chemical compound of the present invention. Binding of the chemical compound to the crystal is then determined by methods standard in the art. Thereby, the co-crystal of MurG and a compound of interest is determined.
  • the present invention includes a method for producing crystals of MurG proteins, comprising: combining MurG protein with a reservoir solution and inducing crystal formation to produce MurG crystals.
  • a method for producing crystals of MurG protein comprises combining MurG protein with UDP-GlcNAc in a 1 :3 ratio and with a reservoir solution and inducing crystal formation to produce MurG crystals.
  • crystals of MurG are formed using a solution containing a range of MurG protein from about 1 mg/ml to about 20 mg/ml, more preferably above 5 mg/ml, limited only by the solubility of the protein, which may vary depending on the specific amino acid sequence.
  • a reservoir solution contains the buffer, the precipitant, and additives if necessary.
  • a suitable reservoir buffer of the present invention comprises NaMES (2-[N- morpholino]ethanesulfonic acid, sodium salt) buffer, NaHEPES (N-[2- hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid, sodium salt) buffer, Tris (tris[hydroxymethyl]aminomethane) buffer, and any buffer which has the PKa between 5.5 and 8.0.
  • a suitable NaMES buffer solution has a pH range from about 5.6-6.5. Most preferably, the NaMES buffer has a pH of about 6.5.
  • the precipitant comprises ammonium sulfate, saturated sodium and potassium tartrate and polyethylene glycol.
  • a suitable concentration of ammonium sulfate can range from 0.8 M to 1.5 M. Most preferably, the ammonium sulfate concentration is about 0.96 M.
  • a suitable additive comprises detergents like Triton X-100 and n-octyl-beta-glucoside. The concentration of Triton X-100 can range from 0.1% to 1%. Most preferably, the concentration of Triton X-100 is 0.4%.
  • MurG crystals are produced by a method comprising concentrating MurG protein in a buffer solution, mixing the protein concentrate with UDP-GlcNAc in a 1:3 molar ratio, mixing equal volumes of protein solution with a reservoir solution, and inducing crystal formation to produce MurG crystals.
  • MurG crystals are produced by a method comprising concentrating MurG protein to 10 mg/ml in a buffer of 20 mM Tris- HCl, pH 7.9/150mM NaCl and 50 mM EDTA; mixing the protein concentrate with UDP-GlcNAc in a 1:3 molar ratio; mixing equal volumes of protein solution with a reservoir solution comprising (0.1 M NaMES, pH 6.5, 0.96 M (NH 4 ) 2 SO 4 , 0.4% TRITON® X-100, and 10 mM dithiolthreitol (DTT)), and inducing crystal formation using hanging drop vapor-diffusion.
  • This preferred method is described in greater detail in Example 1.
  • Supersaturated solutions of MurG protein can be induced to crystallize by several methods including, but not limited to, vapor diffusion, liquid diffusion, batch crystallization, constant temperature and temperature induction or a combination thereof.
  • supersaturated solutions of MurG protein are induced to crystallize by vapor diffusion (i.e., hanging drop method).
  • vapor diffusion i.e., hanging drop method.
  • a MurG protein solution is combined with a reservoir solution of the present invention that will cause the MurG protein solution to become supersaturated and form MurG crystals at a constant temperature.
  • Vapor diffusion is preferably performed under a controlled temperature in the range of from about 15°C to about 30°C, more preferably from about 20°C to about 25°C, and most preferably at a constant temperature of about 22°C.
  • the present invention includes a method to produce crystals of MurG protein comprising the steps of: (a) preparing an about 10 mg/ml solution of MurG protein in a Tris-HCl buffer, (b) mixing UDP-GlcNAc with the MurG protein solution in a 3:1 molar ratio, (c) dropping 2 ⁇ l droplet of this protein sample onto a coverslip, (d) adding an equal volume of reservoir solution to this droplet and inverting this over a well containing about 1 ml of the reservoir solution; and (e) incubating until crystals of MurG form.
  • Any isolated MurG protein can be used with the present method.
  • An isolated MurG protein can be isolated from its natural milieu or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • PCR polymerase chain reaction
  • a nucleic acid molecule encoding a MurG protein can be inserted into any vector capable of expressing the nucleic acid in a host cell.
  • Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein.
  • Such suitable and preferred nucleic acid molecules include numerous MurG encoding genes that have been isolated to date, and that will be isolated in the future.
  • a preferred nucleic acid molecule of the present invention encodes a homologue of MurG. Homologues of MurG can be recognized by the presence of certain conserved amino acid residues or sequences.
  • a sequence alignment for six MurG sequences is shown in fig. 3A. Highlighted residues include those that are invariant or almost invariant across all MurG proteins.
  • a nucleic acid molecule of the present invention can encode any portion of a MurG protein, preferably a full-length MurG protein or either of the two domains.
  • a more preferred nucleic acid molecule to include in a recombinant vector, and particularly in a recombinant molecule includes a nucleic acid molecule encoding a protein having the amino acid sequence represented by amino acid sequences of MurG proteins as deposited in the NCBI database and are identified with Accession Nos.
  • nucleic acid molecules encoding MurG proteins have been deposited in NCBI, Genbank, and have Accession Nos. AL162758, AE002281. D90917, AF 110367, AL 139077, AJ242646, AE000520, AE000511, L42023, U00096, NC-000922, AE000783, AE000657, AE001348, AF099188, AR048673, AR048672, AF179611, AL022602, AL109663, X55034, AE000621, D10602, AE001670, X64259, Y13922, U10879, AE001535, AF068902, AJ235271, AE000118, AE001227, AE001176, U94707, Z95388, U32793, AE000727, D84504, Z99111, D10483,X52644, X52540, and L24773. These sequences are known and are publicly
  • E. coli genomic DNA can be purified from E. coli or purchased from ATCC, or the gene for E. coli MurG is cloned into a plasmid can be obtained from numerous sources. Primers were designed to the portions of the gene corresponding to the N and C termini of the protein. The primers also encoded restriction enzyme sites outside the protein coding region.
  • the gene sequence was amplified; the corresponding double stranded nucleic acid molecule was cut with appropriate restriction enzymes for cloning into a commercially available expression vector (p ⁇ T expression vectors available from Novagen provide for numerous variations of MurG protein - wild-type or fusion proteins or proteins with affinity tags at N or C terminus. We have worked with several constructs but found that MurG with a His-tag at C-terminus crystallized best; the protein sequence contained an extra methionine at N-terminus and eight extra residues at C terminus, six of which were histidines.
  • the vector used was p ⁇ T21b. (as described in Ha et al. J. Am. Chem. Soc. 121, (1999) 8415-8426 hereby incorporated by reference in its entirety).
