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WO1994028139A1 - INHIBITION, DEPENDANTE DE LA FIXATION D'ARNt, DE LA CROISSANCE PATHOGENE MICROBIENNE - Google Patents

INHIBITION, DEPENDANTE DE LA FIXATION D'ARNt, DE LA CROISSANCE PATHOGENE MICROBIENNE Download PDF

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WO1994028139A1
WO1994028139A1 PCT/US1994/005905 US9405905W WO9428139A1 WO 1994028139 A1 WO1994028139 A1 WO 1994028139A1 US 9405905 W US9405905 W US 9405905W WO 9428139 A1 WO9428139 A1 WO 9428139A1
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trna
ala
mutant
gly
leu
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PCT/US1994/005905
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Paul R. Schimmel
Eric T. Schmidt
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Massachusetts Institute Of Technology
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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
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Antibiotics are chemical substances produced by various microorganisms, including bacteria, fungi, and actinomycetes, which suppress the growth of other microorganisms and, in some cases, eventually destroy those microorganisms.
  • the term antibiotic is also used to refer to synthetic antibacterial agents which are not products of microbes, such as sulfona ides and quinolones. Since the introduction of penicillin, antimicrobial therapy has become routine. For example, today, at least 30% of all hospitalized patients receive one or more courses of antibiotic therapy (Sande, M.A. et al. , 1990, In: Goodman and Gilman's The Pharmacological Basis of Therapeutics. Eighth edition, A.G. Gilman et al.
  • the present invention relates to a method of specifically inhibiting growth of microbes, such as bacteria, fungi, or viruses.
  • specific tRNA-dependent inhibition of the growth of microbial pathogens can be achieved through use of tRNA binding molecules.
  • a tRNA binding molecule such as a mutant aminoacyl-tRNA synthetase, capable of binding tRNA, but incapable of aminoacylation, can be selectively toxic to a selected microbial pathogen, leading to inhibition of (i.e., a reduction in or arrest of) growth of the pathogen, while sparing the host cell.
  • tRNA binding molecules such as mutant aminoacyl-tRNA synthetases
  • the activity of tRNA binding molecules is tRNA-binding dependent and may result from the ability of these molecules to sequester specific tRNA, thereby rendering the tRNA unavailable to the translational apparatus.
  • the ability of specific tRNA-binding molecules to sequester microbial tRNA selectively can lead to inhibition of microbial protein synthesis, leading to inhibition of microbial growth (i.e., a reduction in or arrest of growth or death) , with minimal effect on the host.
  • the invention further relates to tRNA binding molecules useful in the method, such as a mutant aminoacyl-tRNA synthetase capable of binding tRNA, a tRNA binding fragment of an aminoacyl-tRNA synthetase, or a tRNA-binding synthetase mimetic.
  • tRNA binding molecules useful in the method such as a mutant aminoacyl-tRNA synthetase capable of binding tRNA, a tRNA binding fragment of an aminoacyl-tRNA synthetase, or a tRNA-binding synthetase mimetic.
  • the method of the present invention provides a new strategy of antimicrobial therapy useful against a large variety of pathogens, and the tRNA binding molecules represent a new class of antimicrobial agents useful in antimicrobial therapy or gene therapy of microbial infection, capable of selective inhibition of growth of pathogens and limited toxicity to the host.
  • FIG. 1 is an illustration of the secondary structure of isoleucyl-tRNA synthetase (IleRS) and of portions of the primary sequences of known IleRS and MetRS proteins.
  • IleRS isoleucyl-tRNA synthetase
  • the structure of IleRS is based on sequence alignments with the three dimensional structure of E. coli MetRS (Starzyk, R. et al., Science. 237:1614-1618 (1987); Brunie, S. et al. , J. Mol. Biol. 216(2) :411-424 (1990)).
  • the N-terminal nucleotide binding fold, C-terminal helical domain, and anticodon binding regions are labeled.
  • CP1 and CP2 refer to connective polypeptide insertions l and 2 in the nucleotide binding fold.
  • the rectangles indicate ⁇ -helices, and the pentagons indicate /3-sheets.
  • the sequence surrounding the mutations introduced into IleRS, and the corresponding sequences for four isoleucyl- and five methionyl-tRNA synthetases are shown (see also SEQ ID NO:2 through SEQ ID NO:9 and SEQ ID NO:11 through SEQ ID NO:18). Residues inside shaded boxes are conserved between Escherichia coli MetRS and the other synthetases.
  • Escherichia coli IleRS (Ec- I) (SEQ ID NO:l and SEQ ID NO:10; Webster, T.A. et al. , Science. 2 ⁇ 6:1315-1317 (1984)), Saccharomyces cerevisiae IleRS (Sc-I) (SEQ ID NO:2 and SEQ ID NO:11; Englisch, U. et al. , Biol. Chem. Hoppe-Seyler, 368:971-979 (1987)), Methanobacteri thermoautrophicum IleRS (Mt-I) (SEQ ID NO:3 and SEQ ID NO:12; Jenal, U. et al. , J. Biol. Chem..
  • Tetrahymena thermophxla IleRS (Tet-I) (SEQ ID N0:4 and SEQ ID N0:13; Csank, C. and D.W. Martindale, J. Biol. Chem.. 267_Q1:4592-4599 (1992)), Thermus thermophilus MetRS (Tmt-M) (SEQ ID NO:5 and SEQ ID N0:14; Nureki, O. et al. , J. Biol. Chem..
  • Saccharomyces cerevisiae mitochondrial MetRS Scm-M
  • Scm-M Saccharomyces cerevisiae mitochondrial MetRS
  • Sc-M Saccharomyces cerevisiae MetRS
  • Sc-M Saccharomyces cerevisiae MetRS
  • Bacillus stearothermophilus MetRS Bacillus stearothermophilus MetRS (Bst- M) (SEQ ID NO:8 and SEQ ID NO:17; Mechulam, Y. et al. , Nucleic Acids Res.. 19(13) :3673-3681 (1991)), and Escherichia coli MetRS (Ec-M) (SEQ ID NO:9 and SEQ ID NO:18; Dardel, F. , J. Bacteriol.. 160(3) :1115-1122 (1984) ) .
  • FIGS 2A-C are illustrations of the growth rates of cultures of E. coli MV1184, harboring plasmids encoding wild-type or mutant IleRS genes.
  • FIG. 2A is a graph illustrating the in vitro aminoacylation activity of the D96A IleRS mutant. The graph shows the extent of aminoacylation of purified E. coli tRNA Dc by wild-type IleRS, D96A IleRS or the mutant IleRS purified from the Mil strain. No activity above background could be detected for either the D96A mutant or the Mil mutant under the conditions of the assay.
