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WO2001055351A1 - Cellule eucaryote exprimant une formylase bacterienne et ses utilisations - Google Patents

Cellule eucaryote exprimant une formylase bacterienne et ses utilisations Download PDF

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WO2001055351A1
WO2001055351A1 PCT/US2001/002720 US0102720W WO0155351A1 WO 2001055351 A1 WO2001055351 A1 WO 2001055351A1 US 0102720 W US0102720 W US 0102720W WO 0155351 A1 WO0155351 A1 WO 0155351A1
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Prior art keywords
formylase
bacterial
cell
growth
inhibited
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Ramesh Vaidyanathan
U. L. Rajbhandary
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Massachusetts Institute of Technology
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • antibiotics during the first half of the tw entieth century revolutionized the treatment of bacterial infections Most antibiotics exploit some difference between the physiology or metabolism of bacterial cells and cells of the host in order to selectively inhibit the growth of bacte ⁇ a while having minimal effects on the host
  • penicillin specifically prevents synthesis of the bacte ⁇ al cell wall, a structure necessary for the survival of many bacte ⁇ al species Since eukaryotic cells lack cell walls, penicillin has little effect on their viability
  • tetracychnes are believed to inhibit bacte ⁇ al protein synthesis by binding to the 3 OS subunit of the bacterial ⁇ bosome and preventing the ammoacyl tRNA from gaming access to the acceptor (A) site on the mRNA- ⁇ bosome complex as described on p 1125 of Hardman, J G et al (eds ), Goodman and Gilman 's The Pharmacological Basis of Therapeutics, 9th ed , New York, 1996) The contents of this work are incorporated herein in their entirety
  • a major problem in the treatment of bacterial infection is the emergence of strains of bacteria that are resistant to currently available antibiotics.
  • Drug resistance can arise due to mutations in the drug's target, e.g., a ribosomal subunit. Bacteria can also develop more effective ways to metabolize the antibiotic to an inactive form or to pump it out of the cell. Drug resistance is a genetic trait, and bacteria can transfer drug resistance genes both between members of the same species and even across species barriers. When bacteria acquire resistance to one member of a given antibiotic class, they are frequently resistant to all members of that class. Therefore, there is an urgent need for new classes of antibiotics that would work by a fundamentally different mechanism.
  • the present invention provides a system for the expression of formylase in a eukaryotic cell and encompasses the inventors' finding that such expression can confer a detectable phenotype on the cells, so that inhibitors of formylase can be identified through assessment of changes in the detectable phenotype.
  • the invention provides an in vivo system for identifying inhibitors of formylase.
  • the invention provides a eukaryotic cell containing formylase or a biologically active analog thereof.
  • the presence of formylase inside the eukaryotic cell alters a detectable characteristic of the cell, e.g., growth rate. In some preferred embodiments the alteration is conditional (dependent upon, for example, temperature, composition of growth media, etc.).
  • the eukaryotic cell can be, for example, a yeast cell or a mammalian cell.
  • the invention further provides a method of identifying inhibitors of bacterial formylase.
  • Inhibitors are identified by contacting test agents with eukaryotic cells containing formylase and determining whether such agents selectively alter a detectable characteristic, e.g., growth, of cells containing formylase. For example, agents that selectively alter a characteristic of a eukaryotic cell containing formylase to more closely resemble that characteristic as manifested in control cells not containing formylase are identified as inhibitors of formylase. Since bacteria lacking formylase grow extremely poorly, inhibitors of formylase have potential as useful antibacterial agents.
  • the invention provides compositions comprising inhibitors of formylase for the inhibition of bacterial growth.
  • the present invention provides pharmaceutical compositions comprising inhibitors of formylase for the treatment of bacterial infections.
  • the pharmaceutical compositions further comprise other antibacterial agents, delivery vehicles, etc.
  • 'Agent' refers to any chemical compound or combination of chemical compounds including biological macromolecules such as nucleic acids, polypeptides, lipids, and polysaccharides.
  • Bioly active analog' refers to a portion of an agent or a modified version of an agent that possesses at least one activity characteristic of that agent.
  • modifications include but are not limited to amino acid changes (addition, deletion, substitution) and glycosylation.
  • 'Constitutive' refers to a cellular event or condition such as expression of an RNA or protein, that occurs regardless of the presence or absence of a specific inducer.
  • Control cell' as used herein is used to refer to a cell that has not undergone a given manipulation or manipulations and/or is not exposed to a given condition.
  • control cells serve as controls for cells that have been subjected to a given manipulation or manipulation(s) and/or been exposed to a given condition.
  • eukaryotic cells of a particular cell type or strain are modified to express bacterial formylase.
  • Eukaryotic cells of the same cell type or strain or population as those so modified, but that do not express bacterial formylase are suitable control cells.
  • the cells that serve as a control for cells that express formylase may be untransformed cells of the same cell type or strain or may be transformed cells containing the same vector as that used to cause expression of formylase, but without the insert that encodes formylase.
  • suitable control cells are substantially identical with respect to at least 90%, more preferably at least 95%, yet more preferably at least 99%, and most preferably 100% of all detectable phenotypic or genotypic characteristics except for those altered either directly or indirectly by the manipulation or condition to which the cells for which the control cells are to act as a control have been subjected.
  • DEFE133A refers to a gene encoding bacterial (E. coli) deformylase containing a mutation that converts the glutamic acid (E) residue at position 133 to alanine (A).
  • DEFE133A also refers to a bacterial deformylase protein containing this mutation, as will be clear from context. It has been reported (Meinnel T., Lazennec C, and Blanquet S., J. Mol. Biol, 254, 175-183, 1995) that the rfe/E133A mutant fails to complement the temperature sensitive phenotype of an E. coli strain deficient in formylase, suggesting that DEFE133A is inactive with respect to deformylase activity.
  • cfe/WT and "de 5"as used herein refers to a gene encoding wild type bacterial (E. coli) deformylase.
  • D ⁇ FWT refers to a wild type bacterial deformylase protein, as will be clear from context.
  • Detectable characteristic' refers to any feature of the phenotype of an organism that can be observed or measured, either quantitatively or qualitatively, using an appropriate assay. Detectable characteristics of particular relevance to this invention include growth rate, rate of synthesis of particular proteins, and rate of metabolism of particular substrates.
  • 'Exogenous' refers to a nucleic acid or polypeptide that is not found in nature in the organism under study. Expression of exogenous genes or proteins is typically achieved by DNA transfer.
  • 'Expression vector' as used herein refers to a vector having a site for insertion of a DNA segment and genetic control elements so positioned as to direct transcription of the inserted DNA segment when the expression vector is introduced into an appropriate cell or cell-free transcription system.
  • 'Formyl group' refers to the chemical structure -COH.
  • 'Formylase' refers to an enzyme that transfers a formyl group to another organic molecule.
  • the preferred formylases referred to herein transfer a formyl group to the amino acid methionine, when the methionine is linked to an initiator tRNA. Transfer of a formyl group to a target molecule is known as formylation.
  • the terms formylase, methionyl-fRNA formyltransferase, 10- formyltetrahydrofolate:L-methionyl-tRNA Met N-formyltransferase, and methionine transformylase will be used interchangeably herein to refer to an enzyme that has this activity.
  • 'Growth' refers to an increase in cell number or an increase in overall cell mass or in any combination of these.
  • High copy vector' refers to a vector that is typically maintained at many copies per cell, e.g., between 10 to 40 or more copies per cell.
  • yeast for example, such vectors typically contain a genetic element including an origin of replication and other sequences from an endogenous episome called the 2 micron circle.
  • High copy vector capable of replicating in mammalian cells typically include a viral origin of replication.
  • vector pREP7 contains the Epstein-Barr virus origin of replication.