  • a recombinant vector of the present invention can be either RNA (probably not) or DNA, and typically includes, but is not limited to, a virus or plasmid. Any recombinant vector and host cell that provides for expression of a MurG protein encoding mucleic acid sequence can be used in the present invention to express MurG protein for crystallization. Preferred vectors are engineered for high level expression in E. coli such as, but not limited to, pET vectors. We have found that over-expression of Murg from either E. coli or E. faecalis in E. coli cells is not toxic and, thus, this approach will work for other MurG proteins.
  • an expression vector is a DNA vector that is capable of transforming a host cell and of affecting expression of a specified nucleic acid molecule.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including bacterial, fungal, and other microorganisms cells.
  • Preferred expression vectors of the present invention direct expression in bacterial cells from a plasmid.
  • a preferred recombinant molecule of the present invention comprises pET21b with E. coli MurG gene cloned into the Ndel and Xhol sites.
  • An expression vector of the present invention can be transformed into any suitable host cell to form a recombinant cell.
  • a suitable host cell includes any cell capable of expressing a nucleic acid molecule inserted into the expression vector.
  • a procaryotic expression vector can be transformed into a bacterial host cell. If the expression vector contains a T7 promoter then a source of T7 RNA polymerase must be provided to induce expression. Some host cells contain the T7 RNA polymerase gene in a repressed state. Expression of T7 RNA polymerase can be induced with a chemical signal such as IPTG or heat.
  • a source of T7 RNA polymerase can be introduced at the appropriate time by infection with a phage containing a copy of T7 RNA polymerase.
  • a wide range of hosts strains can be infected with a suitable phage. Some host strains have been engineered to contain inducible copies of T7 RNA polymerase gene. Such host strains include BL21(DE3) and derivatives thereof.
  • a preferred host strain of the present invention is BL21(DE3)pLysS or BL21(DE3)pLysE, which are commercially available from Novagen and can be readily transformed with a DNA plasmid vector containing a MurG gene under the control of the T7 promoter.
  • a preferred vector is a pET vector, preferably containing a restriction enzyme site permitting cloning of the gene as a fusion containing a C-terminal his tag.
  • one method to isolate MurG protein useful for producing MurG crystals includes recovery of MurG protein having a C-terminal LEHHHHHH (His tag) sequence purified as described in Ha et al. (1999, J. Amer. Chem. Soc. 121 :8415-8426).
  • LEHHHHHH His tag sequence purified as described in Ha et al. (1999, J. Amer. Chem. Soc. 121 :8415-8426).
  • One of skill in the art is able to modify this procedure in order to purify other proteins can be produced as C-terminal histadine (his) tags.
  • the purification conditions for specific MurG proteins will vary depending upon the particular characteristics of the proteins such as their isoelectric point, molecular weight, etc. It is known that the isoelectric points of different Murg homologues vary a bit, although they are generally relatively high. Also, some Murg homologues may be more hydrophobic than others, which will mean differences in amount of detergent necessary for purification. It is likely that all the Murg homologues can be purified over nickel affinity columns using the C-terminal his-tag as a handle. Those skilled in the art of protein purification will know how to modify purification parameters depending upon the protein characteristics, in order to purify the protein for crystallization.
  • One embodiment of the present invention includes a model of a MurG protein, in which the model represents a three dimensional structure of a MurG protein.
  • Another embodiment of the present invention includes the three dimensional structure of a MurG protein.
  • a three dimensional structure of a MurG protein encompassed by the present invention substantially conforms with the atomic coordinates represented in Table 1.
  • the use of the term "substantially conforms" refers to at least a portion of a three dimensional structure of a MurG protein which is sufficiently spatially similar to at least a portion of a specified three-dimensional configuration of a particular set of atomic coordinates (e.g., those represented by Table 1) to allow the three dimensional structure of another MurG protein to be modeled or calculated using the particular set of atomic coordinates defining the three dimensional configuration of the MurG protein.
  • a particular set of atomic coordinates e.g., those represented by Table 1
  • homology modeling can be done using the linear sequence of a different MurG and E. coli coordinates; molecular replacement can allow the solution of a different MurG structure using the E.
  • a three dimensional structure of a given portion or chain of a first MurG protein can substantially conform to at least a portion of the atomic coordinates which represent a three dimensional configuration of a second MurG.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an average root-mean-square deviation (RMSD) of less than about 2.5 A for the ⁇ -carbon or C-alpha backbone atoms in secondary structure elements in each domain, and more preferably, less than about 2.0 A for the C-alpha backbone atoms in secondary structure elements in each domain, and, in increasing preference, less than about 1.5 A, less than about 1.0 A, less than about 0.7 A, and more preferably, less than about 0.5 A for the C-alpha backbone atoms in secondary structure elements in each domain.
  • RMSD average root-mean-square deviation
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited average root- mean-square deviation (RMSD) value, and more preferably, at least about 90% of such structure has the recited average RMSD value, and most preferably, about 100% of such structure has the recited average RMSD value.
  • RMSD root- mean-square deviation
  • the above definition of “substantially conforms” can be extended to include atoms of amino acid side chains.
  • the phrase “common amino acid side chains” refers to amino acid side chains that are common to both the structure which substantially conforms to a given set of atomic coordinates and the structure that is actually represented by such atomic coordinates.
  • a three dimensional structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of the common amino acid side chains have an average RMSD value of less than about 1.5 A, and more preferably, less than about 1.3 A, and in increasing preference, less than about 1.0 A, less' than about 0.7 A, and most preferably, less than about 0.3 A.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of the common amino acid side chains have the recited average RMSD value, and more preferably, at least about 90% of the common amino acid side chains have the recited average RMSD value, and most preferably, about 100% of the common amino acid side chains have the recited average RMSD value.
  • a large number of different "rotamers” or “rotational isomers” of the MurG protein are encompassed by three dimensional structures of the invention in which the amino acid side chains are at a variety of positions in crystalline forms of the protein or for the protein in solution.
  • Different rotamers refer to molecules of identical configuration may be distinguished as having different conformations after rotation about the various molecular bonds. Therefore, while the same or similar amino acids may be present, the exact location will vary depending upon the freedom of rotation of the bonds due to hydrogen bonding, and other molecular forces.
  • the present invention includes the three dimensional structure of the ⁇ -carbon or C-alpha backbone of a MurG protein, in particular the E. coli MurG protein.
  • a three dimensional structure of the C-alpha backbone of the MurG protein encompassed by the present invention substantially conforms with the atomic coordinates represented in Table 2.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an average root-mean-square deviation (RMSD) of less than about 2.5 A for the C-alpha backbone atoms in secondary structure elements in each domain, and more preferably, less than about 2.0 A for the C-alpha backbone atoms in secondary structure elements in each domain, and, in increasing preference, less than about 1.5 A, less than about 1.0 A, less than about 0.7 A, and more preferably, less than about 0.5 A for the C-alpha backbone atoms in secondary structure elements in each domain.
