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WO2000058345A1 - NOUVELLE ARNt SYNTHETASE, COMPOSITIONS CAPABLES DE SE FIXER A CETTE ENZYME ET PROCEDES D'UTILISATION DE CES COMPOSITIONS - Google Patents

NOUVELLE ARNt SYNTHETASE, COMPOSITIONS CAPABLES DE SE FIXER A CETTE ENZYME ET PROCEDES D'UTILISATION DE CES COMPOSITIONS Download PDF

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WO2000058345A1
WO2000058345A1 PCT/US2000/008246 US0008246W WO0058345A1 WO 2000058345 A1 WO2000058345 A1 WO 2000058345A1 US 0008246 W US0008246 W US 0008246W WO 0058345 A1 WO0058345 A1 WO 0058345A1
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synthetase
trna synthetase
active site
coordinates
glycyl trna
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Cheryl A. Janson
Neal Frederick Osborne
Xiayang Qiu
Christine M. Richardson
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • the invention relates to the identification of a novel enzyme active site and methods enabling the design and selection of inhibitors of that active site.
  • Transfer RNA (tRNA) synthetase enzymes are of interest as potential targets for antibacterial agents.
  • Mupirocin a selective inhibitor of bacterial isoleucyl tRNA synthetase, is marketed for the treatment of skin infections and the eradication of nasal carriage of MRSA (methicillin-resistant Staphylococcus aureus) in hospital staff and patients.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Nucleic acid and amino acid sequences for glycyl tRNA synthetases are publicly available, including those of Thermus thermophilus, Mycoplasma genitalium, Homo sapiens, yeast, Bombyx mori and Caenorhabditis elegans, which are all characterized by a2 dimers, and Coxiella burnetti, Escherichia coii, Chlamydia trachomatous, Neisseria gonorrheae, Synechocystis sp., and Haemophilus influenzae, which are all characterized by being a2b2 tetramers.
  • the present invention provides a novel Staphylococcus aureus tRNA synthetase enzyme active site crystalline form.
  • the present invention provides a novel tRNA synthetase composition characterized by a catalytic site of 16 amino acid residues.
  • the invention provides a method for identifying inhibitors of the compositions described above which methods involve the steps of: providing the coordinates of the tRNA synthetase structure of the invention to a computerized modeling system; identifying compounds which will bind to the structure; and screening the compounds identified for tRNA synthetase inhibitory bioactivity.
  • the present invention provides an inhibitor of the catalytic activity of any composition bearing the catalytic domain described above.
  • Another aspect of this invention includes machine readable media encoded with data representing the coordinates of the three-dimensional structure of the tRNA synthetase crystal.
  • Fig. 1 provides the atomic coordinates of the Staph aureus glycyl tRNA synthetase.
  • Fig. 2 illustrates the cloning of the grs gene in pDB575. Briefly, the grs gene was
  • the GRS2 primer contains the Xbal site and stop codons in the three possible reading frames.
  • Fig. 3 illustrates the SDS-PAGE analysis of the GRS production by E. coii. E. coii HB 101 cells, harboring either pDB575 or pDBGRS, were induced with 1 mM IPTG.
  • Fig. 4 provides a projection of the ribbon structure of the Staphylococcus aureus glycyl tRNA synthetase dimer. The two monomers are shaded in dark and light gray.
  • Fig. 5 provides a schematic drawing of the molecular structure of the
  • Staphylococcus aureus glycyl tRNA synthetase dimer The two monomers are shaded in dark and light gray.
  • Fig. 6 provides the ribbon structure of the human glycyl tRNA synthetase monomer.
  • Fig. 7 provides a schematic drawing comparing the active sites of the human and
  • the present invention provides a novel glycyl tRNA synthetase crystalline structure, a novel Staph aureus tRNA synthetase active site, and methods of use of the crystalline form and active site to identify synthetase inhibitor compounds (peptide, peptidomimetic or synthetic compositions) characterized by the ability to competitively inhibit binding to the active site of a glycyl tRNA synthetase.
  • synthetase inhibitor compounds peptide, peptidomimetic or synthetic compositions
  • a novel human glycyl tRNA synthetase crystalline structure This structure can be used as described below for the Staph tRNA synthetase crystal structure.
  • the present invention provides a novel glycyl tRNA synthetase crystalline structure based on the Staph aureus tRNA synthetase.