  • 'Inducer' refers to a substance, typically a small molecule such as a sugar or amino acid, that is able to positively regulate a cellular process, e.g., expression of a gene or protein. For example, it is frequently the case that the presence of a substrate acts as an inducer to induce the synthesis of an enzyme capable of metabolizing the substrate.
  • 'Inducible' refers to a cellular event or condition such as expression of an RNA or protein, that occurs in response to an inducer, e.g., galactose. Inducible also refers to a gene or protein whose expression can be induced or to a genetic control element whose operation is increased in response to an inducer.
  • inducers include compounds ranging from ethanol to drugs such as barbiturates.
  • barbiturates induce enzymes responsible for their own metabolism, including liver oxidases and glucuronyl transferase.
  • 'Low copy vector' refers to a vector that is typically maintained at 1 to 2 copies per cell.
  • yeast such vectors typically contain a genetic element comprising an origin of replication and centromeric sequences.
  • 'MTF' refers to formylase as defined above.
  • Wild DEF or “mutant deformylase” refers to a nucleotide sequence encoding bacterial (E. coli) deformylase that differs in at least one nucleotide from the wild type bacterial deformylase sequence or to an amino acid sequence encoding bacterial deformylase that differs in at least one amino acid from the wild type bacterial deformylase.
  • Wild MTF or “mutant formylase” refers to a nucleotide sequence encoding MTF that differs in at least one nucleotide from the wild type MTF sequence or to an amino acid sequence encoding MTF that differs in at least one amino acid from the wild type MTF.
  • MTFR42L' refers to a gene encoding MTF containing a mutation that converts the arginine at position 42 in the wild type protein to leucine.
  • MTFR42L also refers to a formylase protein containing this mutation, as will be clear from context. This mutant formylase protein (MTFR42L protein) lacks detectable formylase activity in vitro (Ramesh, V., et al., Biochemistry:, 37(45), 15925-15932, 1998).
  • 'Operably linked' refers to a relationship between a DNA sequence and a genetic control element such as a promoter or enhancer, such that the DNA sequence and genetic control element are arranged in three dimensional space so that the genetic control element controls and regulates the transcription and/or translation of the gene.
  • a genetic control element such as a promoter or enhancer
  • the genetic control element and the DNA sequence are covalently linked.
  • the genetic control element is a promoter
  • the promoter directs expression of the DNA sequence.
  • a promoter typically contains sites capable of binding RNA polymerase within a cell, leading to initiation of transcription of a downstream (3') sequence.
  • Transformation refers to a cell into which an exogenous DNA molecule has been introduced, or a cell derived from such a cell that contains one or more copies of the DNA molecule. Transformation may occur under natural conditions or may be artificially induced using a variety of techniques well known in the art. Both prokaryotic and eukaryotic cells may be transformed, and particular transformation methods may be selected based upon the cell type being transformed. Such techniques include electroporation, viral infection, lipofection, lithium-acetate or calcium-phosphate mediated transformation, heat shock, etc.
  • Cells can be stably transformed, in which the introduced DNA replicates either autonomously or as part of the cell's genomic DNA, or transiently transformed, in which the introduced DNA may be maintained for variable periods of time but may eventually be lost.
  • 'Vector' refers to a nucleic acid molecule capable of mediating introduction of another nucleic acid to which it has been linked into a cell.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication.
  • Preferred vectors are those capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell.
  • Vectors capable of directing the expression of inserted DNA sequences are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids". Plasmids are circular, double-stranded DNA molecules which, in their vector form, are not bound to or integrated into the chromosome.
  • vector is sometimes applied in the present specification to refer to particular plasmids, however it should be understood that the term “vector” encompasses additional, non-plasmid, molecules.
  • the invention includes such other forms of expression vector which serve equivalent functions and which become known in the art subsequently hereto.
  • 'Wild type' refers to a gene or protein as it is normally found in nature in the organism from which it was originally isolated. Unless otherwise specified, genes and proteins discussed herein are assumed to be wild type.
  • Figure 1 shows the sequence of the E. coli tRNA 2 fMet , to which the initiator methionine is attached in E. coli, and the yeast tRNA, Met , to which the initiator methionine is attached in S. cerevisiae. Nucleotides playing a major role in the formylation of the bacterial initiator tRNA are indicated by solid boxes while those playing a relatively minor role are boxed by dotted lines.
  • Figure 2A shows a map of YEp352-GALl indicating the modifications made for insertion of the wild type and mutant MTF genes.
  • Figure 2B shows the plasmids used for expression of wild type and mutant bacterial MTF in yeast.
  • Figure 3 shows an immunoblot analysis of extracts from yeast containing vector alone, and yeast expressing wild type and mutant MTF in the absence or presence of the inducer galactose.
  • Figure 4 shows an acid urea Northern blot comparing the mobility of yeast tRNAi Met aminoacylated and formylated in vitro with the mobility of yeast initiator tRNA isolated from yeast expressing wild type or mutant MTF.
  • Figure 5 shows a quantitative measurement of the growth in liquid culture of yeast cells containing vector alone compared growth of yeast expressing wild type or mutant MTF in the absence ( Figure 5 A) and presence ( Figure 5B) of the inducer galactose.
  • Figure 6 shows the growth of yeast colonies containing vector alone compared with growth of yeast expressing wild type or mutant MTF in the absence ( Figure 6 A) and presence ( Figure 6B) of the inducer galactose.
  • Figure 7A shows a plasmid used for expression of bacterial deformylase in yeast and the cloning strategy used to create this plasmid.
  • Figure 7B shows a schematic diagram of the 550 base pair Xli ⁇ - Spin fragment encoding wild type E. coli deformylase and the two Xh ⁇ - Sphl fragments that result after engineering the E133A mutation.
  • Figure 7C shows an agarose gel electrophoretic separation of plasmids pRS425 e 5 (wild type deformylase, lane 2) and pRS425rfe E133A (mutant deformylase. lane 3) digested with Sph 1 and Xhol.
  • Figure 8 shows an immunoblot analysis of extracts from yeast containing vector alone and yeast expressing either wild type MTF, wild type deformylase, or both in the absence or presence of the inducer galactose.
  • Figure 9 shows an immunoblot analysis of extracts from yeast transformed with vector alone and with plasmids encoding various combinations of wild type and mutant bacterial formylase and wild type or mutant bacterial deformylase in the presence of the inducer galactose.
  • Figure 10A shows the growth in liquid culture of yeast strain CKY473 transformed with either YEp352-GALl, YEpMTFWT, or YEpMTFR42L.
  • Figure 10B shows the protein synthesis rates in strains expressing wild type formylase compared to those in strains not expressing formylase.
  • Figure 11 shows the growth of mutant yeast strain UMY543 containing either vector alone or plasmids encoding wild type or mutant formylase.
  • Figure 12 shows the growth of yeast containing vector alone compared with growth of yeast expressing wild type MTF, yeast expressing wild type bacterial deformylase, and yeast expressing both wild type MTF and wild type bacterial deformylase.
  • Figure 13 shows the growth of yeast strain CKY473 containing various combinations of wild type and mutant bacterial formylase and wild type and mutant bacterial deformylase.
  • the present invention provides a system for identifying agents that inhibit bacterial growth, taking advantage of the fact that in bacteria, protein synthesis is initiated with formyl-methionine, whereas in eukaryotic cells the initiating methionine is not formylated. Protein synthesis is initiated with methionine in all organisms investigated thus far, i.e., methionine is the first amino acid incorporated into the polypeptide chain. As described in more detail below, all eubacteria investigated thus far contain a formylase enzyme that carries out the formylation reaction. In bacteria in which the gene for this enzyme is disrupted, growth is greatly inhibited. The inventors have recognized that compounds that inhibit bacterial formylase can inhibit bacterial growth and would therefore be effective antibiotics.