  • Figures 4A-B are illustrations of the inhibition of wild-type E. coli IleRS activity by mutant E. coli IleRS in vitro. Wild-type aminoacylation activity was measured under conditions of increasing mutant protein concentration (o, no added protein; A, 0.1 ⁇ M mutant protein; ⁇ , 0.5 ⁇ M mutant protein; #, 1.0 ⁇ M mutant protein) .
  • Figure 4A illustrates the effect of the D96A mutant and
  • Figure 4B illustrates the effect of " the D96A/K732T double mutant.
  • FIG. 5 is an illustration of the effect of E. coli D96A or D96A/K732T mutant IleRS proteins on the extent of aminoacylation of crude bovine tRNA by a human HeLa cell extract in vitro.
  • Aminoacylation activity of the HeLa cell extract was measured by the incorporation of radioactive isoleucine into charged tRNA under the following conditions: no added inhibitor protein (•) , 1.0 ⁇ M D96A IleRS ( ⁇ ) , or in the presence of 1.0 ⁇ M D96A/K732T IleRS (A) .
  • FIG. 6 is an illustration of the growth rates of cultures of E. coli MV1184, harboring plasmids encoding mutant IleRS genes.
  • the aminoacylation of transfer RNA is catalyzed by enzymes of the aminoacyl-tRNA synthetase family.
  • the fidelity of translation depends upon the incorporation of the correct amino acid in a growing polypeptide chain in response to each codon of the mRNA. Fidelity of translation is determined in part by the correct base pairing of the anticodon of each aminoacylated tRNA with the complementary codon in the mRNA. In addition, the fidelity of translation depends upon the attachment of the proper amino acid to each tRNA, a reaction which i ⁇ catalyzed by aminoacyl-tRNA synthetases specific for each amino acid.
  • the aminoacyl-tRNA synthetase for a selected amino acid is capable of aminoacylating or "charging" each of the isoacceptor tRNAs for that amino acid (i.e. , cognate tRNAs) .
  • the aminoacyl-tRNA synthetases catalyze the esterification of an amino acid to cognate tRNA in a two- step reaction. In the first step of the reaction, the amino acid is activated by condensation with ATP to form an enzyme-bound aminoacyl-adenylate.
  • the amino acid is joined via an ester linkage to the 2'- or 3'-hydroxyl group at the 3' end of a cognate tRNA molecule.
  • the overall reaction can be expressed as follows, where AA* is a selected amino acid, tRNA' is a cognate tRNA, and PP* is pyrophosphate:
  • the aminoacylated or charged tRNA is rapidly bound by translation factors and is subsequently delivered to the ribosomes.
  • tRNA-dependent inhibition of the growth of microbial pathogens can be achieved through use of tRNA binding molecules which appear to influence the efficiency of aminoacylation.
  • tRNA synthetase-induced growth arrest has been achieved through the introduction of a mutant aminoacyl-tRNA synthetase in a microbial cell, indicating that essential functions mediated by aminoacyl-tRNA synthetases and tRNAs provide suitable targets for antimicrobial agents.
  • a tRNA binding molecule such as a mutant aminoacyl-tRNA synthetase, is constructed and introduced into a microbial cell.
  • the tRNA binding molecule When introduced into a selected microbial cell or expressed in a selected microbial cell in sufficient amounts, the tRNA binding molecule is capable of binding a specific microbial tRNA, and can be selectively toxic to the microbial pathogen, leading to inhibition of (i.e., a reduction in or arrest of) growth of the pathogen, while sparing the host cell.
  • tRNA binding molecules such as mutant aminoacyl-tRNA synthetases
  • the toxicity of tRNA binding molecules may result from the ability of these molecules to sequester specific tRNA, thereby rendering the tRNA unavailable to the translational apparatus.
  • This inhibition of microbial protein synthesis results in an inhibition of microbial growth.
  • Different tRNA binding molecules may have different inhibitory effects on microbial growth, including reduction of microbial growth or arrest of microbial growth, and/or induction of death.
  • tRNA-binding molecules are designed or selected to sequester microbial tRNA selectively with minimal effects on host cell tRNA.
  • the tRNA-binding molecules of the present " invention specifically inhibit microbial protein synthesis, thereby specifically inhibiting microbial growth.
  • Active or inactive aminoacyl tRNA-synthetase molecules may have the requisite tRNA binding capability.
  • the invention further relates to tRNA binding molecules useful in the method, such as a mutant aminoacyl-tRNA synthetase capable of binding tRNA, a tRNA binding fragment thereof, or a tRNA-binding synthetase mimetic (e.g., a peptide analog of tRNA synthetase).
  • tRNA binding molecules useful in the method such as a mutant aminoacyl-tRNA synthetase capable of binding tRNA, a tRNA binding fragment thereof, or a tRNA-binding synthetase mimetic (e.g., a peptide analog of tRNA synthetase).
  • a mutant E. coli aminoacyl-tRNA synthetase which is defective in aminoacylation, but is capable of binding tRNA, is toxic to E. coli cells.
  • an aminoacyl-tRNA synthetase defective in aminoacylation is preferred, because retention of in vivo aminoacylation activity by a mutant aminoacyl-tRNA synthetase may increase the concentration of mutant protein required for effective inhibition of microbial growth. Moreover, alterations which relax the specificity of aminoacylation or tRNA binding may result in a molecule with activity detrimental to host cells. Thus, a mutant aminoacyl-tRNA synthetase which retains specificity of binding for one or more cognate tRNAs (i.e., does not significantly bind or a inoacylate non- cognate tRNAs) is preferred.
  • inactive aminoacyl-tRNA synthetase mutants can be used in the present method, provided they retain ability to bind tRNA.
  • an inactive aminoacyl-tRNA synthetase having a defect in amino acid binding, ATP binding, aminoacyl-adenylate formation or transfer of amino acid to tRNA, or a combination of defects is useful in the method (see e.g.. Example 1 and Example 3) .
  • Mutant aminoacyl-tRNA synthetases capable of specific binding of tRNA with increased affinity may be particularly effective; however, as shown herein, a mutant isoleucyl-tRNA synthetase having a dissociation constant quite similar to wild-type isoleucyl-tRNA synthetase (0.33 ⁇ M for wild-type and 0.48 ⁇ M for D96A mutant) displayed antimicrobial activity. Although aminoacyl-tRNA synthetases normally bind to all isoaccepting tRNAs in the cell, effective inhibition of protein synthesis could be accomplished by sequestering a single tRNA isoacceptor.