  • the amino acid sequences of the synthetase are provided in SEQ ID NO: 1.
  • the crystal structure is a tightly associated S. aureus GRS dimer.
  • Each monomer has three structural domains: the N- terminal domain (residues 1-86 of SEQ ID NO: l), the active site domain (residues 150-340 of SEQ ID NO: l) and the C-terminal domain (residues 341-463 of SEQ ID NO:l).
  • the N- terminal domain having three a-helices and three b-strands, wraps around the active site domain with its second a-helix lying in the core of the dimer interface and its third b-strand adding to the central b-sheet of the active site domain to form the 7-stranded anti-parallel b sheet where the enzyme active site locates.
  • the C-terminal domain contains mainly a 5- stranded mixed b-sheet with three flanking helices and is believed to be important to anticodon recognition. While the overall architecture of the S. aureus GRS is similar to that of the T. thermophilus GRS, differences exit in the conformation of a number of surface loops, as well as the relative orientation of between the active site and C-terminal domains. With only 44% sequence identity, many amino acid side chains are also different, including several residues near the active site.
  • the Staph aureus synthetase is a dimer.
  • the present invention provides both a crystalline monomer and dimer structure of Staph aureus synthetase. Inhibitors that perturb or interact with this dimer interface are another target for the design and selection of anti-bacterial agents.
  • the crystal structure of Staph aureus tRNA synthetase has been resolved at 3.5 .
  • the structure was determined using the method of molecular replacement, and refined to an R-factor of 23.4% with goal geometry.
  • R-factor 23.4% with goal geometry.
  • further refinement of the atomic coordinates will change the numbers in Figure 1 and Tables I - III
  • refinement of the crystal structure from another crystal form will result in a new set of coordinates.
  • distances and angles in Figure 1 and Tables I - III will remain the same within experimental errors, and relative conformation of residues in the active site will remain the same within experimental error.
  • Figure 1 provides the atomic coordinates of the Staph aureus glycyl tRNA synthetase dimer, which contains 790 amino acids; with 130 residues disordered in the crystal.
  • the occupancy factor is 1.0 and the B factor is 19.60.
  • the tRNA synthetase is characterized by an active site which preferably contains a binding site for glycyl-adenylate and the receptor stem of tRNA (glycines).
  • the crystal structure described herein was solved in the absence of glycine, ATP or tRNA.
  • the region of the active site can be inferred from that of the homologous aspartyl tRNA synthetase.
  • the crystalline active site consists of 16 amino acid residues.
  • residues include Glul74, Arg206, Glu208, Phe216, Arg217, Thr218, Phe221 , Gln223, Glu225, Asp279, Glu290, Leu291, Arg297, Glu330, Ser332 and Arg337 [SEQ ID NO: l].
  • the atomic coordinates of the active site residues are provided in Table I.
  • Table II provides the distances between (D) atoms of the active site residues that are within 5.0 angstroms of one another.
  • Table III provides the angles (A) between active site atoms at are within 4.0 angstrom of each other. For simplicity, intra-residue angles are omitted.
  • the invention further provides homologues, co-complexes, mutants and derivatives of the Staph aureus tRNA synthetase crystal structure of the invention.
  • homologue means a protein having at least 30% amino acid sequence identity with synthetase or any functional domain of glycyl tRNA synthetase.
  • co-complex means glycyl tRNA synthetase or a mutant or homologue of glycyl tRNA synthetase in covalent or non-covalent association with a chemical entity or compound.
  • mutant refers to a glycyl tRNA synthetase polypeptide, i.e., a polypeptide displaying the biological activity of wild-type glycyl tRNA synthetase activity, characterized by the replacement of at least one amino acid from the wild-type synthetase sequence.
  • a mutant may be prepared, for example, by expression of Staph aureus synthetase cDNA previously altered in its coding sequence by oligonucleotide-directed mutagenesis.
  • Staph aureus glycyl tRNA synthetase mutants may also be generated by site-specific incorporation of unnatural amino acids into glycyl tRNA synthetase proteins using the general biosynthetic method of C. J. Noren et al, Science, 244: 182-188 (1989).
  • the codon encoding the amino acid of interest in wild-type glycyl tRNA synthetase is replaced by a "blank" nonsense codon, TAG, using oligonucleotide-directed mutagenesis.
  • a suppressor tRNA directed against this codon is then chemically aminoacylated in vitro with the desired unnatural amino acid.