  • RMSD average root-mean-square deviation
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited average root-mean-square deviation (RMSD) value, and more preferably, at least about 90% of such structure has the recited average RMSD value, and most preferably, about 100% of such structure has the recited average RMSD value.
  • RMSD root-mean-square deviation
  • the C-alpha backbone of MurG proteins is expected to be more conserved than the location of the particular amino acid residue side chains.
  • the present invention also includes the three dimensional structure of the ⁇ - carbon or C-alpha backbone and conserved or invariant amino acid residue side chains of a MurG protein, in particular the E. coli MurG protein.
  • a three dimensional structure of the C-alpha backbone and conserved amino acid residues of the MurG protein encompassed by the present invention substantially conforms with the atomic coordinates represented in Table 3.
  • the conserved amino acids are highlighted in blue in Figure 3a and include G14, G15, G18, H19, G104, H124, ⁇ 125, G190, G191, S192, G194, A195, R261, G263, A264, E269, P281, Q289, N292 and A293 (as numbered in the E. coli MurG sequence set forth in Figure 3a).
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an average root-mean-square deviation (RMSD) of less than about 2.5 A for the C-alpha backbone and conserved amino acid residue atoms in secondary structure elements in each domain, and more preferably, less than about 2.0 A for the backbone atoms in secondary structure elements in each domain, and, in increasing preference, less than about 1.5 A, less than about 1.0 A, less than about 0.7 A, and more preferably, less than about 0.5 A for the backbone atoms in secondary structure elements in each domain.
  • RMSD average root-mean-square deviation
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited average root- mean-square deviation (RMSD) value, and more preferably, at least about 90% of such structure has the recited average RMSD value, and most preferably, about 100% of such structure has the recited average RMSD value.
  • RMSD root- mean-square deviation
  • An embodiment of the present invention includes the three dimensional structure of a donor nucleotide binding site of a MurG protein, in particular an E. coli MurG protein.
  • a more preferred embodiment of the present invention includes a three dimensional structure of a donor nucleotide binding site of a MurG protein wherein the three dimensional structure of the donor nucleotide binding site substantially conforms to the atomic coordinates in Table 4.
  • the donor nucleotide binding site is a UDP-GlcNAc binding site of a MurG protein. As described in Example 1 , the donor nucleotide binding site is located in the C- terminal domain (see Fig. 4a).
  • This binding site is based on the comparison of ⁇ - glucosyltransferase (BGT) and E. coli MurG and based on experiments done in our laboratory showing that the isolated C domain binds to a UDP-hexose column (See Example 1).
  • the atomic coordinates of Table 4 set forth the donor nucleotide binding site three dimensional structure without a donor nucleotide such as UDP-GlcNAc bound to the MurG protein.
  • the use of the term "substantially conforms" refers to at least a portion of a three dimensional structure of a donor nucleotide binding site of a MurG protein which is sufficiently spatially similar to at least a portion of a specified three-dimensional configuration of a particular set of atomic coordinates (e.g., those represented by Table 4) to allow the three dimensional structure of the donor nucleotide binding domain to be modeled or calculated (i.e., by molecular replacement) using the particular set of atomic coordinates defining the three dimensional configuration of the donor nucleotide binding site of a MurG protein.
  • a particular set of atomic coordinates e.g., those represented by Table 4
  • a three dimensional structure of a given donor nucleotide binding site of a first MurG protein can substantially conform to at least a portion of the atomic coordinates which represent a three dimensional configuration of a second MurG. Since the atomic coordinates of Table 4 were obtained from the E. coli MurG crystal protein without a donor nucleotide bound, there will be some variation from the atomic coordinates of the donor nucleotide binding site when a nucleotide is bound vs. unbound. Therefore, a structure "substantially conforming" to that represented by the atomic coordinates in Table 4, will include a structure obtained from co-crytallization of the protein with a donor nucleotide.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an average root-mean-square deviation (RMSD) of less than about 1.5 A for the C-alpha backbone atoms in secondary structure elements in each domain, and more preferably, less than about 1.3 A for the C-alpha backbone atoms in secondary structure elements in each domain, and, in increasing preference, less than about 1.0 A, less than about 0.7 A, and more preferably less than about 0.5 A for the C-alpha backbone atoms in secondary structure elements in each domain.
  • RMSD average root-mean-square deviation
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited average root-mean-square deviation (RMSD) value, and more preferably, at least about 90% of such structure has the recited average RMSD value.
  • RMSD root-mean-square deviation
  • the above definition of “substantially conforms” can be extended to include atoms of the conserved or invariant amino acid side chains located within the binding site.
  • conserved amino acid side chains refers to amino acid side chains that are conserved between MurG proteins within the donor nucleotide binding site.
  • the conserved amino acid residues of the donor nucleotide binding site have been identified as 1125, R261, G263, A264, E269, P281, Q289, N292 and A293 (as numbered in the E.
  • a three dimensional structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of the conserved amino acid side chains have an average RMSD value of less than about 1.5 A, and more preferably, less than about 1.3 A, and in increasing preference, less than about 1.0 A, less than about 0.7 A, and most preferably, less than about 0.3 A.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of the conserved amino acid side chains have the recited average RMSD value, and more preferably, at least about 90% of the conserved amino acid side chains have the recited average RMSD value, and most preferably, about 100% of the conserved amino acid side chains have the recited average RMSD value.
  • An embodiment of the present invention includes the three dimensional structure of an acceptor binding site of a MurG protein.
  • a three dimensional structure of a acceptor binding site of a MurG protein encompassed by the present invention substantially conforms with the atomic coordinates represented in Table 5.
  • a more preferred embodiment of the present invention includes a three dimensional structure of an acceptor binding site of a MurG protein wherein the three dimensional structure of the acceptor binding site substantially conforms to the atomic coordinates Table 5.
  • the use of the term "acceptors" refers to Lipid I and analogues thereof.
  • the analogues need not be functional acceptors in a MurG assay.
  • the acceptor is selected from the group consisting of, but not limited to Lipid I, and analogs of Lipid I (see compounds described in Ha et al., J. Amer. Chem. Soc. 1999, vol. 121 :8415-26, incorporated by herein by reference in its entirety).
  • the acceptor binding site is located in the N- terminal domain of a MurG protein (see Fig. 3a and 4c).
  • the acceptor binding site or domain is characterized by three highly conserved regions, two of which are glycine-rich loops (also referred to as "G loops") that face the cleft between the C-terminal and N- terminal domains.
  • the conserved residues of the acceptor binding site comprise G14, G15, G 18, HI 9, G 104, HI 24, and El 25 (as numbered in the E. coli MurG sequence set forth in Figure 3 a) and two conserved G loop structures.