  • a mutant aminoacyl-tRNA synthetase can be derived from an aminoacyl-tRNA synthetase from the same microbial organism (i.e., a homologous source) or from a different microbial organism (i.e., a non-homologous source).
  • an aminoacyl-tRNA synthetase obtained from a selected microbial cell e.g.,' E. coli
  • E. coli a selected microbial cell
  • the aminoacyl-tRNA synthetase mutant is used in a homologous system and is said to be a homologous enzyme.
  • an aminoacyl-tRNA synthetase obtained from a selected microbial cell e.g., E. coli
  • a selected microbial cell e.g., E. coli
  • a different type of microbial cell e.g., Shigella
  • the aminoacyl-tRNA synthetase mutant is used in a heterologous system and is said to be a heterologous enzyme.
  • Mutant aminoacyl-tRNA synthetases can be obtained via mutagenesis of cloned aminoacyl-tRNA synthetase genes.
  • a variety of mutagenesis strategies are possible, which may generate one or more point mutations, deletions, insertions, or truncations or combinations thereof.
  • suitable mutants can be generated by random mutagenesis (e.g., chemically induced) and screening for the desired properties.
  • suitable mutants can be obtained by random mutagenesis of a selected region, as, for instance, by cassette mutagenesis (see e.g., Clarke, N.D. et al. , Science. .240.:521-523 (1988)).
  • site- directed mutagenesis can be used to obtain mutant enzymes having a specific alteration at a selected position or within a particular region selected for its known or suspected role in a particular function (see e.g., Example 3, Burbaum, J.J. and P. Schimmel, J. Biol. Chem.. 266(26) : 16965-16968 (1991); Cusack, S. et al., Nucleic Acids Res. , 19: 3489 (1991); Moras, D. , Trends in Biochem. Sci. , 17: 159 (1992); Schimmel, P., Ann. Rev. Biochem.. 56: 125-158 (1987) , and references cited therein) .
  • the properties of a particular mutant can be characterized by a variety of methods, including in vivo complementation assays (see e.g., Examples 1 and 3), or enzymatic assay (see e.g., Examples 1-3).
  • the two steps of the aminoacylation reaction can be characterized; adenylate formation can be monitored in an assay of pyrophosphate exchange and aminoacylation can be monitored by incorporation of amino acid into charged tRNA.
  • Inactive mutants do not display activity in assays of in vitro aminoacylation activity or display little or no activity in complementation assays in vivo. Further biochemical analysis of the kinetics of amino acid, ATP and tRNA binding can be conducted.
  • the synthetase is introduced into the microbial cell and its effect on cell growth in monitored by standard methods (e.g., in culture) .
  • nucleic acid encoding the mutant protein can be inserted into an expression vector, and the vector can be introduced into a microbial cell 'in which the protein is expressed.
  • Suitable expression vectors are available for a variety of microbial systems, including bacterial, mycobacterial, and fungal vectors.
  • mutant synthetase as compared with an appropriate control (e.g., a vector control, or vector carrying wild-type synthetase) can be monitored in the target microbial cell.
  • an appropriate control e.g., a vector control, or vector carrying wild-type synthetase
  • derivatives of a selected mutant which contain one or more additional mutations in tRNA binding can be constructed, and the activity of such mutants can be compared with the original selected mutant to demonstrate the tRNA binding-dependence of inhibition (see e.g., Example 1).
  • the mutant aminoacyl-tRNA synthetase is expressed from an inducible promoter. Upon induction, the mutant synthetase is expressed in the cell. Inducible expression vectors for expression of genes in microbial systems are available. Mutant synthetases which are toxic to the microbial cell will, upon expression, cause inhibition of growth, observable as a reduction in growth rate or arrest of growth or cell death. Microbial cells susceptible to tRNA-binding molecule toxicity are suitable target pathogens.
  • the toxic activity of a mutant tRNA synthetase protein can be further characterized in vitro.
  • An extract containing the mutant tRNA synthetase is obtained.
  • the mutant enzyme may optionally be purified as needed.
  • the inhibitory activity of the mutant protein upon aminoacylation catalyzed by microbial synthetases can be monitored.
  • Microbes which are candidate target pathogens, and whose growth may be inhibited according to the method of the present invention can be identified by the ability of a mutant aminoacyl-tRNA synthetase to inhibit the extent of aminoacylation catalyzed by a microbial aminoacyl-tRNA synthetase or microbial cell extract containing aminoacyl-tRNA synthetases.
  • a tRNA binding molecule such as a mutant tRNA-synthetase to selectively inhibit microbial growth, but to spare the infected host can be assessed in vitro or in vivo.
  • a mutant aminoacyl-tRNA synthetase to inhibit the extent of aminoacylation catalyzed by a host aminoacyl-tRNA synthetase or cell extract containing aminoacyl-tRNA synthetases can be assessed.
  • the mutant aminoacyl-tRNA synthetase can be introduced into cultured host cells and assessed for toxicity.
  • nucleic acid encoding a selected mutant aminoacyl-tRNA synthetase is introduced into a host cell by means of an expression vector, and toxicity of the mutant protein is assessed.
  • Preferable tRNA binding molecules will have minimal effects on aminoacylation by host synthetases and host cell growth. Specificity of action against microbial pathogens, while sparing the host cell, due to the selectivity and specificity of binding interactions and aminoacylation activity of the selected aminoacyl-tRNA synthetase, can thereby be achieved according to the method.
  • a fragment of mutant aminoacyl-tRNA synthetase, which retains tRNA binding function, or a fragment of a wild- type aminoacyl-tRNA synthetase which is inactive but retains ability to bind tRNA can also be used in the method.
  • Suitable fragments of mutant synthetases can be identified by their toxic effect, using the in vitro or in vivo assays described above.
  • the fragments can be obtained by digestion of mutant proteins or by recombinant methods via the expression of a portion of an aminoacyl- tRNA synthetase (e.g., and N- or C-terminal fragment or derivative containing an internal deletion) .
  • site-directed mutagenesis was used to create an inactive, but toxic, E. coli aminoacyl-tRNA synthetase.
  • E. coli the aminoacyl-tRNA synthetases are essential enzymes. Based on conserved sequence and structural motifs, the twenty synthetases have been divided into two classes of ten enzymes each (see Table 1, below; Eriani G. et al. , Nature. 347:203-206 (1990);
  • Class I enzymes have a well conserved N-terminal nucleotide binding fold (Rossmann fold) responsible for amino acid binding, aminoacyl-adenylate formation and tRNA acceptor helix docking, joined to a less conserved C- terminal domain responsible for tRNA anticodon loop binding (Burbaum J.J. and P. Schimmel, Biochemistry. 30X21:319-324 (1991); Shepard A. et al. , Proc. Natl.