  • the aminoacylated tRNA is then added to an in vitro translation system to yield a mutant glycyl tRNA synthetase enzyme with the site-specific incorporated unnatural amino acid.
  • Selenocysteine or selenomethionine may be incorporated into wild-type or mutant tRNA glycyl synthetase by expression of Staph aureus glycyl tRNA synthetase- encoding cDNAs in auxotrophic E. coii strains [W. A. Hendrickson et al, EMBO J., 9(5): 1665- 1672 (1990)].
  • the wild-type or mutagenized tRNA synthetase 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).
  • heavy atom derivative refers to derivates of glycyl tRNA synthetase produced by chemically modifying a crystal of glycyl tRNA synthetase.
  • a crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, e.g., lead chloride, gold thiomalate, thimerosal or uranyl acetate, which can diffuse through the crystal and bind to the surface of the protein.
  • the location(s) of the bound heavy metal atom(s) can be determined by X-ray diffraction analysis of the soaked crystal. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the enzyme [T. L. Blundel and N. L. Johnson, Protein Crystallography, Academic Press (1976).
  • Another aspect of this invention involves a method for identifying inhibitors of a Staph glycyl tRNA synthetase characterized by the crystal structure and novel active site described herein, and the inhibitors themselves.
  • the novel synthetase crystalline structure of the invention permits the identification of inhibitors of synthetase activity.
  • Such inhibitors may be competitive, binding to all or a portion of the active site of the glycyl tRNA synthetase; or non-competitive and bind to and inhibit glycl tRNA synthetase whether or not it is bound to another chemical entity.
  • One design approach is to probe the glycyl tRNA synthetase crystal of the invention with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate glycyl tRNA synthetase inhibitors and the enzyme. For example, high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of where each type of solvent molecule sticks. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their glycyl tRNA synthetase inhibitor activity [J. Travis, Science, 262: 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 or with glycyl tRNA synthetase.
  • the time-dependent analysis of structural changes in glycyl tRNA synthetase during its interaction with other molecules is permitted.
  • the reaction intermediates of glycyl tRNA synthetase can also be deduced from the reaction product in co-complex with glycyl tRNA synthetase.
  • Such information is useful to design improved analogues of known glycyl tRNA synthetase inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the glycyl tRNA synthetase enzyme and glycyl tRNA synthetase inhibitor co-complex.
  • This provides a novel route for designing glycyl tRNA synthetase inhibitors with both high specificity and stability.
  • Another approach made possible 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 glycyl tRNA synthetase 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 [E. C. Meng et al, J. Comp. Chem., . 13:505-524 (1992)].
  • glycyl tRNA synthetase may crystallize in more than one crystal form
  • the structure coordinates of glycyl tRNA synthetase, or portions thereof, as provided by this invention are particularly useful to solve the structure of those other crystal forms of tRNA synthetase. They may also be used to solve the structure of glycyl tRNA synthetase mutant co-complexes, or of the crystalline form of any other protein with significant amino acid -sequence homology to any functional domain of glycyl tRNA synthetase.
  • the unknown crystal structure whether it is another crystal form of glycyl tRNA synthetase, a glycl tRNA synthetase mutant, or a glycyl tRNA synthetase co- complex, or the crystal of some other protein with significant amino acid sequence homology to any functional domain of glycyl tRNA synthetase, may be determined using the glycyl tRNA synthetase structure coordinates of this invention as provided in Figure 1 and Tables I - III.
  • This method will provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
  • the synthetase structure permits the screening of known molecules and/or the designing of new molecules which bind to the synthetase structure, particularly at the active site, via the use of computerized evaluation systems.
  • computer modelling systems are available in which the sequence of the synthetase, and the synthetase structure (i.e., the bond angles, dihedral angles, distances between atoms in the active site region, etc. as provided by Figure 1 and Tables I - III herein) may be input.
  • a machine readable medium may be encoded with data representing the coordinates of Figure 1 and Tables I - III.
  • the computer then generates structural details of the site into which a test compound should bind, thereby enabling the determination of the complementary structural details of said test compound.
  • the design of compounds that bind to or inhibit glycyl tRNA synthetase according to this invention generally involves consideration of two factors.
  • the compound must be capable of physically and structurally associating with glycyl tRNA synthetase.