  • the use of the term "substantially conforms" refers to at least a portion of a three dimensional structure of an acceptor binding site of a MurG protein which is sufficiently spatially similar to at least a portion of a specified three-dimensional configuration of a particular set of atomic coordinates (e.g., those represented by Table 5) to allow the three dimensional structure of the acceptor binding site to be modeled or calculated (i.e., by homology modeling) using the particular set of atomic coordinates defining the three dimensional configuration of the acceptor binding site of a MurG protein.
  • a three dimensional structure of a given acceptor binding site of a first MurG protein can substantially conform to at least a portion of the atomic coordinates which represent a three dimensional configuration of a second MurG.
  • the above definition of “substantially conforms” can be extended to include atoms of the conserved amino acid side chains.
  • conserved amino acid side chains refers to the conserved or invariant amino acid side chains that are common to MurG proteins.
  • a three dimensional structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of the conserved amino acid side chains have an average RMSD value of less than about 1.5 A, and more preferably, less than about 1.3 A, and in increasing preference, less than about 1.0 A, less than about 0.7 A, and most preferably, less than about 0.3 A.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of the conserved amino acid side chains have the recited average RMSD value, and more preferably, at least about 90% of the conserved amino acid side chains have the recited average RMSD value, and most preferably, about 100% of the conserved amino acid side chains have the recited average RMSD value.
  • An embodiment of the present invention includes the three dimensional structure of a membrane association site of a MurG protein.
  • a three dimensional structure of a membrane association site of a MurG protein encompassed by the present invention substantially conforms with the atomic coordinates represented in Table 6.
  • a more preferred embodiment of the present invention includes a three dimensional structure of an acceptor binding site of a MurG protein wherein the three dimensional structure of the acceptor binding site substantially conforms to the atomic coordinates in Table 6.
  • membrane association site refers to the region of a MurG protein that associates with cytoplasmic surface of bacterial membranes where it performs the reaction of coupling a soluble donor sugar to the membrane anchored acceptor sugar, Lipid I.
  • Analysis of the E. coli MurG protein structure shows a hydrophobic patch consisting of residues 175, L79, F82, W85, and W116 in the N-domain.
  • the membrane association site is where the MurG protein associates with the bacterial membranes, and that it is target for inhibitors if we find that a) we can bind to it with another molecule; b) we can disrupt membrane association by binding to it; or c) disrupting membrane association inhibits activity.
  • the membrane association site is located in the N- terminal domain of a MurG protein (see Fig. 4c).
  • the location of the membrane association site is in close proximity to the acceptor binding site and membrane association in this patch would bring the two M-terminal G-loops close to the membrane surface where the diphosphate portion of the acceptor is located.
  • the use of the term "substantially conforms" refers to at least a portion of a three dimensional structure of a membrane association site of a MurG protein which is sufficiently spatially similar to at least a portion of a specified three-dimensional configuration of a particular set of atomic coordinates (e.g., those represented by Table 6) to allow the three dimensional structure of the membrane association site to be modeled or calculated (i.e., by molecular replacement) using the particular set of atomic coordinates defining the three dimensional configuration of the membrane association site of a MurG protein.
  • a three dimensional structure of a given membrane association site of a first MurG protein can substantially conform to at least a portion of the atomic coordinates which represent a three dimensional configuration of a second MurG.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an average root-mean-square deviation (RMSD) of less than about 1.5 A for the structural elements in the site, and more preferably, less than about 1.3 A for the structure elements in each site, and, in increasing preference, less than about 1.0 A, less than about 0.7 A, less than about 0.5 A, and more preferably, less than about 0.3 A for the structural elements in each site.
  • RMSD average root-mean-square deviation
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited average root-mean-square deviation (RMSD) value, and more preferably, at least about 90% of such structure has the recited average RMSD value, and most preferably, about 100% of such structure has the recited average RMSD value.
  • RMSD root-mean-square deviation
  • ⁇ 'substantially conforms can be extended to include atoms of ⁇ -carbon backbone and conserved amino acid side chains.
  • conserved amino acid side chains refers to amino acid side chains that are conserved between MurG proteins.
  • a three dimensional structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of the conserved ⁇ -carbon backbone and conserved amino acid side chains have an average RMSD value of less than about 1.5 A, and more preferably, less than about 1.3 A, and in increasing preference, less than about 1.0 A, less than about 0.7 A, and most preferably, less than about 0.3 A.
  • a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of the ⁇ -carbon backbone and conserved amino acid side chains have the recited average RMSD value, and more preferably, at least about 90% of the ⁇ -carbon backbone and conserved acid side chains have the recited average RMSD value, and most preferably, about 100% of the ⁇ -carbon and conserved amino acid side chains have the recited average RMSD value.
  • Another embodiment of the present invention relates to a computer-readable medium encoded with, a set three dimensional coordinates seleceted from the group consisting of the three dimensional coordinates represented in Table 1, the three dimensional coordinates represented in Table 2, the three dimensional coordinates represented in Table 3, the three dimensional coordinates represented in Table 4, the three dimensional coordinates represented in Table 5, or the three dimensional coordinates represented in Table 6, wherein using a graphical display software program, the three dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three dimensional image.
  • the three dimensional image is of a MurG protein, the ⁇ -carbon backbone of MurG, the ⁇ -carbon backbone and conserved amino acid residue sidechains of MurG, the donor nucleotide binding site of MurG, the acceptor binding site of MurG, or the membrane association site of MurG.
  • Yet another embodiment of the present invention relates to a computer-readable medium encoded with a set of three dimensional coordinates of a three dimensional structure which substantially conforms to the three dimensional coordinates* represented in Table 1, wherein using a graphical display software program, the three dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three dimensional image.
  • the present invention relates to a computer-readable medium encoded with a set of three dimensional coordinates of a three dimensional structure which substantially conforms to the three dimensional coordinates represented in Table 2, Table 3, Table 4, Table 5 or Table 6, wherein using a graphical display software program, the three dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three dimensional image.
  • the three dimensional image is of a MurG protein, the ⁇ -carbon backbone of MurG, the ⁇ -carbon backbone and conserved amino acid residue sidechains of MurG, the donor nucleotide binding site of MurG, the acceptor binding site of MurG, or the membrane association site of MurG.
  • One embodiment of the present invention relates to a two dimensional image of an E. coli MurG protein including those illustrated in Figures 3-4. Most of these figures were drawn with the MOLSCRIPT program.
  • the two dimensional image is of a MurG protein, the ⁇ -carbon backbone of MurG, the ⁇ -carbon backbone and conserved amino acid residue sidechains of MurG, the donor nucleotide binding site of MurG, the acceptor binding site of MurG, or the membrane association site of MurG.
  • Another embodiment of the present invention includes a three dimensional computer image of the three dimensional structure of a MurG protein, preferably the E. coli MurG protein. Suitable structures of which to produce three dimensional computer images are disclosed herein. Preferably, a computer image is created to a structure substantially conforming with the three dimensional coordinates represented in Table 1.