  • the amino acid is attached to the initiator tRNA, resulting in a species known as Met-tRNA Met , in a process catalyzed by an aminoacyl tRNA synthetase enzyme.
  • Met-tRNA Met a species known as Met-tRNA Met
  • an aminoacyl tRNA synthetase enzyme an aminoacyl tRNA synthetase enzyme.
  • f-Met-tRNA ⁇ 1 a transformylation reaction
  • Such formylation also occurs in mitochondria and chloroplasts of eukaryotic cells.
  • the formylation reaction is catalyzed by methionyl-fRNA transformylase (MTF).
  • MTF methionyl-fRNA transformylase
  • yeast initiator tRNA is the only cytoplasmic yeast tRNA that can be formylated in vitro by E. coli MTF (RajBhandary, U.L. and Ghosh, H.P., J. Biol. Chem., 244. 1104- 1113, 1969).
  • the formyl group allows the selection of fMet-fRNA above all other tRNAs by IF2, one of the proteins involved in the initiation of protein synthesis.
  • Other possible functions of formylation have also been postulated (RajBhandary, U. and Chow, C.)
  • the formyl group and, in many cases, the methionine at the N- terminus of the protein are subsequently removed through the successive action of the enzymes peptide deformylase (Mazel, D., Pochet, S. and Marliere, P., "Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation" EMBOJ.
  • MTF genes have been identified in every sequenced eubacterial genome.
  • E. coli the gene that encodes MTF is known as fmt.
  • the E. colifmt gene and its flanking regions have been cloned, and their complete sequence is known (described in GuiUon et. al, "Disruption of the Gene for Met-tRNA 61 Formyltransferase Severely Impairs Growth of Eschenchia coir, J. Bad., 174(13): 4294-4301, 1992, which is incorporated herein by reference; GenBank accession number of E.
  • the invention provides a eukaryotic cell containing MTF or a biologically active analog thereof.
  • the formylase can be a formylase that is naturally found in any bacterial species, in mitochondria, or in chloroplasts. Expression of the formylase alters a detectable characteristic of the cell. One such detectable characteristic is the growth rate. The inventors have found that expression of bacterial formylase in yeast inhibits growth. However, other detectable characteristics may also be altered. The alteration may be detected when cells are maintained under any of a number of conditions, which may be either optimal or nonoptimal for growth, e.g.. different temperatures, media compositions, liquid or solid media.
  • the invention comprises a eukaryotic cell containing a genetic expression system including a DNA sequence that encodes bacterial MTF or a biologically active analog thereof, operably linked to a promoter so that expression of the formylase is achieved.
  • the eukaryotic cell can be, for example, a yeast cell, an insect cell, or a mammalian cell.
  • the cell is a Saccharomyces cerevisiae cell.
  • the cell is a mammalian cell from any of a variety of cell lines adapted for growth in tissue culture. Such cells lines are well known in the art.
  • Representative mammalian cell lines include CHO cells, HeLa cells, NIH3T3 cells, and COS cells.
  • Representative insect cell lines include Sf9 cells.
  • the promoter is appropriately selected so that it directs transcription in the eukaryotic cell.
  • suitable constitutive promoters include the ADH1 (alcohol dehydrogenase), PGK (phosphoglycerate kinase), or CYC1 (cytochrome Cl) promoters.
  • appropriate promoters include, for example, viral promoters such as those SV40 virus, cytomegalovirus, or adenovirus, etc.
  • Appropriate promoters for use in insect cells include baculovirus promoters such as the IE1, polyhedrin, or P10 promoters.
  • the promoter is inducible so that cells can be maintained under noninducing conditions in order to avoid deleterious effects due to expression of formylase.
  • cells are used to screen for inhibitors of formylase, as described below, they are grown under inducing conditions so that formylase is expressed.
  • Suitable inducible promoters for use in S. cerevisiae include the GAL1 promoter as described herein and the PH05 promoter (regulated by the level of inorganic phosphate in the growth media).
  • Suitable inducible promoters for use in mammalian cells include the DHFR promoter and tetracycline-inducible promoters.
  • the formylase can come from any bacterial species.
  • the formylase is naturally found in a well-characterized laboratory strain of bacteria such as E. coli.
  • the formylase occurs naturally in a pathogenic bacteria, for example any of the bacterial pathogens discussed in Levinson, W., and Jawetz, E., Medical Microbiology and Immunology, 4th ed., McGraw-Hill Professional Publishing, New York, 1996, which is incorporated herein by reference.
  • Genes exhibiting a high degree of homology with E. coli formylase have been identified in a number of pathogenic bacteria, and the sequences are available from GenBank. Putative formylase genes have been found, for example, in H.
  • expression of the formylase in the eukaryotic cell is achieved by (i) introducing into the eukaryotic cell a DNA vector including a marker gene and a DNA sequence encoding a formylase protein, both operably linked to promoters operable in the cells and (ii) identifying cells that have a trait conferred by the marker gene.
  • the promoter is inducible such that cells do not express formylase when maintained in the noninduced state and express formylase when maintained under inducing conditions.
  • the DNA vector can be either a high copy vector or a low copy vector.
  • the formylase gene can be integrated into the host cell genome to provide stable expression in the absence of selection.
  • expression vectors including promoters and selectable marker genes are well known in the art.
  • markers include genes encoding biosynthetic enzymes (e.g., URA3, TRP1).
  • markers include the neo gene, which confers resistance to the antibiotic G418.
  • Expression vectors for a variety of different cell types are commercially available, e.g., from companies such as Stratagene (La Jolla, CA), Clontech (Palo Alto, CA), or Invitrogen (Carlsbad, CA).
  • the formylase is modified to include an additional moiety that can be used, e.g., for detection or purification of the formylase.
  • additional moiety e.g., for detection or purification of the formylase.
  • moieties, or tags are well known in the art and include, for example, the 6-His tag, the HA epitope tag, the c-Myc epitope tag, etc.
  • Modification of the formylase can be performed by modifying the DNA sequence encoding the formylase so that it includes additional sequences encoding the moieties.
  • the DNA sequence encoding the formylase can be inserted into an expression vector that already contains sequences encoding the moiety.
  • the vector can be introduced into the cell in any of a variety of ways well known in the art.
  • the vector is introduced into a yeast cell using a lithium acetate transformation procedure or using electroporation.
  • the vector is introduced into a mammalian cell using calcium phosphate transfection or using a cationic lipid transfection system.
  • the formylase gene is integrated into the cell genome so that it will be maintained in a stable fashion in the absence of selection.
  • transfection and selection protocols for obtaining transfectants with stably integrated genes are well known in the art.
  • Techniques for integrating genes into the yeast genome are also well known in the art (see, e.g., Guthrie, C. and Fink, G.
  • Yeast in which the formylase gene is integrated into the genome could be grown in rich liquid media, which might make the growth defect more dramatic and easier to detect, and might eliminate fluctuations in expression level due to variable plasmid copy number.
  • the expression level of formylase in the eukaryotic cell is measured.
  • Measurement of formylase in the cells can be used, for example, to confirm that the vectors containing the formylase gene are correctly constructed and are maintained in the cells and that the selected promoter is active in the selected cell type under the selected growth conditions.
  • a variety of techniques well known in the art can be employed to measure the expression level of formylase, e.g., immunoblotting or ELISA assays.
  • the formylase activity of wild type or mutant formylase expressed in the eukaryotic cell is measured by determining the extent to which initiator tRNA, Met isolated from the cell is formylated.
  • the formylation assay is based upon the observation that the addition and subsequent formylation of methionine alters the mobility of the initiator tRNA.
  • the present invention further provides a method of identifying inhibitors of bacterial formylase.