  • FIG. 1 illustrates the predicted secondary structure of the N-terminal domain of E. coli isoleucyl- tRNA synthetase. This representation is based on sequence alignments with the E. coli methionyl-tRNA synthetase; the three-dimensional structure of E.
  • coli methionyl-tRNA synthetase has been solved (Brunie, S. et al. , J. Mol. Graphics , 5(1) :18-21 (1987); Brunie, S. et al., J. Mol. Biol., 216(2) :411-424 (1990)).
  • the structure is comprised of alternating 0-strands and ⁇ -helices arranged in a Rossmann nucleotide binding fold.
  • a large and variable insertion designated CPl connective polypeptide l occurs between the third ⁇ -strand ( ⁇ c ) and ⁇ -helix ( ⁇ c ) and a second insertion CP2 occurs between the fourth ⁇ -stand (3 D ) and ⁇ -helix ( ⁇ D ) .
  • CPl connective polypeptide l
  • CP2 occurs between the fourth ⁇ -stand (3 D ) and ⁇ -helix ( ⁇ D )
  • aligned' sequences of the known synthetases specific for isoleucine and methionine are also shown.
  • the high degree of N-terminal sequence homology between the subgroup of class I synthetases has made possible alignments of conserved and potentially critical residues (Ghosh, G. et al. , Biochemistry, 10:9569-9575 (1991); Hou, Y-M. et al., Proc. Natl. Acad. Sci. USA. j38.:976-980 (1991);
  • An Asp52 ⁇ Ala mutation in the N-terminal domain of E. coli methionyl-tRNA synthetase results in a stable protein characterized by a greatly reduced k.,. for aminoacyl- adenylate formation (Ghosh, G. et al. , Biochemistry. 3__:9569-9575 (1991)).
  • An Asp ⁇ Ala mutation was introduced into the analogous location (i.e., at position 96) in the nucleotide-binding fold of the class I E. coli isoleucyl- tRNA synthetase (IleRS) (See Figure 1, SEQ ID NO:l; see also, the DNA sequence of E.
  • E. coli IleRS SEQ ID NO:19, an predicted protein sequence, SEQ ID NO:20, as reported by Webster, T.A. et al . , Science. 226:1315-1317 (1984;; the DNA and predicted protein sequence of E. coli IleRS (isoleucyl-tRNA ligase, ileS) , SEQ ID NO:21 and SEQ ID NO:22, as reported by Yura et al . , Nucl. Acids Research. 20(13) : 3305-3308 (1992) and entered on the Entrez Database, National Center of Biotechnology Information,
  • the mutant protein referred to herein as D96A IleRS
  • D96A IleRS was stable, ye devoid of activity, as measured by its inability to complement an ileS null tester strain (IQ844/pRMS711) .
  • IQ844/pRMS711 ileS null tester strain
  • the mutant D96A IleRS gene was inserted into an inducible expression vector for introduction into E. coli .
  • the vector was introduced into in E. coli containing a wild-type ileS chromosomal allele. Induction of expression of the inactive D96A mutant revealed a strong toxic effect (Example 1, Figure 2B) .
  • tRNA binding molecule such as an aminoacyl-tRNA synthetase mutant capable of binding charged or uncharged tRNA, i ' s that tRNA may be captured and sequestered prior to aminoacylation by the endogenous synthetase or subsequent to aminoacylation.
  • An alternative or additional contribution to toxicity is the tRNA-binding dependent deacylation or "editing" activity of IleRS (Schreier, A.A. and P.R. Schimmel, Biochemistry r JL1:1582-1589 (1972)). This hydrolytic activity causes a slow deacylation of charged tRNA De in the absence of the products (AMP and PP*) of the aminoacylation reaction.
  • the hydrolytic activity is apparently independent of the aminoacylation function.
  • the capacity of the D96A mutant to deacylate Ile-tRNA ⁇ e was demonstrated.
  • Deacylation of the non-binding D96A/K732T mutant was reduced at least 75%, presumably because of a lack of tRNA binding.
  • Expression of an aminoacylation-deficient IleRS capable of deacylating tRNA could reduce cellular levels of aminoacylated tRNA De available for protein synthesis.
  • deacylation activity may provide an additional or alternative mechanism of tRNA binding dependent toxicity of mutant synthetases. This phenomenon could be observed with other aminoacyl-tRNA synthetases which possess a deacylation function.
  • the aspartic acid residue targeted for mutagenesis in the examples is conserved in all known isoleucyl- and methionyl-tRNA synthetases and in all but one (N . cra ⁇ sa leucyl-tRNA synthetase) of the known subgroup class I synthetases (Shiba, K. and P. Schimmel, Proc. Natl Acad. Sci. USA. 89: 1880-1884 (1992); Hou, Y.-M. et al. , Proc. Natl. Acad. Sci. USA. 88: 976-980 (1991)).
  • Lys732 is conserved as a basic residue in all published isoleucyl-tRNA synthetase sequences ( Figure 1) .
  • double mutants analogous to the D96A/K732T IleRS mutant can be constructed in these IleRS genes.
  • Derivatives of a selected tRNA binding mutant, such as the D96A/K732T IleRS double mutant, which are unable to bind tRNA are useful in demonstrating the tRNA-binding dependent nature of toxicity of the selected mutant.
  • recent reports of single point mutations or deletions which selectively inactivate either tRNA binding or amino acid activation (Clarke, N.D. et al. , Science.
  • tRNA binding molecules of the present invention can be used as antimicrobial agents in antimicrobial therapy or gene therapy against microbial infection of a selected host.
  • tRNA binding-dependent reduction in growth rate or arrest of growth or cell division of microbial pathogens can ultimately lead to inviability, or can aid host defenses in effectively controlling and clearing infection.
  • the induction of microbial cell death is particularly desirable when host defenses are impaired.
  • a variety of hosts are susceptible to microbial infection, including eukaryotic hosts such as plants
  • Suitable microbial targets include bacteria, mycobacteria, Chlamydia species, Pneumocy ⁇ tis species, spirochetes, actinomycetes and fungi.
  • gram- positive and gram-negative bacteria such as Streptococcus , Staphylococcus, Neisseria , Listeria , Clostridium, Enterobacter , Proteus , Pseudomonas , Klebsiella , Salmonella, Shigella, Serratia, and Bacteroides species, Escherichia coli , Mycobacterium tuberculosis , and Mycobacterium leprae can be suitable targets.