  • Another embodiment of the present invention includes an image of an MurG protein that is generated when a set of three dimensional coordinates comprising the three dimensional coordinates represented in Table 1 are analyzed on a computer using a graphical display software program to create an electronic file of the image and visualizing the electronic file as a three dimensional image.
  • Suitable structures to image are disclosed herein.
  • the three dimensional structures are of a MurG protein, the ⁇ -carbon backbone of MurG, the ⁇ -carbon backbone and conserved amino acid residue sidechains of MurG, the donor nucleotide binding site of MurG, the acceptor binding site of MurG, or the membrane association site of MurG.
  • the MurG protein is the E. coli MurG protein described herein.
  • a computer image of the present invention can be produced using any suitable software program, including, but not limited to, MOLSCRIPT 2.0 (Avatar Software AB, Helenebrgsgatan 21C, SE-11713, Swiss, Sweden), the graphical display program O (Jones et al, Acta Crystallography, vol. A47, p. 110, 1991), or the graphical display program GRASP.
  • suitable computer hardware useful for producing an image of the present invention are known to those of skill in the art.
  • Preferred computer hardware includes a Silicon Graphics Workstation.
  • a three dimensional structure of the E. coli MurG protein and its binding sites of the present invention can be used to derive a model of the three dimensional structure of another MurG protein and its binding sites (i.e., a structure to be modeled).
  • a "structure” of a protein refers to the components and the manner of arrangement of the components to constitute a protein or binding site.
  • model refers to a representation of a tangible medium of the three dimensional structure of a protein, polypeptide or peptide, or binding site of a protein.
  • a model can be a representation of the three dimensional structure in a electronic file, on a computer screen, on a piece of paper (i.e., on a two dimensional medium), and/or as a ball-and-stick figure.
  • Physical three- dimensional models are tangible and include, but are not limited to, stick models and space-filling models.
  • imaging the model on a computer screen refers to the ability to express (or represent) and manipulate the model on a computer screen using appropriate computer hardware and software technology known to those skilled in the art. Such technology is available from a variety of sources including, for example, Evans and Sutherland, Salt Lake City, Utah, and Biosym Technologies, San Diego, CA.
  • providing a picture of the model refers to the ability to generate a "hard copy" of the model.
  • Computer screen images and pictures of the model can be visualized in a number of formats including space-filling representations, ⁇ carbon traces, ribbon diagrams and electron density maps.
  • Suitable target MurG proteins and their associated binding sites to model using a method of the present invention include any MurG protein and binding sites that are at least in part structurally related to the E. coli MurG protein or its binding sites.
  • a preferred target MurG structure that is at least in part structurally related includes a target • MurG structure having an amino acid sequence that is at least about 25%, preferably at least about 30%, more preferably at least about 36%, more preferably at least about 40%, even more preferablye at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, and more preferably at least about 90% identical to an amino acid sequence of the E.
  • coli MurG protein across the full-length of the target MurG structure sequence when using, for example, a sequence alignment program such as DNAsisTM program (available from Hitachi Software, San Bruno, CA) or the MacVectorTM program (available from the Eastman Kodak Company, New Haven, CT) or the GC ⁇ TM program (available from the "GC ⁇ ", University of Wisconsin, Madison, WI), such alignment being performed for example, using the standard default values accompanying such alignment programs.
  • a sequence alignment program such as DNAsisTM program (available from Hitachi Software, San Bruno, CA) or the MacVectorTM program (available from the Eastman Kodak Company, New Haven, CT) or the GC ⁇ TM program (available from the "GC ⁇ ", University of Wisconsin, Madison, WI), such alignment being performed for example, using the standard default values accompanying such alignment programs.
  • MurG proteins and their binding sites are set forth in the amino acid sequences of MurG proteins as deposited in the NCBI database and are identified with Accession Nos. CAB51993, A71316, E70579, C71699, F70195, A43727, JC1275, BVECMG, CEECAM, 083535, Q9ZK59, CAB85280, AAF39020, BAA18775, AAD26629, CAB73295, P37585, Q9ZHA9, Q9ZHDC0, Q9ZBA5, Q9X4H4, Q9WY74, P74657, O06224, Q9Z702, 084766, 069552, 067238, 051708, O25770, O07670, O07109, P45065, CAB66324, AAC68356, AAF06830, P18579, P17443, P17952, P16457, P07862, AAE23178, AAD53936, CAA18668, CAA388
  • MurG proteins from numerous organisms can be used to prepare models of MurG proteins and binding sites, including but not limited to, microorganisms such as bacteria, higher-order bacteria, thermal stable bacteria, spirochetes, small pathogenic organisms, fungi, protozoa, cyanobacteria, and trypanosomes.
  • microorganisms such as bacteria, higher-order bacteria, thermal stable bacteria, spirochetes, small pathogenic organisms, fungi, protozoa, cyanobacteria, and trypanosomes.
  • bacteria such as but not limited to, Escherichia coli, Bacillus subtilis, Aquefex aeolicus, Borrelia burgdorferi, Chlamydia pneumoniae, Chlamydia trachomatis, Enterococcus faecais, Enterococcus hirae, Haemophilus influenzae, Helicobacter pylori J99, Helicobacter pylori, Mycobacterium tuberculosis, Porphyromonas gingivalis, Rickettsia prowazekii, Streptomyces coelicolor, Streptomyces collinus, Streptococcus pneumoniae, Synechocystis sp. (strain PCC6803), Thermotoga maritime, and Treponema pallidum. It is noted that nucleotide and amino acid sequences for many of the above identified organisms are known and publicly available.
  • Preferred target MurG proteins and binding site structures to model also include, but are not limited to, derivatives of MurG proteins, such as a MurG protein having one or more amino acid residues substituted, deleted or added (referred to herein as MurG mutants), or proteins encoded by natural variants of a nucleic acid molecule encoding a MurG.
  • derivatives of MurG proteins such as a MurG protein having one or more amino acid residues substituted, deleted or added (referred to herein as MurG mutants), or proteins encoded by natural variants of a nucleic acid molecule encoding a MurG.
  • the process of building a homology model for a protein is divided into the following steps:
  • MurG specific and related sequences are obtained for use for building homology models by text-based or sequence similarity searching.
  • SCRs for MurG is the entire protein, considering the E. coli MurG crystal structure is the only similar sequence with structural data.
  • Alignment of the sequences using an appropriate alignment program and algorithm, such as Clustal W, allows appropriate assignment of the E. coli protein coordinates to a MurG sequence of unknown structure.
  • the Modeler program performs the conformational predictions for the peptide chain and side chains. Dynamics and minimization using an appropriate program and algorithm, such as Discover.