  • Non-covalent molecular interactions important in the association of glycyl tRNA synthetase 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 glycyl tRNA synthetase. Although certain portions of the compound will not directly participate in this association with glycyl tRNA synthetase, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency.
  • Such 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 glycyl tRNA synthetase, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with glycyl tRNA synthetase.
  • the potential inhibitory or binding effect of a chemical compound on glycyl tRNA synthetase 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 glycyl tRNA synthetase, 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 glycyl tRNA synthetase and inhibit using a suitable assay. In this mannei, synthesis of inoperative compounds may be avoided.
  • An inhibitory or other binding compound of glycyl tRNA synthetase 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 glycyl tRNA synthetase.
  • One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with glycyl tRNA synthetase and more particularly with the individual binding pockets of the glycyl tRNA synthetase active site or accessory binding site.
  • This process may begin by visual inspection of, for example, the active site on the computer screen based on the glycyl tRNA synthetase coordinates in Figure 1 and Tables I - III. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within a binding pocket of glycyl tRNA synthetase.
  • Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER. Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:
  • GRID [P. J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 28:849-857 (1985)]. GRID is available from Oxford University, Oxford, UK.
  • MCSS [A. Miranker and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method", Proteins: Structure, Function and Genetics. H:29-34 (1991)]. MCSS is available from
  • DOCK [I. D. Kuntz et al, "A Geometric Approach to Macromolecule- Ligand Interactions", J. Mol. Biol., 161:269-288 (1982)]. DOCK is available from University of California, San Francisco, CA.
  • 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 glycyl tRNA synthetase. This would be followed by manual model building using software such as Quanta or Sybyl.
  • CAVEAT [P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the
  • 3D Database systems such as MACCS-3D (MDL Information Systems, San Diego, Calif.).
  • inhibitory or other glycyl tRNA synthetase 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).
  • LEGEND [Y. Nishibata and A. Itai, Tetrahedron, 47:8985 (1991)]. LEGEND is available from Molecular Simulations, Burlington, MA.
  • the synthetase inhibitor may be tested for bioactivity using standard techniques.
  • structure of the invention may be used in binding assays using conventional formats to screen inhibitors.
  • One particularly suitable assay format includes the enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Other assay formats may be used; these assay formats are not a limitation on the present invention.
  • the synthetase structure of the invention permit the design and identification of synthetic compounds and/or other molecules which are characterized by the conformation of the synthetase of the invention.
  • the coordinates of the synthetase structure of the invention may be provided in machine readable form, the test compounds designed and/or screened and their conformations superimposed on the structure of the synthetase of the invention.
  • suitable candidates identified as above may be screened for the desired synthetase inhibitory bioactivity, stability, and the like. Once identified and screened for biological activity, these inhibitors may be used therapeutically or prophylactically to block synthetase activity, and thus, bacterial replication.
  • the present invention also provides inhibitors of glycyl tRNA synthetase activity identified or designed by the methods of the invention. These inhibitors are useful as antibacterial agents.
  • One particularly desirable inhibitor is glycylsulfamoyladenosine.
  • the structure of this compound is as follows.
  • Glycylsulfmoyladenosine is an analogue of the Gly-AMP reaction intermediate and inhibits GRS catalytic activity as measured by any of the techniques described in the examples below.
  • Estimates of the potency of inhibition are obtained by performing enzyme assays in the presence of a range of inhibitor concentrations, and fitting the effect of inhibitor concentration on enzyme velocity to a four parameter logistic function that allows calculation of an IC 50 (the inhibitor concentration at which GRS activity is reduced by half).
  • This parameter is directly related to the dissociation constant for inhibitor binding (Kj or K d ) and has a value of around 2.4 mM for glycylsulfamoyladenosine when tested against the S. aureus GRS.
  • Binding of glycylsulfamoyladenosine to GRS can also be measured directly using stopped-flow fluorescence techniques because enzyme:inhibitor binary complex has around 5% higher tryptophan fluorescence than the free enzyme.
  • Example 1 The Expression of the Glycyl t-RNA Synthetase from Staphylococcus aureus in Escherichia coii.
  • the strategy for the expression of the glycyl t-RNA synthetase (GRS) from Staphylococcus aureus, using Escherichia coii as a host was based on the PCR amplification of the grs gene and the introduction of suitable restriction sites that allowed the cloning of the gr?-containing DNA fragment in the pDB575 expression vector. After the PCR amplification the insert of the resultant recombinant plasmid, (pDBGRS hereafter), was sequenced to verify the absence of artefacts introduced by the Taq polymerase. Expression was monitored by SDS-polyacrylamide gel analysis.