  • inhibitors are identified by contacting test agents with eukaryotic cells containing the formylase and with control cells not containing the formylase and determining whether such agents alter a detectable characteristic, e.g., growth, of cells containing the formylase.
  • Agents that selectively alter a characteristic of eukaryotic cells containing formylase so that it more closely resembles that characteristic as manifested in the control cells are identified as inhibitors of formylase.
  • suitable control cells are eukaryotic cells of the same cell type or strain that do not contain formylase.
  • appropriate control cells will be cells from the same population of cells that was used to create the formylase-containing cells.
  • appropriate control cells are substantially identical phenotypically or both genotypically and phenotypically to the formylase-containing cells, with the exception of those genotypic and phenotypic changes that result directly or indirectly from the modifications performed to create the formylase-containing cells.
  • different cells will be appropriate controls in different situations, depending upon the particular characteristic or parameter for which the cells are to serve as a control.
  • One skilled in the art will recognize that it is possible to select control cells that differ from the formylase- containing cells, provided that the differences do not affect the detectable characteristic being assessed.
  • a particularly preferred embodiment of the inventive method for identifying inhibitors of formylase includes the following steps:
  • test agent Identifying the test agent as a candidate inhibitor of bacterial formylase if contact with the test agent alters the detectable characteristic, e.g., growth rate, of the eukaryotic cells that contain formylase so that it more closely resembles the characteristic as manifested in control cells that do not contain formylase.
  • detectable characteristic e.g., growth rate
  • any characteristic of a eukaryotic cell that is altered by expressing bacterial formylase within the cell could be assayed.
  • the growth rate is used as the detectable characteristic.
  • the growth rate may be reduced in eukaryotic cells containing formylase as compared with control cells not containing formylase.
  • the inventors have found that expressing bacterial formylase in yeast cells reduces the growth rate of the cells.
  • coexpressing bacterial deformylase along with bacterial formylase in yeast substantially restores the ability of yeast to grow.
  • coexpressing an inactive mutant of bacterial deformylase i.e., a mutant that lacks deformylating activity
  • bacterial deformylase by removing the formyl group methionine within polypeptides, behaves like a formylase inhibitor in that it apparently counters the activity of formylase.
  • the growth defect in yeast expressing formylase is due to initiation of proteins with formylmethionine rather than to a nonspecific toxic effect of formylase.
  • the invention is not limited to characteristics that are due explicitly to the ability of formylase to add formyl groups to methionine but rather encompass any effect caused by the presence of formylase within a eukaryotic cell.
  • a test agent may be identified as an inhibitor of formylase if it enhances the growth of eukaryotic cells that contain formylase to a greater extent than it enhances the growth of control cells not containing formylase.
  • growth is enhanced by a factor of at least 1.5. More preferably growth is enhanced by a factor of 2, yet more preferably by a factor of 5, and yet more preferably by a factor of 10 or greater. In certain embodiments growth is enhanced by a factor of 100 or more.
  • cells are grown in liquid medium, and growth is monitored by measuring the turbidity of the culture using a colorimeter or spectrophotometer.
  • cell number can be monitored by counting cell number, e.g., using a Coulter counter or a hemocytometer.
  • incorporation of a radiolabeled metabolic precursor such as tritiated thymidine may be measured.
  • a preferred means of assessing the growth of yeast is to streak a small number of cells on a plate containing solid medium using a toothpick. By making successive streaks a very low concentration of cells is achieved on a portion of the plate. Each cell gives rise to a colony, thus providing a very sensitive means of assessing growth.
  • One skilled in the art will know how to select and practice appropriate techniques for measuring growth of the eukaryotic cells of the present invention.
  • Cells can be contacted with the test agent in a variety of ways.
  • the cells are maintained on plates containing solid (agar) media.
  • Discs of filter paper impregnated with the test agent are applied to the plates.
  • the test agent diffuses into the surrounding media.
  • the growth of cells in the area adjacent to the filter paper is compared with the growth of cells further from the filter paper.
  • the presence or size of colonies e.g. yeast colonies
  • the cells are grown in liquid media to which the test agent is added. The growth of the cells is monitored using any suitable method, e.g., those mentioned above.
  • Cells may be grown in multiwell plates (e.g., 96-well or 384-well plates) to facilitate high throughput screening for inhibitors as described in Hammonds, T., et al., Antimicrob. Agents Chemother., 42(4), 889-894, 1998, which is incorporated herein by reference.
  • Automated plate readers may be used to identify wells in which a detectable characteristic, e.g., turbidity of the wells, is altered.
  • the detectable characteristic is tested in cells grown under different growth conditions.
  • the growth defect of formylase- expressing yeast cells may be enhanced if such cells are grown at elevated temperature, e.g, at 37 degrees C or if such cells are grown using rich media rather than minimal media. Enhancing the growth defect may make it easier to identify formylase inhibitors.
  • the detectable characteristic used in the identification of formylase inhibitors is cell growth
  • detectable characteristics include, for example, production of a specific biological macromolecule, e.g., a protein whose activity can be easily detected.
  • proteins include, e.g., beta-galactosidase, green fluorescent protein, luciferase, and chloramphenicol acetyltransferase.
  • the formylase-expressing eukaryotic cell can be modified by introduction of a gene encoding the protein whose activity is to be detected, operably linked to appropriate genetic control elements so that expression of the protein is achieved. Activity of the protein can serve as a surrogate marker for cell growth.
  • the detectable characteristic is production of an endogenous macromolecule such as a protein or nucleic acid or metabolism of a substrate.
  • assays can be established in which the detectable characteristic is metabolism of a colored substrate to a noncolored metabolite or metabolism of a noncolored substrate to a colored substrate.
  • the cell is a yeast cell, e.g,. a Saccharomyces cerevisiae cell.
  • a S. cerevisiae cell of strain CKY473 is employed.
  • different strains of yeast are used.
  • strain JEL1 the GAL4 activator protein is induced by galactose, and this improves expression genes under the control o ⁇ GALl promoters between 5 and 10 fold (Lindsley, J.E. and Wang, J.C., J. Biol.
  • yeast strains that exhibit increased permeability can also be used for the expression of bacterial formylase. Such strains are described in Hammonds, T. et al., 1998. Candidate formylase inhibitors would more readily enter the cytoplasm of such yeast, thus making their growth-enhancing effect more evident. Thus yeast strain JEL4 and yeast strains exhibiting increased permeability are employed in certain preferred embodiments of the invention.
  • libraries of compounds are screened, preferably in a high throughput format.
  • Libraries of compounds are well known in the art and include, for example, libraries of natural compounds and libraries obtained through synthetic approaches including combinatorial chemistry. See, for example, Tan, et al., "Stereoselective Synthesis of Over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assays", Am. Chem Soc.120, 8565-8566, 1998. Libraries are commercially available, e.g., from AnalytiCon USA Inc., P.O. Box 5926, Kingwood, TX 77325.
  • Inventive formylase inhibitors may inhibit formylase to varying degrees or levels.
  • the present invention provides inhibitors that inhibit formylase activity to varying degrees, e.g., by 10%, 33%, 50%, 75%, 90%, 95%, 97%, or 99%.
  • the inhibiting activity of formylase inhibitors can be measured by any technique.
  • the activity of inventive formylase inhibitors is quantified by adding the inhibitors to an in vitro assay in which the ability of purified bacterial formylase (MTF) to formylate bacterial tRNA is measured.
  • MTF purified bacterial formylase
  • inventive formylase inhibitors are employed to inhibit bacterial cell growth. Therefore, the invention further provides both compositions for inhibiting the growth of bacteria comprising inhibitors of bacterial formylase and methods for inhibiting bacterial growth by administering inhibitors of formylase.