  • Fungal pathogens such as Candida , Coccidioides , Histoplasma , Aspergillus , and Cryptococcus species can also be suitable targets.
  • Plant pathogens include bacterial pathogens such as Pseudomonas species and funga pathogens, such as the blight fungus Phytophthora infestans .
  • Viral infections are often considered to be microbial.
  • therapy according to the present invention may be possible. Effective therapy of selected microbe depends upon obtaining the requisite therapeutic benefit (e.g., inhibition of microbial growth without damaging toxicity to the host (i.e., specificity of action against the microbe) .
  • a given microbial infection can be treated according to the present invention when a tRNA binding molecule has the requisite toxicity against the microbial pathogen, while sparing th host.
  • One application of the invention is in controlling o killing microbial contaminants present in mammalian cell culture.
  • specific inhibition of growth of the microbial pathogen is attained with minimal toxicity to the cultured cells.
  • the therapeutic approach of the present invention has other applications.
  • the approach can be applied in the treatment of parasitic diseases (e.g., protozoan infections, such as amebiasis, giardiasis, leish aniasis, malaria, trypanosomiasis or metazoan nematode, cestode or trematod infections, such as schistosomiasis) , where a tRNA bindin molecule with appropriately selective activity is available.
  • parasitic diseases e.g., protozoan infections, such as amebiasis, giardiasis, leish aniasis, malaria, trypanosomiasis or metazoan nematode, cestode or trematod infections, such as schistosomias
  • a tRNA binding molecule is introduced into target cells which have been introduced into the host for a selected purpose
  • cells may be introduced into a host for immunization (i.e., to stimulate an immune response) or cells may be engineered for purposes of gene therapy and introduced into a host cell. Subsequently, it may desirable to eradicate these cells.
  • a tRNA binding molecule capable of selective toxicity to the introduced target cells in the host, is introduced into the target cells.
  • the introduced cells may be cells of the same species as the host (e.g., lymphocytes engineered for purposes of gene therapy, tumor cells, fetal cells, allografts) , growth of these cells may be inhibited according to the present method, given a tRNA binding molecule with appropriately selective activity.
  • the cell introduced into the host is engineered to contain a tRNA whose function is essential to viability of the cell (e.g., because it is required for translation in general or because it is required for translation of a particular protein, such as one containing a nonsense mutation, which is essential to viability of the cell) , yielding a target cell against which a selected tRNA binding molecule may be selectively toxic.
  • a tRNA binding molecule such as an inactive mutant aminoacyl-tRNA synthetase or tRNA binding synthetase fragment is introduced into the microbial cell.
  • a nucleic acid encoding the mutant protein can be introduced into the microbial cell. Expression of the nucleic acid and production of the toxic tRNA binding molecule will inhibit growth of the microbial cell.
  • the nucleic acid can be introduced by means of an expression vector capable of directing the expression of the nucleic acid encoding the mutant protein. Suitable expression vectors, which may be inducible or constitutive, are available for a variety of microbial systems, including bacterial, mycobacterial, and fungal vectors.
  • the vector may be delivered to the microbe by packaging in a phage or virus capable of delivering the vector to the microbial cell.
  • synthetically or recombinantly produced tRNA binding molecules can be administered to the host by a suitable route, either alone or in combination with another drug.
  • routes of administration are possible including, but not necessarily limited to oral, dietary, topical, parenteral (e.g., intravenous, intramuscular, subcutaneous injection) routes of administration.
  • routes of administration are possible including, but not necessarily limited to oral, dietary, topical, parenteral (e.g., intravenous, intramuscular, subcutaneous injection) routes of administration.
  • Formulation of a tRNA binding molecule such as a mutant aminoacyl-tRNA synthetase or a tRNA binding fragment of a synthetase, will vary according to the route of administration selected (e.g.
  • composition can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science. 16th Edition, Mack, Ed. 1980) .
  • the molecule can be fused to a moiety (e.g., a moiety capable of binding a specific surface molecule or receptor) which facilitates uptake by microbial cells.
  • a moiety e.g., a moiety capable of binding a specific surface molecule or receptor
  • vesicles e.g., microparticulates or colloidal carriers composed of proteins or lipids, such as liposomes
  • vesicles can facilitate delivery of drug to the microbial pathogen (see Langer, R. , Science. 249: 1527- 1533 (1990) .
  • Vesicles may be targeted passively, in which case, they a taken up naturally by cells that scavenge foreign microparticulates, such as reticuloendothelial cells (macrophages) .
  • Active targeting of vesicles is accomplished by placing a recognition sequence (e.g., an antibody or other molecule capable of binding the target microbial cell) onto the vesicle to obtain efficient uptake by the microbial cell.
  • a recognition sequence e.g., an antibody or other molecule capable of binding the target microbial cell
  • Controlled release systems e.g., polymeric systems
  • protein- based tRNA binding therapeutics can also be used with protein- based tRNA binding therapeutics.
  • a tRNA binding molecule of the present invention can be introduced into host phagocytic cells by means of a suitable vector (e.g., a retroviral vector) .
  • the vector will direct the constitutive or inducible expression of the tRNA binding molecule, which is then taken up by the intracellular pathogen.
  • Various strategies to facilitate uptake by the pathogen are possible, including fusion of the tRNA binding molecule to a moiety which mediates uptake of the molecule by the pathogen.
  • introduction of the tRNA binding molecule by engineering the host cells would be an appropriate strategy.
  • a variety of plant vectors are available for transformation of plants.
  • the target is a viral pathogen
  • the tRNA binding molecule is also introduced into host cells by administering the tRNA binding molecule to the host or by introducing it into host cells via gene therapy.
  • a therapeutically effective amount of a tRNA binding molecule of the present invention is administered or introduced into cells.
  • a therapeutically effective amount is one which results in a level of tRNA binding molecule in the microbial pathogen sufficient to achieve the desired effect of inhibiting microbial growth, without undue toxicity to the host (e.g., which results in a level of tRNA binding molecule in the microbial pathogen sufficient for the molecule to be selectively toxic to the microbial pathogen present in the host) .
  • a therapeutically effective amount is one which results in a level of tRNA binding molecule in the host cell sufficient to achieve the desired effect of inhibiting viral growth, without undue toxicity to the host.
  • E. coli K-12 strain TGI (supE, h ⁇ dA5 , thi, A(lac- proAB) /F' [traD36 , proAB + , 2acJ q , IacZ ⁇ M15], from Amersham, UK) was used as a host strain for site-directed mutagenesis.