  • the Escherichia coii strains used were: DH5a (supE44, D/ ⁇ cU169 (f 80 / ⁇ cZDM15), hsdRXl, recAX, endAX, gyrA96, thi-X, relA X) and HB101 (thi-X, hsdS,20(r ,m B ), supE44, recAX3, ara-X4, leuB ⁇ , proA2, lacYX, rpsL20(st ⁇ ⁇ ), xyl-5, mtl-X).
  • E. coii cells were grown at 37°C in Luria Bertani broth (LB). These strains may all be obtained from commercial sources.
  • plasmids used were pBluescript SK- [Stratagene], pUC18 [J. Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)] and pDB575.
  • pDB575 A detailed description of pDB575 is provided in A.F. Chalker et al, Gene, 141:103-108 (1994).
  • pDB575 is a expression vector of E.coli based on pKK223-3 [Pharmacia] with the following modifications: (i) the polylinker between EcoRl and Hind ⁇ ll has been replaced with a longer one (EcoRX, NcoX, Kpnl, Ndel, SstX, Sstll, XbaX, ClaX, SmaX, BgllX, XmaXXX, Hindlll) (ii) it has a lacl q gene inserted; (iii) it is non-mobilizable, the pBR322 portion of pKK223-3 has been replaced by the equivalent fragment from pATIS3.
  • pDB575 allows the selection of the recombinant clones by ampicillin resistance and the gene expression is driven by the tac promoter.
  • LB Medium Per litre:
  • Plasmid DNA was prepared by the rapid alkaline method (Sambrook et al, 1989). Transformations of E. coii cells were carried out using the RbCl methods (Sambrook et al, 1989). DNA fragments were purified using the Geneclean Kit [BIO 101 Inc., La Jolla, CA, USA]. The plasmids for sequencing were purified using QIAGEN plasmid kit [QIAGEN]. DNA sequencing was carried out on supercoiled plasmid DNA by the dideoxy chain-termination method using the Thermo Sequenase cycle sequencing kit [Amersham Life Science, Inc. USA]. DNA was also sequenced by the Automated Sequencing Service of Pharmacy Faculty in the Complutense University of Madrid. Universal or synthetic oligonucleotides [MedProbe, Norway] were used as primers. Restriction enzymes and T4 DNA ligase were obtained from Promega and Boehringer respectively and the experiments were carried out following the instructions provided by the suppliers.
  • the grs gene from S. aureus cloned in the pBluescript SK- was amplified by PCR using the primers GRS 1 : (5'-GGGGTACCGCTAGCAGGAGAGGTAATTATGGCAAAAGATATG-3' ; SEQ ID NO:2) and GRS2: (5 -GCTCTAGATTAGTCATTTAATTAGAATTTTGTTTTTTCAGTTAAG- 3'; SEQ ID NO:3).
  • Kpn I and Xba I restriction sites were incorporated at the 5' and 3' ends respectively of each primer to facilitate ligation of the amplified DNA into vectors.
  • Plasmid DNA (100 ng) was amplified in 100 ml of PCR mixture containing 250 mM deoxynucleotide triphosphates (dNTPs), 0.9 mM oligonucleotide primers, the manufacturer's buffer and 2U of Taq polymerase (Promega). The following cycling parameters were used:
  • PCR Polymerase chain reaction
  • the cloning strategy is shown in Figure 2.
  • PCR amplification of the grs gene from S. aureus using the primers GRS1 and GRS2 resulted in a DNA fragment of 1.4 kb.
  • This fragment was purified and ligated to the Kpnl, Xbal sites of pDB575 to obtain the recombinant plasmid pDBGRS and the ligation mix was used to transform E. coii DH5a competent cells.
  • the construction of pDBGRS was initially confirmed by restriction analysis of the plasmid purified from the transformants.
  • the amplification with Taq DNA polymerase made the sequencing of the grs of pDBGRS an obligatory step to confirm that no changes were introduced due to the low fidelity of this enzyme. Sequence analysis was accomplished by using grs gene introduced in the expression plasmid pDB575 and/or in pUC18. The sequencing of both strands showed that no artefacts had been introduced during the amplification process
  • the plasmid pDBGRS and the negative control pDB575 were used to transform the E. coii HB 101 host strain.