  • inhibiting bacterial growth is meant inhibiting bacterial replication and/or metabolism such that the bacterial mass does not increase.
  • inventive formylase inhibitors substantially prevent bacterial replication.
  • inventive formylase inhibitors reduce bacterial survival, i.e., kill existing bacteria, in addition to preventing bacterial replication.
  • bacterial growth is inhibited by at least 10%. In more preferred embodiments bacterial growth is inhibited to a greater extent, e.g., to at least 25%, at least 50%), at least 66%, or at least 80%>. In particularly preferred embodiments bacterial growth is substantially inhibited, e.g. to at least 90%, 95%, 97%, or 99%. Bacterial growth may be assessed using any available method including in vitro tests, as described below, or in vivo tests, e.g., in experimental animals.
  • formylase inhibitors may be combined with a variety of compounds, agents, or materials in delivery compositions to be administered to an organism in which bacterial growth is to be inhibited.
  • inventive formylase inhibitors are administered in a pharmaceutically acceptable formulation.
  • Pharmaceutically acceptable refers to agents and compositions that are physiologically tolerable and do not generally produce undesirable effects such as allergic reactions, nausea, or the like.
  • Particularly preferred pharmaceutically acceptable agents are approved by a Federal or state regulatory agency (e.g., the U.S. Food and Drug Administration) and/or listed in a publication such as the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, preferably in humans.
  • ingredients typically used in pharmaceutically acceptable formulations include carriers such as sterile liquids (e.g., water or oils).
  • sterile liquids e.g., water or oils
  • a suitable liquid e.g., buffered aqueous saline, aqueous dextrose solution
  • ingredients suitable for use in tablets or capsules include excipients (e.g., lactose, corn starch, sucrose), binders (e.g., starch, methylcellulose, dextrin, pullulan), stabilizers (e.g., magnesium or calcium carbonate), flavoring agents, and coloring agents.
  • excipients e.g., lactose, corn starch, sucrose
  • binders e.g., starch, methylcellulose, dextrin, pullulan
  • stabilizers e.g., magnesium or calcium carbonate
  • inventive formylase inhibitors are optionally administered in combination with other antibiotics. While any antibiotics can be used in conjunction with formylase inhibitors of the present invention, including antibiotics such as penicillins, cephalosporins, quinolones, etc., that have a very different mechanism of action to formylase inhibitors, in certain preferred embodiments antibiotics for use in combination with inventive formylase inhibitors are selected from among those known to act by inhibiting bacterial protein synthesis. Examples are antibiotics from various classes including macrolides, aminoglycosides, or chloramphenicol that act by inhibiting certain steps of the bacterial protein synthesis pathway. Formylase inhibitors may act synergistically with such antibiotics.
  • antibiotics act on a different step in the protein synthesis pathway to the inventive formylase inhibitors.
  • Many such antibiotics are known and are described, e.g., in Goodman and Gilman, 1996.
  • macrolides a category that includes the antibiotics erythromycin, clarithromycin, and azithromycin, inhibit bacterial protein synthesis by binding reversibly to the 50S ribosomal subunits where they may inhibit the translocation of the growing polypeptide from the A site to the P site.
  • Aminoglycosides a class of antibiotic that includes gentamicin, tobramycin, amikacin, and kanamycin, bind to the 30S subunit and disrupt bacterial protein synthesis at multiple steps.
  • Chloramphenicol inhibits protein synthesis in bacteria (and to a lesser extent in eukaryotic cells) by binding to the 5 OS ribosomal subunit at the peptidyltransferase (P) site and inhibiting the transpeptidation reaction.
  • an inventive formylase inhibitor when used in the treatment of a bacterial infection will depend upon the bacterial species causing the infection, the condition of the organism (e.g., human being) being treated, the route of administration, and other factors.
  • a therapeutically effective amount is an amount that reduces or prevents by at least 10%, preferably by at least 50%, more preferably by at least 90%, more preferably by at least 95%, the symptoms and/or signs of a disease or an appropriate marker thereof (e.g., positive blood culture in the case of certain bacterial infections).
  • an appropriate marker thereof e.g., positive blood culture in the case of certain bacterial infections.
  • guidelines for determining appropriate therapeutic dose are obtained by measuring the effect of inventive formylase inhibitors on bacterial growth using in vitro and in vivo assays.
  • the effect of formylase inhibitors on bacterial growth can be assessed by adding the inhibitors to bacteria growing in liquid culture and comparing the turbidity of the culture with the turbidity of a culture to which the inhibitor has not been added.
  • the inhibitor can be spread on the surface of solid media or otherwise added to the solid media, and the growth of bacterial colonies can be assessed.
  • Dilution tests in which serially diluted concentrations of the inhibitory agent are added to liquid or solid media can be used to determine the minimum inhibitory concentration (MIC), i.e., the lowest concentration that prevents visible bacterial growth after 18 to 24 hours of incubation.
  • the MIC is an important indicator of antibacterial activity. Methods of determining the MIC are well known to one of ordinary skill in the art.
  • Formylase inhibitors that have a MIC in the range of standard antibacterial agents e.g., those described in Goodman and Gilman, 1996) are particularly preferred for use as antibiotics to treat disease. Note that the tests described above need not be performed using the species of bacteria that naturally produce the formylase used to identify the inventive formylase inhibitors.
  • the effect of inventive formylase inhibitors on bacterial growth can also be expressed in terms of the LD 50 , i.e., the concentration needed to kill 50% of the organisms.
  • inventive formylase inhibitors on bacterial growth as well as the range of doses necessary to achieve clinical efficacy for treating bacterial infection can also be assessed in vivo.
  • different amounts of inventive formylase inhibitors can be administered (e.g., intravenously or orally) to animals that are infected with pathogenic bacteria.
  • Samples e.g., blood or tissues, can be taken from treated animals and untreated controls. These samples can be assessed for the presence of the pathogenic bacterium, e.g., by culturing or by microscopic examination using techniques that are known in the art. Length of time to recovery and/or overall survival of treated animals versus untreated controls can also be assessed.
  • mitochondria and chloroplasts organelles present within the cytoplasm of animal and plant cells respectively
  • formylases very similar to those found in eubacteria (Stryer, 1995). This similarity is likely to reflect the prokaryotic origin of these organelles.
  • Protein synthesis in mitochondria and chloroplasts is similar to that in eubacteria in that it utilizes formylated methionine as the initiating amino acid.
  • Mitochondrial and chloroplast formylase are likely to be active only within the organelles themselves and do not play a role in cytoplasmic protein synthesis carried out by the cell's protein synthesis machinery.
  • the present invention encompasses embodiments in which mitochondrial or chloroplast formylase is expressed in the cytoplasm of eukaryotic cells.
  • the descriptions contained herein are intended to illustrate certain embodiments of the invention and are not intended to limit its scope. Those of ordinary skill in the art will readily appreciate that various modifications and refinements are possible without departing from the spirit and scope of the appended claims.
  • plasmids used for expression of MTFWT and MTFR42L in yeast The complete coding sequences of the wild type and R42L mutant fmt genes were amplified from plasmids pQE16FMTp and pQE16FMTpR42L respectively using PCR. These plasmids, which are fully described in Ramesh, et al., 1997, include a tag encoding six histidine residues at the C terminus for use in protein purification and detection using antibodies. His-tagged MTF proteins are as active as their unmodified counterparts in formylating the E. coli initiator tRNA. The DNA was amplified with 20 cycles of denaturation at 95 degrees C for 45 seconds, annealing at 45 degrees C for 45 seconds, and extension at 70 degrees C for 2 minutes. The forward primer for both genes, SEQ ID NO: 1,
  • FIG. 1 shows a map of YEp352-GALl indicating the sites at which the wild type and mutant MTF genes (fmt) were inserted and showing the sequence immediately surrounding the start site (ATG codon) and stop codon of the genes.