  • coli K-12 strain MV1184 (ara , A[lac- proAB ] , rspL, thi , [ 80 lacZA l ⁇ ] , A[srl-recA] , 306: :Tnl0[tet r 7/F' [traD36, proAB + , lad , la ⁇ Z ⁇ Ml ⁇ ]) was used as a host in tRNA synthetase expression experiments. These cells contain an F' episome carrying the lacl q gene which gives low levels of lac promoter activity in the absence of IPTG. E.
  • the ileS mutation present in this strain decreases amino acid binding of the chromosomally encoded protein to undetectable levels at low isoleucine concentrations ( ⁇ 100 ⁇ M) , thus allowing for biochemical analysis of phagemid- encoded IleRS proteins expressed in this background.
  • the pKS21 phagemid (Shiba, K. and P. Schimmel, Proc. Natl. Acad. Sci. USA. 89:1880-1884 (1992)), a derivative of pTZ19R (Pharmacia) , encodes wild-type IleRS and was used as a template for site-directed mutagenesis.
  • the phagemid allows for inducible expression of IleRS from the lac promoter and contains the ⁇ -lactamase gene conferring ampicillin resistance.
  • Phagemids were transformed into MV1184 cells and overnight cultures were grown from single colonies and then diluted 1:150 into LB/ampicillin. Two separate cultures were grown at 37°C for each IleRS mutant. One of the cultures was induced with 1 mM IPTG (Sigma) at O. O . 6O0 K 0.250. An equal volume of water was added to the other. Growth was monitored for up to 11 hours by measuring cell densities by OD ⁇ on a Beckman DU-64 spectrophotometer.
  • Standard aminoacylation reactions were performed as described by Shepard et al. (Proc. Natl. Acad. Sci. USA. i8_9:9964-9968 (1992)), except that assays were performed at ambient temperature using 1 nM enzyme and 5 ⁇ M purified tRNA De (Subriden RNA) .
  • the aminoacylation reaction employing various amounts of mutant inhibitory IleRS was performed similarly using 2 nM wild-type IleRS, 100 nM purified tRNA ⁇ and 0 to 1 ⁇ M mutant IleRS.
  • the reaction cocktail (everything except wild-type and mutant IleRS) was pre-incubated at ambient temperature. Mutant proteins were added and the sample was mixed. Wild-type protein was added soon after followed by further mixing.
  • the filter binding reaction mixture (100 ⁇ L) contained 20 mM HEPES (pH 7.5), 0.1 mM EDTA, 0.15 M NH 4 Cl, 100 ⁇ g/ml bovine serum albumin, 4 mM MgCl 2 , and 25 nM IleRS. After pre-incubation at ambient temperature [ 3 H]-Ile-tRNA ⁇ e wa s added at 25 to 5000 nM. The entire reaction was filtered through a 24 mm nitrocellulose disk (Schleicher and
  • the D96A IleRS mutant is inactive
  • the activity of the D96A mutant was tested in a complementation assay in the ileS null tester strain
  • This tester strain carries a chromosomal null mutation in the ileS gene, and contains a copy of ileS on a temperature-sensitive maintenance plasmid. Mutant or wild-type phagemid was introduced into the tester strain by electroporation. Transformants were plated directly at the permissive (30°C) or nonpermissive (42°C) temperature and scored for growth. Although mutant phagemid readily produced transformants on plates at the permissive temperature, no transformants were produced at 42°C. In contrast, wild-type phagemid produced equal numbers of transformants at nonpermissive and permissive temperatures.
  • mutant or wild-type IleRS genes were introduced by transformation into strain MV1184, which contains a wild-type chromosomal ileS allele.
  • the growth of MV1184 cultures containing plasmids encoding the wild-type or D96A mutant were monitored under induced or uninduced conditions.
  • Induction of wild-type IleRS had little or no effect on the growth of MV1184 cells, demonstrating that over-production of the enzyme per se is not toxic ( Figure 2A) .
  • induction of D96A IleRS stopped cell growth within two hours ( Figure 2B) .
  • the dominant lethality of the D96A mutant is most likely related to the ability of its intact C-terminus to bind tRNA De . If the mutant tRNA synthetase, unable to catalyze aminoacylation, still bound its cognate tRNA ⁇ e with near wild-type affinity, the mutant could sequester tRNA from the endogenous wild-type synthetase and the cellular protein synthesis machinery. Further experiments were conducted to determine whether tRNA De is the in vivo target responsible for the toxicity of D96A IleRS. A lysine to threonine mutation at position 732
  • K732T was placed in the C-terminal domain of the D96A mutant enzyme in order to disrupt the ability of the mutant to bind tRNA De .
  • the K732T mutation increases the K,-, for tRNA Dc in the aminoacylation reaction by 225-fold (Shepard, A. et al. , Proc. Natl. Acad. Sci. USA. 9:9964- 9968 (1992)).
  • the resulting double mutant D96A/K732T IleRS also accumulated in vivo but was unable to complement the IQ844/pRMS711 null strain.
  • Mutant IleRS proteins were expressed from multiple copy plasmids (15-20 copies/cell) under the control of the inducible lac promoter. Under these conditions, a concentration of mutant protein relative to the chromosomally encoded wild-type protein sufficient to be toxic to the microbial cell was obtained. Toxicity due to sequestration of tRNA De probably requires overexpression of the mutant synthetase, because in vivo ratios of tRNA ⁇ e :IleRS for E. coli are about 4 to 1 (Jakubowski, H. and E. Goldman, J. Bacteriol.. 158(3) :769-776 (1984)).
  • a mutant IleRS with similar tRNA binding capacity, would need to be overexpressed at least 4-fold relative to endogenous E. coli synthetase in order to titrate a significant amount of tRNA ⁇ e in vivo sufficient to achieve the desired effect of inhibiting microbial growth.
  • the pKS21-encoded D96A and D96A/K732T mutant enzymes were expressed and purified from Mil cells, which contain a mutant chromosomal ileS allele (see above) .
  • the Mil- encoded IleRS has a high Kön. for isoleucine (>100 ⁇ M) and is able to maintain cell viability in vivo only when cell media are supplemented with isoleucine (laccarino, M. and P. Berg, J. Bacteriol.. 105:527-537 (1971)).
  • the activity of the Mil mutant enzyme is not detectable under normal in vitro assay conditions which employ 20 ⁇ M isoleucine. This feature of the strain permits the direct biochemical analysis of mutant proteins upon over-production and purification from Mil cells.