  • Modeler is an automated homology-modeling scheme designed to find the most probable three-dimensional structure of a protein, given its amino acid sequence and its alignment with related structures. It derives 3D protein models without the time consuming separate stages of core region identification and loop region building or searching that is inherent to manual homology modeling schemes.
  • the related or reference protein structures are used to derive spatial restraints expressed as probability density functions (PDFs) for each of the restrained features of the model.
  • PDFs probability density functions
  • the main chain conformation of a given residue in the model will be described by restraints that depend upon the residue type, the main chain conformation of equivalent residues in the reference proteins and the local sequence similarity.
  • the probability distribution functions that are used in restraining the model structure are derived from correlations between structural features in a database of families of homologous proteins aligned on the basis of their 3D structure. These functions are used to restrain C-C distances, main chain N-O distances, main chain and side chain dihedral angles, etc.
  • the individual restraints are assembled into a single molecular probability density function (MPDF).
  • MPDF molecular probability density function
  • the three-dimensional protein model is then obtained by an optimization of this MPDF.
  • the optimization procedure itself consists of a variable target function method (Braun and Go, 1985) with conjugate gradient minimization scheme followed by an optional restrained simulated annealing molecular dynamics scheme.
  • Modeler is able to simultaneously incorporate structural data from one or more reference proteins.
  • Structural features in the reference proteins are used to derive spatial restraints which in turn are used to generate model protein structures using conjugate gradient and simulated annealing optimization procedures.
  • Clustal W aligns multiple sequences using a progressive pairwise alignment algorithm. It first generates all possible pairwise alignments for a list of sequences and then builds the guide tree based on their pairwise sequence identity, aligning the sequences following the order of the guide tree.
  • the Discover program performs energy minimization, template forcing, torsion forcing, and dynamic trajectories and calculates properties such as interaction energies, derivatives, mean square displacements, and vibrational frequencies. It provides tools for performing simulations under various conditions including constant temperature, constant pressure, constant stress, periodic boundaries, and fixed and restrained atoms.
  • GenBank database InteUiGenetics, Inc., 700 El Camino Real East, Mountain View, CA
  • the present invention relates to the use of the crystal structure of the E. coli MurG protein represented by the atomic coordinates in Table 1 to make models of MurG proteins and binding sites thereof.
  • the present invention also relates to the use of the crystal structure, ⁇ -carbon backbone, ⁇ -carbon backbone plus conserved amino acid residue side chains or binding sites of the E. coli MurG protein to construct models of these structures in other MurG proteins.
  • the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including inhibitory compounds, capable of binding to the active site or accessory binding site of MURG, in whole or in part.
  • this invention On approach enabled by this invention, is to use the structure coordinates of MURG to design compounds that bind to the enzyme and alter the physical properties of the compounds in different ways, e.g., solubility.
  • this invention enables the design of compounds that act as inhibitors of the MURG enzyme by binding to, all or a portion of, the active site of MURG.
  • a second design approach is to probe a MurG crystal with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate MURG inhibitors and the enzyme. For example, high resolution X- ray diffraction data collected from crystals saturated with solvent allows the determination of where each tpe of solvent molecule sticks. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their MURG inhibitor activity. Travis, J., Science, 262, p. 1374 (1993).
  • This invention also enables the development of compounds that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that binds to MURG, with MURG.
  • the reaction intermediates of MURG can also be deduced from the reaction product in co- complex with MURG.
  • Such information is useful to design improved analogues of known MURG inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the MURG enzyme and MURG-inhibitor co-complex. This provides a novel route for designing MURG inhibitors with both high specificity and stability.
  • Another approach made possible and enabled by this invention is to screen computationally small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to the MURG enzyme.
  • the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity or by estimated interaction energy.
  • MURG may crystallize in more than one crystal form
  • the structure coordinates of MURG, or portions thereof, as provided by this invention are particularly useful to solve the structure of those other crystal forms of MURG. They may also be used to solve the structure of MURG mutants, MURG co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of MURG.
  • One method that may be employed for this pu ⁇ ose is molecular replacement.
  • the unknown crystal structure whether it is another crystal form of MURG, a MurG mutant, or a MurG co-complex, or the crystal of some other protein with significant amino acid sequence homology to any functional domain of MURG, may be determined using the MURG structure coordinates of this invention as provided in Tables 1-6.
  • This method will provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
  • MURG mutants may be crystallized in co-complex with known MURG inhibitors.
  • the crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of wild-type MURG. Potential sites for modification within the various binding sites of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between MURG and a chemical entity or compound.
  • All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 2-3 .ANG. resolution X-ray date to an R value of about 0.20 or less using computer software, such as X-PLOR (Yale University, .COPYRGT.1992, distributed by Molecular Simulations, Inc.). See, e.g., Blundel & Johnson, supra; Methods in Enzvmoloav, vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985). This information may thus be used to design, synthezie and optimize novel classes of MURG inhibitors.
  • the structure coordinates of MURG mutants provided in this invention also facilitate the identification of related proteins or enzymes analogous to MURG in function, structure or both, thereby further leading to novel therapeutic modes for treating or preventing UDP-glycosyltransferase mediated diseases.
  • the design of compounds that bind to or inhibit MURG according to this invention generally involves consideration of two factors.
  • the compound must be capable of physically and structurally associating with MURG.
  • Non-covalent molecular interactions important in the association of MURG with its substrate include hydrogen bonding, van der Waals and hydrophobic interactions.
  • the compound must be able to assume a conformation that allows it to associate with MURG. Although certain portions of the compound will not directly participate in this association with MURG, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency.
  • conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., active site or accessory binding site of MURG, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with MURG.
  • the potential inhibitory or binding effect of a chemical compound on MURG may be analyzed prior to its actual synthesis and testing by the use of computer modelling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and MURG, synthesis and testing of the compound is obviated. However, if computer modelling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to MURG and inhibit using the assay of Walker et al. patents (cited supra). In this manner, synthesis of inoperative compounds may be avoided.
  • An inhibitory or other binding compound of MURG may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding pockets or other areas of MURG.
  • One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with MURG and more particularly with the individual binding pockets of the MURG donor nucleotide binding site, acceptor binding site or membrane association site.
  • This process may begin by visual inspection of, for example, the binding sites on the computer screen based on the MURG coordinates in Tables 1-6. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within an individual binding pocket of MURG as defined supra. 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.
  • GRID Goodford, P. J., "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules" J. Med. Chem., 28, pp. 849-857 (1985)). GRID is available from Oxford University, Oxford, UK.
  • MCSS (Miranker, A. and M. Ka ⁇ lus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, Burlington, Mass.
  • DOCK (Kuntz, I. D. et al., "A Geometric Approach to Macromolecule-Ligand Interactions" J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is available from University of California, San Francisco, Calif.
  • Assembly may be proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of MURG. This would be followed by manual model building using software such as Quanta or Sybyl.