  • Figure 2B shows the plasmids YEpMTF and YEpMTFR42L used for expression of wild type and mutant MTF in yeast.
  • YPD is rich media, providing all amino acids and dextrose (glucose).
  • SM is minimal media, which may be prepared with the omission of one or more amino acids to allow selection of yeast transformants and maintenance of plasmids.
  • SMD is minimal media with dextrose (2% by weight).
  • SMG is minimal media with galactose (2% by weight).
  • SMRG is minimal media with raffinose (2%> by weight) and glycerol (3% by weight). Unless otherwise specified, yeast were grown at 30 degrees.
  • yeast cells (strain CKY473 of genotype ura3-52, leu2,2-112) were transformed with plasmids YEpMTFWT and YEpMTFR42L as described in Tan et al., Biotechniques, 25, 792-796, 1988. Briefly, yeast cells were made competent for the uptake of DNA by exposure to lithium acetate. Transformation was accomplished by incubating cells with plasmid DNA in the presence of polyethylene glycol (PEG), lithium acetate, and carrier DNA. Transformants were selected on SMD plates lacking uracil.
  • PEG polyethylene glycol
  • Yeast transformed with YEpMTFWT and YEpMTFR42L were inoculated into SMD liquid media and allowed to grow until saturation (OD 60 o approximately 10-15) was reached. Cells were harvested by centrifugation and washed with sterile water. Cells were then inoculated into either 10 ml SMD media (uninduced) or SMG media
  • Yeast cells were collected from 1.5 ml of culture and resuspended in 50 micro liters of sterile water. 50 micro liters of lysis buffer (4% SDS, 10 mm EDTA) and glass beads were added, followed by vigorous vortexing for 2 minutes. The lysate was spun in a tabletop centrifuge for 5-10 minutes, and clarified supernatant was collected.
  • lysis buffer 4% SDS, 10 mm EDTA
  • Yeast lysate (10 to 15 micro liters) was loaded onto a 12% SDS polyacrylamide gel. After electrophoresis, proteins were transferred to PVDF membranes. The membrane was blocked in TBST (20 mM Tris.Cl pH7.5, 140 mM NaCl, 0.05% Tween-20) containing 5%> nonfat dry milk for 1 hour or overnight. The blot was probed with polyclonal antiserum against MTF. The antiserum was raised against the full length MTF polypeptide according to standard protocols (see, for example, Harlow, E., and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988). Detection was by ECL (NEB) using appropriate secondary antibodies and was performed in accordance with the instructions accompanying the ECL kit. Results
  • Figure 3 shows an immunob lotting analysis used to determine qualitatively the levels at which MTFWT and MTFR42L are expressed under noninducing and inducing conditions in yeast cells transformed with YEpMTF and YEpMTFR42L.
  • Lanes 1 and 2 of Figure 3 show that yeast transformed with the plasmid YEp352-GALl do not express MTF under noninducing or inducing conditions, i.e., in the absence or presence of galactose.
  • Lanes 3 and 4 compare the levels of MTF protein in yeast transformed with YEpMTFWT in the absence (lane 3) or presence (lane 4) of the inducer galactose.
  • Lanes 5 and 6 similarly compare the levels of the mutant protein, MTFR42L, in yeast transformed with MTFR42L in the absence (lane 5) or presence (lane 6) of galactose.
  • Lane 7 is a positive control in which pure bacterial MTF was loaded on the gel, confirming that the antibody used for detection recognizes MTF. From this blot it may readily be seen that yeast transformed with plasmids encoding wild type or mutant MTF express, in an inducible manner, approximately equivalent levels of MTFWT or MTFR42L respectively. Under noninducing conditions there is no detectable expression of MTFWT of MTFR42L.
  • Detection of MTF confirms that plasmids YEpMTF and YEpMTFR42L were correctly constructed and are maintained in the cells and that the GAL1 promoter is active under inducing conditions. Furthermore, the lack of detectable MTF under noninducing conditions confirms that GAL1 is functioning as expected.
  • the pellet was washed with 70% ethanol and dissolved in 2 ml TE.
  • High molecular weight nucleic acids were precipitated by the addition of 0.5 ml of 5 M NaCl and centrifugation.
  • Total tRNA was recovered from the supernatant by ethanol precipitation. The yield was approximately 100 OD 26 o units.
  • Yeast containing YEpMTFWT were grown to late log phase in SMRG supplemented with 2% galactose (approximately 70 hours) for analysis under inducing conditions.
  • Total tRNA was isolated from a 3 ml culture as described for isolation of total tRNA from E. coli (Varshney, U., Lee, C.P., and RajBhandary, U, "Direct Analysis of Aminoacylation Levels of tRNAs in vivo, J. Biol. Chem., 266(36), 24712-24718, 1997) except that the cells were subjected to 3 min vortexing followed by 2 min on ice repeated 4 to 5 times.
  • Acid urea gels and Northern blotting tRNAs isolated from yeast under acidic conditions and tRNAs aminoacylated and formylated in vitro were subjected to gel electrophoresis and Northern blotting as described for E. coli tRNA in Ramesh et al., 1997.
  • the sequence of the 5' end-labelled oligonucleotide probe used for detection of yeast tRNA was 5 GCGCGCTTCCACTGCGCCACGGC3' (S ⁇ Q ID NO: 3). The probe is complementary to positions 8 to 24 of yeast cytoplasmic initiator tRNA.
  • the Northern blot was quantitated using a Phosphorlmager. Results
  • the formylase activity of MTFWT and MTFR42L in yeast was investigated by determining the extent to which the initiator tRNA, Met is formylated when these proteins are expressed.
  • the formylation assay is based upon the observation that the addition and subsequent formylation of methionine alters the mobility of the initiator tRNA. It has been shown that under conditions of electrophoresis in acid urea gels, the aminoacylated E. coli initiator tRNA (i.e., the tRNA to which methionine has been linked) migrates more slowly than the nonaminoacylated species.
  • the formylated species migrates in between the aminoacylated and nonaminoacylated species because formylated methionine is uncharged whereas unformylated methionine bears a positive charge. Similar relationships hold for the yeast initiator tRNA, i.e., the formylated, aminoacylated initiator tRNA migrates in between the nonformylated, aminoacylated tRNA and the nonaminoacylated tRNA.
  • the mobility assay was used to analyze tRNA isolated from yeast cells expressing MTFWT or expressing the inactive, mutant formylase, MTFR42L.
  • Figure 4 shows a Northern blot that compares the mobility of yeast initiator tRNA, aminoacylated and formylated in vitro with that of yeast tRNA, isolated from yeast expressing MTFWT or MTFR42L.
  • the first three lanes of Figure 4 show total tRNA, isolated from yeast under conditions in which tRNA is in its nonaminoacylated state.
  • Lane 1 shows untreated tRNA
  • Lane 2 shows tRNA that has been aminoacylated in vitro using E. coli methionine tRNA synthetase (MetRS).
  • Aminoacylated tRNA migrates more slowly than the uncharged form.
  • Lane 3 shows aminoacylated tRNA, that has been formylated in vitro using E. coli MTF.
  • formylated aminoacyl yeast tRNA migrates between the aminoacylated (but unformylated) and nonaminoacylated species. This analysis allows the bands in the remainder of the figure to be unambigously assigned to the various species of yeast initiator tRNA.
  • Lanes 4 to 7 of Figure 4 show the in vivo levels of yeast initiator tRNA isolated from yeast containing plasmids YEpMTFWT (lanes 4 and 5) and YEpMTFR42L (lanes 6 and 7) grown in the presence (lanes 5 and 7) or absence (lanes 4 and 6) of galactose.