  • Single clones of HB101 :pDBGRS and HB 101:pDB575 cells were grown overnight at 37°C in 2 ml of LB medium in the presence of 0.1 mg/ml ampicillin. The cells were then diluted 100-fold in 30 ml LB with ampicillin. When the cultures reached a value of 0.5 at OD 6 oothe grs expression was induced by addition of isopropyl-thio-galactoside (IPTG) at ImM of final concentration. After this induction 2 ml samples were taken at different times (2, 3 and 4 hours).
  • IPTG isopropyl-thio-galactoside
  • the cells were harvested in a microfuge for 3 min, the pellets were washed with 20 mM Tris-HCl pH 8 / ImM PMSF and resuspended in 300 ml of SDS-PAGE gel-loading buffer. The cells were broken by sonication (15 seconds). The samples were then boiled 10 minutes and after one spin, 10 ml fractions were analyzed by SDS-PAGE according to the methods of Laemmli [U. K. Laemmli, Nature 227, 680-685 (1970)]. The 12% polyacrylamide gels were stained with Coomassie blue. As shown in Figure 3 good expression levels were detected from the early stages after induction with IPTG.
  • the evidence was the presence of a prominent band (lanes 2, 4 and 6 in Figure 3) that was in good agreement with the M r predicted from the primary sequence.
  • the GRS protein has a theoretical molecular weight of about 53.7 kDa.
  • a 300 litre fermentation of E.coli HB 101 :pDB575GRS was carried out in double strength Luria Bertani medium (LB), containing 50 ug/ml ampicillin.
  • the vessel was inoculated at 2% (v/v) from a 15 hour secondary seed culture in single strength LB medium, containing 50 mg/ml ampicillin.
  • the production vessel was incubated at 37°C, agitated at 1.5 msec "1 and aerated at 1.0 VVM.
  • the OD at 550nm was monitored, and at 2.5 absorbance units, GRS expression was induced with the addition of isopropyl- thiogalactosidase to 1.0 mM and the cells harvested by centrifugation in a Westfalia CSA- 19, 2 hours post induction. A total of 990 grams of cell paste was recovered.
  • LB Medium per litre, contains the following components. The medium ingredients were supplied by Difco Laboratories, West Molesey, Surrey UK. Double strength Single strength Bacto Tryptone 20 g Bacto Tryptone 10 g
  • the supernatant from 1 was loaded onto a Q-Sepharose high performance (Pharmacia) column of 200ml packed into a Pharmacia XK-50 column.
  • the column was equilibrated with buffer A prior to loading.
  • the column is then washed with buffer A (1000ml) at 40 ml/min, and eluted with a linear gradient of buffer A to IM NaCl in buffer A over 140 minutes at lOml/min.
  • the eluate was fractionated into 5 minute fractions using a Pharmacia Superfrac.
  • the eluted fractions were assayed for GRS activity by measurement of aminoacylation of tRNA Gly , and for protein by the Bradford method. Active fractions were analyzed by reducing SDS PAGE (Pharmacia Phast System 10-15% gradient gel)
  • Eluted fractions are collected ( 1 minute fraction) and assayed for GRS activity and protein. Active fractions are pooled and diafiltered against (1,000 fold buffer exchange) buffer A using an Amicon ultrafiltration cell (350ml) under nitrogen. A final volume of 33 ml of protein was obtained containing 4.2 mg/ml of protein (by amino acid analysis). This product was greater than 95% purity by SDS PAGE and the activity showed an overall process yield of 60 % from 1). N-terminal amino acid analysis confirmed identity.
  • Glycyl tRNA Synthetase (GRS) activity The enzyme catalyses the aminoacylation of tRNA Gly , which proceeds through a two step mechanism. The first step involves the formation of a stable enzyme:glycyl adenylate complex resulting from the specific binding and reaction of ATP and L-glycine. Subsequently, the 3' terminal adenosine of enzyme-bound tRNAGly reacts with the aminoacyladenylate, leading to the esterification of the tRNA and release of AMP. These steps are summarized below. a) L-Gly + ATP.Mg + GRS P GRS: Gly-AMP + PPi.Mg b) GRS:Gly-AMP + tRNA Gly I GRS + Gly-tRNA Gly + AMP
  • This reaction can be assayed in order to characterize the enzyme or identify specific inhibitors of its activity in a number of ways: (1) Measurement of the formation of Gly-tRNA Gly can be specifically determined using radiolabelled glycine and separating free glycine from Gly-tRNA using precipitation/filtration techniques (e.g. in cold trichloroacetic acid; see, Calender & Berg (1966) Biochemistry 5, 1681-1690; Toth MJ & Schimmel P (1990) J. Biol. Chem. 265, 1000-1004].