  • Figure 3 shows that, under the conditions of the aminoacylation assay, no activity could be detected for either the D96A mutant or the Mil endogenous mutant enzyme. Additional experiments showed that the D96A mutant had a severely reduced k c . t for aminoacyl-adenylate formation. ' -
  • a nitrocellulose filter binding assay was employed to determine the apparent dissociation constant of the IleRS'Ile-tRNA ⁇ e complex at pH 7.5.
  • the aminoacylated form of tRNA De was used for these experiments because it is the predominant form of tRNA in vivo (Yegian, D. et al.. Proc. Natl. Acad. Sci. USA. 55:839-846 (1966)) and the amino acid provided a convenient radiolabel.
  • These experiments indicated similar dissociation constants of 0.33 ⁇ M and 0.48 ⁇ M for the wild-type and D96A mutant binding complexes, respectively.
  • the double mutant D96A/K732T IleRS had a K d for complex dissociation which was too high (> 3 ⁇ M) to be measured by this assay.
  • HeLa cell extracts were prepared after the method of Pearson et al. (Pearson et al. , Biochim. Biophvs. Acta. 294:236 (1973); describing preparation of crude extracts from calf liver) .
  • the HeLa cell pellet from a one liter culture was resuspended in 10 ml of 0.5 M Tris-Cl (pH 7.5), 10 mM MgCl 2 , 10 mM S-mercaptoethanol, 0.1 M KCl, 1 mM EDTA, 0.25 M sucrose, and 15% glycerol. The suspension was centrifuged at 30,000 rpm for 45 minutes.
  • the resulting supernatant was diluted 5-fold with phosphate buffer (10 mM potassium phosphate (pH 7.5), 20 mM ⁇ - mercaptoethanol, 1 mM EDTA, 4 mM MgCl 2 , 15% glycerol, and 0.1% PMSF) and loaded onto a DEAE-cellulose column equilibrated in phosphate buffer. The column was washed with 60 ml of the same buffer.
  • phosphate buffer 10 mM potassium phosphate (pH 7.5), 20 mM ⁇ - mercaptoethanol, 1 mM EDTA, 4 mM MgCl 2 , 15% glycerol, and 0.1% PMSF
  • Proteins were eluted from the column with 0.25 KCl, lOmM potassium phosphate (pH 6.5), 1 mM /3-mercaptoethanol, 5 mM MgCl 2 , l mM EDTA, 0.1 mM ATP, 50% glycerol, and 0.1% PMSF. Fractions were collected and OD 280 was measured for each fraction. Fractions containing a high concentration of protein were used for charging assays. Fractionated extracts were stored at -20°C.
  • Aminoacylation assays were performed essentially as described in Example 1, except that a HeLa cell extract and crude ⁇ calf liver (bovine) tRNA were used. To determine the effect of mutant E. coli IleRS proteins on aminoacylation, the assay was performed in the presence of 1.0 ⁇ M D96A IleRS protein or 1.0 ⁇ M D96A/K732T IleRS protein.
  • coli IleRS did not aminoacylate calf liver tRNA under the conditions of the assay. Additional aminoacylation assays were performed using crude E. coli tRNA and E. coli IleRS under identical conditions. In these assays, the D96A IleRS mutant inhibited aminoacylation by 65% and the D96A/K732T IleRS mutant inhibited aminoacylation by 15%, indicating that specific inhibition can be observed using crude or fractionated tRNA in the E. coli system. These results indicate that species specific inhibition of microbial tRNA function can be achieved with tRNA binding molecules.
  • IleRS E . coli isoleucyl-tRNA synthetase
  • the enzyme must discriminate between the amino acids isoleucine and valine based solely on the extra ethylene group of isoleucine.
  • affinity labeling of active site peptides and mutagenesis of possible critical residues in these peptides was carried out.
  • a mutation of IleRS which confers isoleucine auxotrophy in an E. coli strain has been identified. Affinitv labeling and site-directed mutagenesis
  • the affinity label bromoacetylated-Ile-tRNA 110 (Santi, D. et al. , Biochem. Biophys. Res. Commun.. 51: 370-375) was used to probe for residues interacting with the isoleucyl moiety of charged tRNA De . Two tryptic peptides beginning with Thr 50 and lie 452 were identified as being reactive toward the affinity label.
  • the peptide beginning with Thr 50 contains the highly conserved signature sequence ending in HIGH (His-Ile-Gly- His, according to the standard single-letter amino acid code) found in class I synthetases, as well as a conserved proline previously identified as important in ethionine binding by the class I methionyl-tRNA synthetase (Burbaum, J. and P. Schimmel, Protein Science. 1:575-581 (1992)). Mutagenesis of other residues in the peptide has identified a Gly56-Ala mutation (G56A) which increases the K,-, for amino acid binding by 1750-fold (Table 2) . Further mutagenesis of residues in the tryptic peptide beginning with Thr 50 may identify additional residues involved in isoleucine binding.
  • the peptide beginning with lie 452 contains a conserved peptide sequence DWCISR (Asp-Trp-Cys-Ile-Ser-Arg, according to the standard single-letter amino acid code) , which contains a cysteine residue previously affinity labeled by an isoleucine analog (Rainey, et al. , Eur. J. Biochem. , .63.: 419-426) . Extensive mutagenesis of the region of the IleRS gene encoding this peptide has led to the identification of several residues critical for amino acid binding and transfer of the activated amino acid to tRNA ⁇ e . Several conservative point mutations in the
  • DWCISR sequence significantly reduced enzymatic activity. Residues especially sensitive to change include Trp 462 , Arg 466 , and Arg 468 .
  • the results of the biochemical characterization of Trp462 ⁇ Phe (W462F) , Arg466 ⁇ Gln (R466Q) , and Arg468 ⁇ Gln (R468Q) IleRS mutant proteins are shown in Table 2 below.
  • Trp462 ⁇ Phe (W462F) , Arg466 ⁇ Gln (R466Q) , and Arg468 ⁇ Gln (R468Q) IleRS mutant proteins retain the deacylation or editing function which can contribute to toxicity, suggesting that they bind tRNA, and are candidate toxic tRNA binding molecules with antimicrobial utility.
  • Val-AMP enzyme-bound misactivated valyl-adenosine monophosphate
  • Mutant or wild-type enzyme was isolated as described in Example 1. Reactions contained 150 mM tris-HCl (pH 7.5), 10 mM MgCl 2 , 200 mM valine, 3 mM [7 ⁇ 32 P]ATP (10-20 cpm/pmol) , pyrophosphatase (2 U/ml) , 14 ⁇ M tRNA De , and 2.8 ⁇ M enzyme. Reactions were assayed at 25 °C for up to 20 minutes and were quenched with four volumes of 7% HCI0 4 . Activated charcoal containing 10 mM sodium pyrophosphate was added, and ATP was separated by filtration through glass fiber pads (Schleicher and Schuell) .