  • Lanes 4 to 7 demonstrate that in yeast containing plasmid YEpMTFWT grown under inducing conditions, approximately 70% of the initiator tRNA is formylated, whereas there is no detectable formylation under noninducing conditions or in yeast containing YEp352-GALl or YEpMTFR42L.
  • This result confirms that the cytoplasmic yeast tRNA is formylated in vivo by MTFWT expressed from plasmid YEpMTFWT, i.e., MTFWT functions in the cytoplasm of yeast.
  • This result further confirms that, as noted above, the mutant MTFR42L lacks detectable formylase activity.
  • Solid media (plates) growth assav S. cerevisiae cells of strain CKY473 containing YEp352-GALl, YEpMTFWT, or YEpMTFR42L were streaked on agar plates containing either SMRG (uninduced) or SMRG+2% galactose (induced) and incubated at 30 degrees C for at least 3 days. Growth of colonies was assessed visually.
  • Figure 5 shows quantitative measurements of the growth rate of yeast strains containing YEp352-GALl, YEpMTFWT, or YEpMTFR42L under noninducing or inducing conditions.
  • Figure 5 A the doubling time of all strains in SMRG medium (noninduced) was approximately 4 hours, and there is little difference between strains. However, a significant difference is seen upon induction.
  • Figure 5B the doubling time of cells containing YEp352-GALl or YEpMTFR42L in SMRG+2% galactose medium (induced) is approximately 2 hours.
  • the doubling time of the strain containing YEpMTFWT (and therefore expressing wild type formylase) is at least 5 hours.
  • Figure 6 shows the growth on solid medium of yeast strains containing YEp352- GAL1, YEpMTFWT, or YEpMTFR42L under noninducing (left) or inducing (right) conditions.
  • the name of the plasmid contained in each strain is indicated adjacent to the sector on which the strain was streaked.
  • noninducing conditions in which none of the strains express formylase, all three strains grow equally well as measured by size of colonies and time for colonies to become visible.
  • the plasmid pAS565 is a yeast expression vector containing the GALl promoter ( Figure 7A).
  • the def gene, encoding deformylase was amplified by PCR from E. coli genomic JM109 DNA using the following primers, which incorporate restriction enzyme sites as indicated below (the second primer also contains a six-His tag):
  • pRS425 is a high copy number plasmid containing the yeast LEU2 gene to allow selection of transformants on media lacking leucine (Christianson et al., Gene, 110, 119-122, 1992).
  • the plasmid pRS425 e E133A was constructed by engineering a GAG (E133) to GCG (A133) change in the def 5 coding sequence in plasmid pRS425de 5, which introduces a restriction site for Sphl.
  • the following primers (Sphl site bold italics) were used to create the El 33 A mutation using the Quik-change site-directed mutagenesis procedure (Stratagene, La Jolla, CA) and 100 ng pRS425 e 5 as template:
  • Figure 7B shows a schematic diagram of the 550 base paired - Sphl fragment encoding E. coli deformylase.
  • the wild type gene contains no internal Sphl sites, and therefore an XhoUSphl double digest releases a 550 base pair fragment.
  • the E133A mutant contains an engineered internal Sphl site, and therefore an XhollSphl double digest releases two fragments of 425 and 125 base pairs.
  • Figure 7C shows an agarose gel electrophoretic separation of plasmids pRS425 e/5 (wild type deformylase, lane 2) and pRS425 e E133A (mutant deformylase, lane 3) digested with Sphl andXhol indicating that the El 33 A change was successful.
  • Figure 8 shows an immunoblotting analysis used to determine qualitatively the levels at which bacterial deformylase is expressed under noninducing and inducing conditions in yeast cells containing YEp352-GALl or YEpMTFWT and subsequently transformed with pRS425 or pRS425de/5.
  • Lane 1 is a positive control in which pure his-tagged bacterial deformylase was loaded on the gel, confirming that the antibody used for detection recognizes the his-tagged protein.
  • Lanes 2, 4, and 6 show that yeast transformed with pRS425 or pRS425 e/5 do not express detectable levels of MTF or deformylase under noninducing conditions, i.e., in the absence of galactose.
  • Lane 3 shows that MTF but not deformylase is expressed in yeast transformed with YEpMTFWT and pRS425 in the presence of galactose.
  • Lane 7 shows that deformylase but not MTF is expressed in yeast transformed with YEp352-GALl and pRS425cfe 5 in the presence of galactose.
  • Lane 5 shows that both MTF and deformylase are expressed in yeast transformed with both YEpMTFWT and pRS425 e 5 in the presence of galactose. From this blot it may readily be seen that under inducing conditions yeast transformed with plasmids encoding MTF and/or deformylase express the respective proteins and that both proteins accumulate in yeast.
  • Figure 9 shows an immunoblotting analysis used to determine qualitatively the relative levels at which wild type and mutant bacterial deformylase are expressed under inducing conditions in cells containing YEp352-GALl or YEpMTFWT and subsequently transformed with pRS425, pRS425 e 5, or pRS425 e E133A.
  • Lanes 1 and 2 show that wild type and mutant bacterial deformylase are expressed at approximately equal levels in cells not expressing bacterial formylase.
  • Lanes 4, 5, and 6 show that wild type (lane 4) and mutant (lanes 5 and 6) bacterial deformylase are expressed at similar levels in cells expressing either wild type (lanes 4 and 5) or mutant (lane 6) bacterial formylase.
  • deformylase both wild type and mutant
  • the expression levels of deformylase are similar in cells expressing or not expressing formylase. This result indicates that both wild type and mutant deformylase accumulate in yeast and that the failure of mutant deformylase to rescue the slow growth phenotype of yeast expressing wild type formylase (see Examples 4 and 9) is not due to absence or instability of the mutant deformylase protein.
  • Synthetic medium was prepared by first preparing a 20X amino acid solution containing the following amino acids: Leu 0.8 g/L, Tyr 0.6 g/L, He 0.6 g/L, Phe 1 g/L, Glu 2 g/L, Asp 2g/L, Val 3g/L, Thr 4 g/L, Ser 8g/L, His 0.4 g/L, Lys 0.6 g/L, Try 0.4 g/L, Arg 0.4 g/L.
  • the final medium contained IX amino acids, 0.67% nitrogen base without amino acids, 2% raffmose, 3% glycerol, and 2% galactose.
  • Yeast strains (CKY473 transformed either with YEp352-GALl, YEpMTFWT, or YEpMTFR42L) were grown in the above medium (50 ml) lacking methionine until early log phase, and at various times between 15 and 20 hours after inoculation of the culture, cells were harvested and resuspended in 300 ⁇ l of the same medium. 50 ⁇ l of Easy Tag Express Protein labeling mix [ S] 11 mCi/ml was added, and the culture was incubated for 5 min at 30 degrees C. To stop incorporation, 1 ml of a solution containing 0.5 mg/ml of cycloheximide was added, and the mixture immediately frozen in dry ice.
  • the suspension was vigorously vortexed for 2 min, boiled for 5 min, and centrifuged. The clear supernatant after centrifugation was used for determining the TCA precipitable radioactivity.
  • Total protein was estimated using BCA reagents (Pierce). The rate of protein synthesis is expressed as counts/min of radioactivity incorporated into 1 ⁇ g of TCA-precipitable protein/min incubation.
  • Figure 10A shows the growth (measured in Klett units as described above) of yeast strain CKY473 transformed with either YEp352-GALl, YEpMTFWT, or YEpMTFR42L and grown under inducing conditions so that formylase is expressed.
  • cells expressing wild type formylase grow significantly slower than cells containing either vector alone or expressing mutant formylase. Cells within the indicated window (15 to 20 hours) were used for measuring protein synthesis rates.