  • CAVEAT Bartlett, P. A. et al, "CAVEAT: A Program to Facilitate the Structure- Derived Design of Biologically Active Molecules". In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989)). CAVEAT is available from the University of California, Berkeley, Calif. 2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif). This area is reviewed in Martin, Y. C, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992)).
  • inhibitory or other MURG binding compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some portion(s) of a known inhibitor(s). These methods include, but are not limited to:
  • LUDI Bohm, H.-J., "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. ComR. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from Biosym Technologies, San Diego, Calif.
  • LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations, Burlington, Mass.
  • a compound that has been designed or selected to function as a MurG-inhibitor must also preferably traverse a volume not overlapping that occupied by the active site when it is bound to the native substrate.
  • An effective MURG inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding) .
  • the most efficient MURG inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, preferably, not greater than 7 kcal/mole.
  • MURG inhibitors may interact with the enzyme in more than one conformation that is similar in overall binding energy.
  • the deformation energy of binding is taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the inhibitor binds to the enzyme.
  • a compound designed or selected as binding to MURG may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme.
  • Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge- dipole interactions.
  • the sum of all electrostatic interactions between the inhibitor and the enzyme when the inhibitor is bound to MURG preferably make a neutral or favorable contribution to the enthalpy of binding.
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.
  • substituted chemical compounds may then be analyzed for efficiency of fit to MURG by the same computer methods described in detail, above.
  • One embodiment of the present invention is a compound that is capable of binding to a MurG protein, inhibiting the activity of a MurG protein, or stimulating the activity of a MurG protein.
  • Suitable inhibitory compounds of the present invention can: (1) inhibit (i.e., prevent or block) the activity of MurG enzyme by binding to a MurG donor nucleotide binding site and interfering with the binding of the donor nucleotide molecule; (2) inhibit the activity of MurG enzyme by binding to the MurG acceptor binding site and interfering with the binding of the acceptor molecule; (3) inhibit the activity of a MurG enzyme by binding to the membrane association site and interfering with the association of the protein with the bacterial membrane and/or acceptor molecule.
  • Another embodiment of the present invention is a compound that is capable of stimulating MurG activity.
  • Suitable stimulatory compounds of the present invention can stimulate the activity of a MurG enzyme by binding to the protein at a binding site and causing an increase in enzymatic activity, for example, by increasing the enzymes affinity to bind a donor nucleotide, an acceptor molecule or improve the enzymes stability or increasing the binding affinity of a molecule to MurG.
  • Such compounds that bind to, inhibit or stimulate activity of a MurG protein include, for example, compounds that mimic donor nucleotide molecules.
  • the compound includes, for example, pyrimidine nucleoside analogues.
  • the compounds include compounds comprising a pyrimidine nucleoside with a substituent containing at least one heteroatom attached to the C5 hydroxyl.
  • pyrimidine derivatives make complementary hydrogen bonding contacts to the amide backbone segment containing He 245 and also contact glutamate 269.
  • acceptor analogs refers to a compound that interacts with (e.g., binds to, associates with, modifies) the acceptor binding site of a MurG protein.
  • An acceptor analog for example, is a compound that mimics the natural acceptor molecule, Lipid I. Examples of such acceptor analogs are set forth in Ha et al., J. Amer. Chem. Soc 1999, and PCT/US99/02187, U.S. Provisional Application No. 60/073,376 filed February 2, 1998, inco ⁇ orated herein by reference.
  • Lipid II analogs refers to a compound that interacts with (i.e. , binds to, associates with, modifies) the acceptor binding site of a Mur G protein which mimics the product of the transglycosylase reaction.
  • Inhibitory and stimulatory compounds of the present invention can be identified by various means known to those of skill in the art. For example, binding of an inhibitory compound ' to, or otherwise interaction with, a MurG protein, can be determined with MurG in solution, for example, using assays described in PCT/US99/02187, U.S. Provisional Application No. 60/073,376 filed February 2, 1998, and PCT/US 00/ 0555 , U.S. Provisional Application Nos. 60/122,966 and 60/137,696, inco ⁇ orated herein by reference.
  • suitable compounds of the present invention include peptides or other organic molecules, and inorganic molecules.
  • Suitable organic molecules include small organic molecules.
  • a compound of the present invention is not harmful (i.e., toxic) to an animal when administered to an animal.
  • compounds are suitable for use in the inhibition of bacterial or microbial growth in an animal, and for example, function as an antibiotic for treatment of bacterial infections in animals.
  • compositions comprising compounds of the present invention that inhibit or stimulate MurG activity which function as antibiotics or antimicrobial agents in animals.
  • Compositions of the present invention can be used therapeutically or diagnostically in an animal.
  • Compositions of the present invention comprises at least one compound of the present invention.
  • compositions of the present invention further comprise a carrier. More particularly, a suitable carrier is a pharmaceutically acceptable carrier known to those skilled in the art. TABLE 1- ATOMIC COORDINATES OF E. COLI MURG PROTEIN
  • REMARK FILENAME "minimize5. pdb"REMARK DATE : 14-Jan-( DO 15:25:; 36 created by user: shal REMARK VERSIO
  • ATOM 58 CA GLY A 14 12, .287 4 , .762 15. .757 1, .00 40. .54 AAAA
  • ATOM 73 CA GLY A 17 -7, ,190 0. ,268 11, .109 1, .00 40. , 71 AAAA