  • precipitation/filtration techniques e.g. in cold trichloroacetic acid; see, Calender & Berg (1966) Biochemistry 5, 1681-1690; Toth MJ & Schimmel P (1990) J. Biol. Chem. 265, 1000-1004].
  • the full acylation reaction can also be measured by analyzing production of either PPi or AMP which are produced in stoichiometric ratio to the tRNA acylation. This may be achieved in a number of ways, for example using colorimetric [Hoenig (1989) J. Biochem. Biophys. Meth. 19, 249-252]; or enzyme coupled [Webb TM (1994) Anal. Biochem. 218, 449-454] measurement of Pi after addition of excess inorganic pyrophosphatase or using enzyme coupled assays to directly measure AMP or PPi production [Sigma Chemicals Catalogue, 1986].
  • the partial reaction (a) can be assayed through radiolabel isotopic exchange between ATP and PPi, since each of the steps in this part of the reaction are freely reversible.
  • This reaction is independent of tRNA binding, typically has a k CM around 20-fold higher than the full acylation reaction (a+b), and is readily measured using chromatographic principles which separate PPi from ATP (i.e. using activated charcoal; see, Calender & Berg, cited above; Toth & Schimmel, cited above).
  • D Ligand binding to GRS. It is also possible to define ligand interactions with GRS in experiments that are not dependent upon enzyme catalyzed turnover of substrates. This type of experiment can be done in a number of ways:
  • the ligands could either be inhibitors or variants of the natural ligands (i.e. fluorescent ATP derivatives or tRNAGly labelled with a fluorophore).
  • Assays were performed either using purified S. aureus GRS overexpressed in E. coii, or using crude cell lysate from E. coii overexpressing GRS. The latter contained around 10% of total protein as GRS.
  • Enzyme was stored at -70°C in 50 mM Tris-HCl buffer (pH 7.8), 10 mM MgCl 2 and 10 mM B-mercaptoethanol after flash freezing in liquid ⁇ 2 . In experiments to determine the activity of enzyme samples, these stocks were diluted over a wide range (100 fold to 10,000 fold) in 50 mM Tris pH 7.8, 10 mM MgCl 2 , 1 mM Dithiothreitol and stored on ice prior to assay.
  • the assay procedure was as follows. 50 ml of enzyme prepared and diluted as described above was mixed with reaction mixture (100 ml), comprising: 0.15 mCi L-[U- 14 ]-Glycine (Amersham International), 4 mg/ml E. coii MRE600 mixed tRNA (Boehringer Manheim), 5 mM ATP, 15 mM MgS0 4 , 3 M DTT, 75 mM KC1 and 50 mM Tris-HCl, pH 7.8. Unless otherwise states, all reagents were obtained from Sigma Chemical Company Ltd. Concentrations are given as in the final reaction mix.
  • Example 3 Crystallization of Staphylococcus aureus Glycyl tRNA Synthetase A. Crystallization A large crystal (0.25 x 0.25 x 0.18 mm 3 ) was formed using the following conditions. The protein used for the crystallization was supplied @ 5.8 mg/ml in a solution of 20mM tris, 5mM MgCl 2 , ImM DTT, ImM EDTA, 10% glycerol, pH 7.5). The crystal was obtained from a 1 : 1 mixture of the protein solution and a solution of 10% PEG 8000, 0.1M imidazole pH 8.0 and 0.2M calcium acetate using the hanging drop method, grown at room temperature.
  • the Staph aureus synthetase crystal was mounted in a sealed glass capillary with a small amount of mother liquor in each end of the capillary.
  • the CuK a X- ray having a wavelength of 1.54 A, was generated by a Rigaku-RU200 rotating anode machine operating at 100 mA x 50 kV electric power.
  • the crystal was exposed to the CuK a X-ray, and the diffracted X-ray was collected by a Siemens multiwire area detector.