  • Figure 10B protein synthesis rates in strains expressing wild type formylase are very similar to those in strains not expressing formyase.
  • Yeast strain UMY543 (MATaura3-52 trp ⁇ l imt2::TRPl imt3 TRP1 imt4::TRPl) was obtained from the laboratory of Dr. Anders Bystrom, Umea University, Sweden. UMY543 was transformed with YEp352-GALl, YEpMTFWT, or YEpMTFR42L as described above for strain CKY473. Transformed cells were streaked onto selective plates either lacking (noninducing conditions) or containing (inducing conditions) 2% galactose and incubated at 30 degrees C. Growth was assessed visually. Transformed cells were also grown in liquid medium (SMRG) under inducing (+galactose) or noninducing (SMRG-galactose) conditions, and doubling time was assessed as described above.
  • SMRG liquid medium
  • Yeast strain UMY543 has a single functional copy of the initiator tRNA gene as compared with the 4 copies present in its isogenic parent (Bystrom and Fink, Mol. Gen. Genet., 216, 276-286, 1989).
  • the initiator tRNA level in this strain is -2.3 fold lower than in the parent strain (Francis and RajBhandary, Mol. Cell. Biol, 10, 4486- 4494, 1990), and the strain has a doubling time that is 2.5 to 3 times longer, possibly due to effects on initiation of protein synthesis.
  • S. cerevesiaie cells of strain CKY473 containing various combinations of plasmids were streaked on plates containing SMRG+2% galactose.
  • the combinations tested were: (1) YEp352-GALl and pRS425; (2) YEp352-GALl and pRS425 e 5; (3) YEpMTFWT and pRS425; and (4) YEpMTFWT and pRS425 e 5.
  • the plates were maintained at 30 degrees C.
  • Figure 12 shows the growth on solid medium of yeast strains expressing either no exogenous proteins (sector 1), wild type bacterial MTF (sector 2), wild type bacterial deformylase (sector 3), or wild type bacterial MTF and bacterial deformylase (sector 4) under inducing conditions.
  • yeast strains expressing either no exogenous proteins (sector 1), wild type bacterial MTF (sector 2), wild type bacterial deformylase (sector 3), or wild type bacterial MTF and bacterial deformylase (sector 4) under inducing conditions.
  • Yeast expressing only deformylase grew normally whereas cells expressing only MTF grew much more slowly than cells either cells expressing no exogenous proteins or cells expressing only deformylase.
  • Cells expressing both MTF and deformylase grew much faster than cells expressing only MTF.
  • deformylase largely rescues the growth defect of cells expressing MTF suggests that the growth-inhibiting effect of MTF arises due to its formylase activity.
  • deformylase By removing the formyl group from formylmethionine in polypeptides, deformylase apparently counteracts the effect of MTF.
  • yeast expressing bacterial formylase synthesize protein at an overall rate approximately equal to that of yeast not expressing formylase. Furthermore, yeast cells with reduced amounts of initiator tRNA were not more severely affected by expression of formylase than yeast with normal levels of initiator tRNA. Taken together these results suggest that the slow growth of yeast expressing formylase may be due to the retention of formyl methionine at the N-terminus of proteins, which may adversely affect protein function or stability.
  • S. cerevesiaie cells of strain CKY473 containing various combinations of plasmids were streaked on plates containing SMRG+2% galactose.
  • the combinations tested were: (1) YEp352-GALl and pRS425 defWT; (2) YEp352-GALl and pRS425 e E133A; (3) YEpMTFWT and pRS425; (4) YEpMTFWT and pRS425 e WT; (5) YEpMTFWT and pRS425 e E133A; and (6) YEpMTFR42L and pRS425 e E133A.
  • the plates were maintained at 30 degrees C.
  • Figure 13 shows the growth on solid medium of yeast strains expressing either
  • DEFWT (sector 1), DEFE133A (sector 2), MTFWT (sector 3), MTFWT and DEFWT (sector 4), MTFWT and DEFE133A (sector 5), or MTFR42L and DEFE133A (sector 6) under inducing conditions.
  • DEFE133A is a mutant deformylase protein that has been shown to be defective in deformylase activity. Yeast expressing only wild type or mutant bacterial deformylase grew normally (sectors 1 and 2) whereas cells expressing only wild type MTF (sector 3) grew much more slowly than cells expressing deformylase (wild type or mutant).
  • Sector 6 confirms that the growth defect of cells expressing wild type bacterial formylase is likely to be a result of the protein's formylating activity rather than some other property of the protein since cells containing mutant bacterial formylase lacking formylase activity display no growth defect.

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Abstract

L'invention concerne une cellule eucaryote exprimant formylase et un système pour l'expression de formylase dans une cellule eucaryote. L'expression formylase confère un phénotype détectable sur les cellules, à savoir une modification de la vitesse de croissance, de telle sorte que des inhibiteurs de formylase peuvent être identifiés grâce à l'évaluation de modifications dans le phénotype détectable. L'invention concerne également des procédés et des systèmes (c.-à-d. des écrans) destinés à identifier des inhibiteurs de formylase. De tels inhibiteurs sont utiles en tant qu'agents antibactériens.
PCT/US2001/002720 2000-01-28 2001-01-26 Cellule eucaryote exprimant une formylase bacterienne et ses utilisations Ceased WO2001055351A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008959A1 (fr) * 1996-08-29 1998-03-05 Massachusetts Institute Of Technology PROCEDE DE SYNTHESE DE PROTEINES $i(IN VIVO)
WO1999052926A1 (fr) * 1998-04-14 1999-10-21 Versicor, Inc. Expression regulee de cibles a des fins de criblage

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008959A1 (fr) * 1996-08-29 1998-03-05 Massachusetts Institute Of Technology PROCEDE DE SYNTHESE DE PROTEINES $i(IN VIVO)
WO1999052926A1 (fr) * 1998-04-14 1999-10-21 Versicor, Inc. Expression regulee de cibles a des fins de criblage

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHAN K-K ET AL: "SYNTHESIS AND ACTIVITY OF A TETRAHYDROFOLATE INHIBITOR OF THE ENZYME N-10 FORMYLTETRAHYDROFOLATE-METHIONYL TRANSFER RNA TRANSFORMYLASE", HETEROCYCLES (TOKYO), vol. 24, no. 11, 1986, pages 3079 - 3086, XP000995957, ISSN: 0385-5414 *
GUILLON J -M ET AL: "DISRUPTION OF THE GENE FOR MET-TRNAFMET FORMYLTRANSFERASE SEVERELY IMPAIRS GROWTH OF ESCHERICHIA COLI", JOURNAL OF BACTERIOLOGY,US,WASHINGTON, DC, vol. 174, no. 13, 1 July 1992 (1992-07-01), pages 4294 - 4301, XP002043974, ISSN: 0021-9193 *
MAZEL D ET AL: "GENETIC CHARACTERIZATION OF POLYPEPTIDE DEFORMYLASE, A DISTINCTIVE ENZYME OF EUBACTERIAL TRANSLATION", EMBO JOURNAL,GB,OXFORD UNIVERSITY PRESS, SURREY, vol. 13, no. 4, 15 February 1994 (1994-02-15), pages 914 - 923, XP002043973, ISSN: 0261-4189 *
NEWTON D TREVOR ET AL: "Formylation is not essential for initiation of protein synthesis in all eubacteria.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 32, 6 August 1999 (1999-08-06), pages 22143 - 22146, XP002166129, ISSN: 0021-9258 *
P T RAVI RAJAGOPALAN ET AL: "Peptide Deformylase: A New Type of Mononuclear Iron Protein", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC,US, vol. 119, 1997, pages 12418 - 12419-21419, XP002106376, ISSN: 0002-7863 *

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