  • ATOM 81 CA HIS A 19 -6. ,454 4, ,664 14. ,110 1 .00 32. , 14 AAAA
  • ATOM 110 CA PRO A 22 1 259 2 396 16 139 1 00 30 01 AAAA
  • ATOM 140 CA ALA A 27 1. 371 -0 215 24 382 1 00 29 32 AAAA
  • ATOM 181 CA ALA A 32 .262 -4 .871 29 .044 1 .00 37, .61 AAAA
  • ATOM 186 CA GLN A 33 5, .408 -2 .275 31, .548 1, .00 38. .14 AAAA
  • ATOM 195 CA GLY A 34 2, ,738 -2. .755 34. ,170 1. ,00 35. ,52 AAAA
  • ATOM 199 CA TRP A 35 0. ,121 -0, .435 32. ,661 1, ,00 33. , 63 AAAA
  • ATOM 201 CG TRP A 35 1. ,150 1, ,722 31. ,753 1, ,00 35. ,09 AAAA
  • ATOM 262 CA GLY A 41 -14, .946 2, ,553 20, .821 1, .00 25, .95 AAAA
  • ATOM 266 CA THR A 42 -18. .185 1, .641 19, .059 1, .00 31, .41 AAAA
  • ATOM 273 CA ALA A 43 -19. 920 -1. 760 19. 127 1. .00 37. ,23 AAAA
  • ATOM 314 CA ALA A 48 -15, .342 -4, .066 18, .700 1, .00 36. .98 AAAA
  • ATOM 324 C ASP A 49 -12. ,524 -7. .519 17. ,426 1. ,00 40. ,10 AAAA
  • ATOM 342 CD PRO A 52 -12. ,141 -6. .700 20. .932 1. .00 38. .92 AAAA
  • ATOM 343 CA PRO A 52 -10. ,959 -8. .237 22. .373 1. .00 39, .32 AAAA
  • ATOM 353 CE LYS A 53 -11. ,280 -12. .102 16, ,133 1. .00 45. .87 AAAA
  • ATOM 358 CA HIS A 54 -5 .790 -8 .642 21 .727 1 .00 37. .71 AAAA
  • ATOM 368 CA GLY A 55 -6. .948 -9. ,477 25. .240 1. .00 36. .70 AAAA
  • ATOM 372 CA ILE A 56 -8, .009 -5. .885 25. .900 1. .00 34. ,03 AAAA
  • ATOM 389 CA ILE A 58 -13. .454 -2. ,454 25. .529 1. .00 29. ,92 AAAA
  • ATOM 402 C ASP A 59 -16. ,723 0. ,608 25, .378 1 .00 30, .39 AAAA
  • ATOM 405 CA PHE A 60 -18. ,612 1. ,183 24, .000 1. .00 32. ,36 AAAA
  • ATOM 416 CA ILE A 61 -20, ,326 4. ,500 23, .421 1. .00 36, .08 AAAA
  • ATOM 435 CA ILE A 63 -24 .537 6. .912 19, .378 1. .00 49. .26 AAAA
  • ATOM 449 CA GLY A 65 -28. .774 8. ,196 14, .090 1, .00 57, .42 AAAA
  • ATOM 461 CA ARG A 67 -25. ,923 8. ,224 9. .089 1, .00 61. .57 AAAA
  • ATOM 506 CA ALA A 73 -23, .282 15 .650 12, .954 1 .00 39. .79 AAAA
  • ATOM 508 C ALA A 73 -23, .575 15 .524 14, .438 1. .00 39, .28 AAAA
  • ATOM 538 CA PRO A 78 -27. 577 18. 653 21. 514 1. ,00 31. 07 AAAA
  • ATOM 582 CA ASN A 83 -25, .648 11, .640 26. .045 1, .00 22, .62 AAAA
  • ATOM 590 CA ALA A 84 -22, ,383 10. ,480 24. ,401 1. .00 19. .25 AAAA
  • ATOM 612 CD ARG A 86 -25. 318 13. .357 31. ,154 1. ,00 19. .61 AAAA
  • ATOM 645 CA ALA A 90 -19 .719 7. .063 33. .386 1. .00 23. .31 AAAA

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Abstract

L'invention concerne des cristaux de la protéine MurG de Escherichia coli, une UDP-glycosyltransférase à membrane associée impliquée dans la biosynthèse du peptidogylcane, ainsi que des coordonnées atomiques tridimensionnelles de la protéine MurG, des structures tridimensionnelles de la protéine, et des images de ceux-ci. L'invention concerne en outre les coordonnées atomiques et les structures tridimensionnelles du squelette α-carbone, le squelette α-carbone, ainsi que les chaînes latérales des restes d'acides aminés conservés de la protéine MurG, et des images de ceux-ci. L'invention concerne encore des coordonnées atomiques tridimensionnelles du site de liaison de nucléotide donneur, du site de liaison accepteur et du site d'association de la membrane de la protéine MurG, des structures tridimensionnelles des domaines de liaison, et des images de ceux-ci. L'invention concerne aussi des supports lisibles par ordinateur, codés au moyen d'ensembles des coordonnées tridimensionnelles susmentionnées, des procédés de cristallisation des protéines MurG, des modèles des structures tridimensionnelles des UDP-glycosyltransférases et, notamment des protéines MurG, en fonction de la structure tridimensionnelle des cristaux de la protéine MurG de Escherichia coli. L'invention concerne encore des modèles des structures tridimensionnelles du squelette α-carbone, le squelette α-carbone, les chaînes latérales des restes d'acides aminés conservés des UDP-glycosyltransférases et des protéines MurG et des sites de liaison de ceux-ci, ainsi que des procédés de conception de médicaments mettant en oeuvre des modèles selon l'invention, les composés identifiés mettant en oeuvre des modèles selon l'invention qui lient, inhibent ou stimulent des UDP-glycosyltransférases ou des protéines MurG, de même que des compositions renfermant des composés identifiés au moyen des modèles selon l'invention, aux fins d'applications thérapeutiques ou diagnostiques. L'invention concerne enfin des procédés de préparation de modèles selon l'invention.
PCT/US2001/011500 2000-05-17 2001-04-09 Procedes de preparation de modeles, procedes d'utilisation des modeles de la proteine murg, composes liant, inhibant ou stimulant des proteines murg et des compositions therapeutiques associees WO2001090301A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2385328B (en) * 2001-12-19 2005-09-21 Hoffmann La Roche Crystals of glucokinase and methods of growing them
US7778779B2 (en) 2002-10-16 2010-08-17 Isis Innovation Limited Method of identifying a chemical entity which is a hydroxylase modulator
WO2012068053A1 (fr) * 2010-11-16 2012-05-24 Vertex Pharmaceuticals Incorporated Structure cristalline de l'enzyme murg de pseudomonas aeruginosa

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5068191A (en) * 1989-08-31 1991-11-26 The Biomembrane Institute Purified histo-blood group a glycosyltransferase and antibodies thereto
WO1999038958A1 (fr) * 1998-02-02 1999-08-05 Princeton University ANALOGUES DU SUBSTRAT DE LA MurG, PROCEDES DE MARQUAGE DE CEUX-CI ET ANALYSES DANS LESQUELLES ILS SONT UTILISES

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5068191A (en) * 1989-08-31 1991-11-26 The Biomembrane Institute Purified histo-blood group a glycosyltransferase and antibodies thereto
WO1999038958A1 (fr) * 1998-02-02 1999-08-05 Princeton University ANALOGUES DU SUBSTRAT DE LA MurG, PROCEDES DE MARQUAGE DE CEUX-CI ET ANALYSES DANS LESQUELLES ILS SONT UTILISES

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2385328B (en) * 2001-12-19 2005-09-21 Hoffmann La Roche Crystals of glucokinase and methods of growing them
US7534593B2 (en) 2001-12-19 2009-05-19 Hoffmann-La Roche Inc. Crystals of glucokinase and methods of growing them
US7778779B2 (en) 2002-10-16 2010-08-17 Isis Innovation Limited Method of identifying a chemical entity which is a hydroxylase modulator
WO2012068053A1 (fr) * 2010-11-16 2012-05-24 Vertex Pharmaceuticals Incorporated Structure cristalline de l'enzyme murg de pseudomonas aeruginosa

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