  • ATP consumption (p ol ATP remaining/pmol enzyme) in the presence or absence of tRNA ⁇ e was determined over time (minutes) . It was determined that the mutant enzyme was as active as the wild-type enzyme in overall pre- and post-transfer ATP hydrolysis induced by the addition of tRNA" 6 . In particular, the tRNA ⁇ °-dependent rate of hydrolysis of Val-AMP was measured as 2.7 s" 1 in both cases. (Although the G56A mutant showed more ATP hydrolysis in the absence of tRNA De than did the wild-type enzyme when the assay was carried out for longer time periods, this hydrolysis represented only 15% or less of the tRNA ⁇ c -dependent hydrolysis by the mutant.)
  • misacylated [ 3 H]valine-tRNA De was purified through a series of phenol-chloroform extractions and ethanol precipitations. Deacylation reactions, performed at 25 °C, contained 150 mM tris-HCl (pH 7.5), 10 mM MgCl 2 , 3.25 ⁇ M [ 3 H]valine-tRNA De , (2170 cpm/pmol) , pyrophosphatase (4 U/ml) , and 5.2 nM enzyme.
  • E. coli strain Mil contains a chromosomal mutation in the JleS gene which is responsible for the isoleucine requirement of the strain.
  • the IleS mutant gene was amplified using PCR, and a single amino acid substitution (Phe570 ⁇ Ser or F570S) has been identified as the likely cause of auxotrophy.
  • Phe 570 is within the nucleotide-binding fold at the beginning of the fourth (or D) helix, and, on the basis of the alignment of this region with methionine tRNA synthetase (Shiba, K. and P. Schimmel, Proc. Natl. Acad. Sci. U.S.A., 89: 1880 (1992)), also contributes to the formation of the amino acid binding site (Ghosh, G. et al . , Biochemistry. 30: 9569 1991) ) .
  • the resulting mutant enzyme has a K,-, for isoleucine which is elevated -2000-fold as compared with the wild-type enzyme (Table 3).
  • the K bulk. for valine was greater than 200 mM.
  • coli IleRS can be identified by sequence similarity to other IleRS enzymes; phenylalanine is strictly conserved among all known isoleucyl-tRNA synthetases and is not found in any other class I tRNA synthetase. It is likely that Phe 570 forms part of the amino acid binding pocket of IleRS that specifically accommodates the isoleucine side chain. Construction of similar substitutions (e.g. , Phe to Ser) at the corresponding residue in other IleRS proteins is predicted to cause analogous amino acid binding defects. Construction of mutations close to or adjacent to positio 570 in E. coli IleRS, or to the corresponding residue in other IleRS proteins, may yield other mutants defective i tRNA binding.
  • genes encoding the Gly56->Ala (G56A) mutant or the Phe570 ⁇ Ser (F570 ' S) mutant were introduced by transformation into strain MV1184, which contains a wild-type chromosomal ileS allele.
  • the growth of MV1184 cultures containing plasmids encoding the G56A or F570S mutants were monitored essentially as described in Example 1, under both induced (+ 500 ⁇ M IPTG induction at 3.0 hours) or uninduced conditions. Induction of expression of either the G56A or the F570S IleRS mutant stopped cell growth, although the effect of the G56A mutant was more pronounced ( Figure 6) .
  • Toxicity to microbial cells by over-expression of the F570S mutant may be enhanced under conditions where, for example, isoleucine is limiting.
  • Trp Tyr lie Arg Leu lie Arg Ser Arg Thr Trp 1 5 10
  • Asn Trp Tyr lie Arg Leu Asn Arg Asn Arg Leu Lys 1 5 10
  • ACG CCA AAG CGG ACA GTG TGG GCG CGT CGT AGC TGC CAG ACT GCG CTA 225 Thr Pro Lys Arg Thr Val Trp Ala Arg Arg Ser Cys Gin Thr Ala Leu 740 745 750
  • GGT GGC TCG CTG GAA GCG GCA GTA ACC TTG TAT GCA GAA CCG GAA CTG 2544 Gly Gly Ser Leu Glu Ala Ala Val Thr Leu Tyr Ala Glu Pro Glu Leu 835 840 845
  • MOLECULE TYPE protein

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Abstract

La présente invention concerne un procédé pour inhiber spécifiquement la croissance de microbes, tels que des bactéries, des champignons, ou bien des virus, et des compositions utiles pour ledit procédé. Selon ce dernier, l'inhibition spécifique, ARNt-dépendante, de la croissance d'agents pathogènes microbiens peut s'effectuer à l'aide de molécules fixant l'ARNt. Par exemple, une molécule fixant l'ARNt, telle qu'une aminoacyl-ARNt synthétase mutante, qui est susceptible de fixer l'ARNt, mais non susceptible d'aminoacylation, peut présenter une toxicité sélective vis-à-vis d'un agent pathogène microbien choisi, entraînant l'inhibition (c'est-à-dire une réduction ou un arrêt) de la croissance de l'agent pathogène, tout en épargnant la cellule hôte.
PCT/US1994/005905 1993-05-28 1994-05-25 INHIBITION, DEPENDANTE DE LA FIXATION D'ARNt, DE LA CROISSANCE PATHOGENE MICROBIENNE WO1994028139A1 (fr)

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US5985630A (en) * 1993-10-06 1999-11-16 Zeneca Limited Assay for detecting inhibitors of aminoacyl-tRNA synthetases
WO1997026354A1 (fr) * 1996-01-19 1997-07-24 Smithkline Beecham Plc SYNTHETASE D'HISTIDYL-ARNt ISSUE DE STAPHYLOCOCCUS AUREUS
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EP0785269A1 (fr) * 1996-01-19 1997-07-23 Smithkline Beecham Plc Histidyl-tRNA synthétase de Staphylococcus Aureus
EP0785272A1 (fr) * 1996-01-19 1997-07-23 Smithkline Beecham Plc Prolyl-ARNt synthétase de Staphylococcus Aureus
EP0785261A1 (fr) * 1996-01-19 1997-07-23 Smithkline Beecham Plc Glutamyl-ARNt synthétase de Staphylococcus Aureus
EP0785265A1 (fr) * 1996-01-19 1997-07-23 Smithkline Beecham Plc Synthetase d'aspartyl ARNt de Staphylococcus Aureus
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