  • the crystal diffracted to 3.5 .
  • the crystal has been determined to be an orthorhombic crystal system and P2,2,2, space group.
  • an asymmetric unit was calculated to have one protein molecule.
  • the crystal contains an estimated 60% solvent.
  • Solvent flattening and 2-fold non-crystallographic averaging was then used to improve the phases [Collaborative Computational Project, Number 4, Acta Crystaliogr. D50, 760-763 (1994)], which introduced about 30°C phase shifts and improved the averaged figure of merit from 0.4 to 0.8 and Rfree from 47% to 28%. An improved electron density map was then calculated.
  • the GRS model was subjected to one round of Xplor [A. Brunger et al, Science, 235:458-460 (1987) refinement using the standard positional, slowcool and overall B factor refining protocols.
  • the GRS was refined as a tightly contained dimer without any solvent molecules.
  • the R factor of the model is 23.9% with satisfactory geometry.
  • the rms deviations are 0.017 A for bond lengths, 2.0° for bond angles, 25.4 for dihedrals and 1.8°C for impropers.
  • the structure contains residues 1-86, 150-161 , 164-352 and 356-463 [SEQ ID NO:l], while the other 68 residues (15%) are disordered in the crystal and not included in the model.
  • the characterization of the compound as an inhibitor of the catalytic activity of GRS was performed using a procedure similar to that described in Example 2E above, except that multiple assays were performed in the presence of inhibitor concentrations ranging (in two-fold dilution steps) from 100 mM down to 0.1 mM (final concentrations). These were added from stocks prepared at 10-fold higher concentrations and added to each reaction mix. The stock of inhibitor was prepared freshly from a solid sample and dissolved in dimethylsulfoxide. The enzyme concentration used for these assays was selected so that around 50% of the tRNA available was acylated during the reaction time course.
  • acylation activity (relative to controls in the absence of inhibitor) were plotted as a function of inhibitor concentration and fitted to a four-parameter logistic function (using the Grafit package; Erithacus Software Ltd.) to yield IC 50 , the inhibitor concentration required to inhibit half the enzyme activity.
  • a model of the human glycyl tRNA synthetase was constructed using Quanta version 4.1 [Molecular Simulations Inc, Burlington, MA].
  • the human enzyme contains a number of large surface loops (see Fig. 6).
  • a comparison of the human and Staph enzyme aminoacylation sites is shown in Figure 7.
  • One of the most significant differences is that a glutamine in the prokaryotic enzyme is replaced by a methionine. The glutamine is believed to be capable of hydrogen bonding to the acyl phosphage moiety of glycyl adenylate.

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Abstract

L'invention concerne une nouvelle structure cristalline de Staphilococcus glycyl ARNt-synthétase. L'invention concerne également des techniques d'identification d'inhibiteurs de ces synthétases et/ou de sites actifs, ainsi que des inhibiteurs identifiés selon ces techniques.
PCT/US2000/008246 1999-03-29 2000-03-29 NOUVELLE ARNt SYNTHETASE, COMPOSITIONS CAPABLES DE SE FIXER A CETTE ENZYME ET PROCEDES D'UTILISATION DE CES COMPOSITIONS Ceased WO2000058345A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026340A1 (fr) * 1996-01-19 1997-07-24 Smithkline Beecham Plc GLYCYL-ARNt SYNTHETASE OBTENUE A PARTIR DE STAPHYLOCOCCUS AUREUS
US6037117A (en) * 1997-01-31 2000-03-14 Smithkline Beecham Corporation Methods using the Staphylococcus aureus glycyl tRNA synthetase crystalline structure

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026340A1 (fr) * 1996-01-19 1997-07-24 Smithkline Beecham Plc GLYCYL-ARNt SYNTHETASE OBTENUE A PARTIR DE STAPHYLOCOCCUS AUREUS
US6037117A (en) * 1997-01-31 2000-03-14 Smithkline Beecham Corporation Methods using the Staphylococcus aureus glycyl tRNA synthetase crystalline structure

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NIYOMPORN ET AL.: "Biosynthesis of the peptidoglycan of bacterial cell walls. IX. Purification and properties of glycyl ribonucleic acid synthetase from Staphylococcus aureus", JOURNAL OF BIOLOGICAL CHEMISTRY,, vol. 243, no. 4, 1968, pages 773 - 778, XP000900608 *

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