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WO1999023244A1 - Procedes et compositions utiles pour une modulation d'enzymes de topoisomerase bacterienne - Google Patents

Procedes et compositions utiles pour une modulation d'enzymes de topoisomerase bacterienne Download PDF

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WO1999023244A1
WO1999023244A1 PCT/US1998/023384 US9823384W WO9923244A1 WO 1999023244 A1 WO1999023244 A1 WO 1999023244A1 US 9823384 W US9823384 W US 9823384W WO 9923244 A1 WO9923244 A1 WO 9923244A1
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bacterial
strains
dna
strain
test
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PCT/US1998/023384
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English (en)
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Rolf Menzel
David A. Elsemore
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Small Molecule Therapeutics, Inc.
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Priority to AU13770/99A priority Critical patent/AU1377099A/en
Publication of WO1999023244A1 publication Critical patent/WO1999023244A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase

Definitions

  • the present invention relates to methods and compositions for the modulation of bacterial topoisomerase enzymes within bacterial cells. More specifically, the present invention relates to bacterial assays wherein the levels of bacterial topoisomerase enzymes or the levels of target sites within the enzymes are varied within bacterial
  • test strains in order to screen for compounds that target, i.e., interact with the topoisomerase enzymes, thereby causing DNA damage and hence, bacterial growth inhibition and/or cell death.
  • the present methods and compositions are useful for the identification and characterization of novel target, i.e., interact with the topoisomerase enzymes, thereby causing DNA damage and hence, bacterial growth inhibition and/or cell death.
  • DNA topoisomerases are enzymes that affect the topology or structure of DNA. More specifically, these enzymes have 0 the ability to introduce supercoils into DNA molecules or relax the DNA molecules; they can catenate or decatenate circular DNA or they can knot or unknot DNA (Schmid et al., 1993, BioEssay ⁇ .15. (No. 7): 445-449).
  • the DNA topoisomerases act by catalyzing the breakage and rejoining of the DNA __ phosphodiester backbone (Wang, 1985, Annu. Rev. Biochem. 54: 665-697; Vosberg, 1985, Curr. Top. Microbiol. Immunol. 114: 19-102) . These reactions, together with an intervening strand passage event, allow the topoisomerases to alter DNA topology. In fact, correct topoisomerase function is 3 necessary for such basic cellular processes as DNA replication and transcription.
  • Type I topoisomerases act by causing a transient break in one strand of the double-stranded DNA and
  • the Type II DNA topoisomerases alter DNA topology by causing transient breaks in both strands of a double-stranded DNA, allowing the passage of one double- stranded DNA molecule through another.
  • the Type II topoisomerases also allow the relaxation and decatenation of DNA; however, one bacterial Type II topoisomerase, DNA gyrase, also has the ability to introduce negative supercoils into relaxed DNA. In either type of topoisomerase, after the strand passage event, the final step of the reaction is the rejoining or ligation of the DNA break (s) .
  • Topo I encoded by the topA gene, is responsible for the major DNA relaxing activity of the cell, and catalyzes the relaxation of DNA through the seguential breakage-strand-passage-religation cycle common to all topoisomerase enzymes.
  • Topo III represents a minor activity encoded by the topB gene, is less well-characterized and its role in vivo is less clear, although the molecule appears to possess decatenation activity (Scmid et al. , supra at p. 447) .
  • Bacteria also have two Type II topoisomerases: DNA gyrase (Topo II) and Topoisomerase IV (Topo IV) . As noted supra .
  • DNA gyrase catalyzes negative supercoiling in DNA; Topo IV has been found to catalyze decatenation of interlinked DNA, e.g., subseguent to DNA replication, as well play a role in DNA relaxation. Although Topo IV shares extensive ho ology with DNA gyrase (40-50% a ino acid identity) , it differs from DNA gyrase in the activities it can catalyze. For example, Topo IV cannot couple the hydrolysis of ATP to the process of DNA supercoiling as gyrase can, but it is able to relax DNA in an ATP-stimulated fashion.
  • eukaryotes In contrast to the bacteria, eukaryotes, such as yeast and humans, possess a single Type I DNA topoisomerase (see, e.g., Wang, J.C., 1987, J. Biochem. Biophys. Acta. 909: 1-9). Furthermore, this eukaryotic topoisomerase is distinct from bacterial Topo I in both structure and function. For example, the bacterial and eukaryotic Topo I enzymes share no amino acid seguence homology. In addition, the eukaryotic enzyme shows a preference for binding double-stranded DNA and catalyzes its reaction by forming a covalent 3'- phosphodiester intermediate.
  • the bacterial Topo I enzyme shows a preference for binding at the junction of double and single-stranded regions and catalyzes its reaction by forming a 5 ' -phosphodiester intermediate (see, e.g., Taylor and Menzel, 1995, Gene 167: 69-74).
  • the human Topo I enzyme is the target of the antitumor drug camptothecin (CPT)
  • the bacterial enzyme is resistant to that drug.
  • Eukaryotes also possess a Type II DNA topoisomerase that demonstrates some structural and evolutionary relationship to bacterial gyrase (Wang, 1994, Advances in Pharmacology, Volume 29A, Liu (ed.). Academic Press) .
  • Topo III While the bacterial Type I topoisomerase, Topo III, appears to be non-essential to the viability of the cell (Hiasa, et al. , 1994, J. Biol. Chem. 269: 2093-2099), Topo I is essential in an otherwise normal bacterial cell. Nevertheless, the topA gene may be deleted or otherwise rendered inactive, provided that certain second site compensatory mutations are present in the topA" strains, leading some to question the essential nature of the bacterial Topo I enzyme (Pruss et al., 1982, Cell 3_1: 35-42; DiNardo et al., 1982, Cell 3_1: 43-51).
  • Second site mutations that compensate for the loss of topA do so by altering the activity or levels of other topoisomerase enzymes in the mutant host cell.
  • mutations that compensate for the loss of bacterial Topo I have been mapped to the two genes (gyrA and gryrB) that encode the bacterial Type II DNA topoisomerase, DNA gyrase. These mutations have been shown to reduce the levels and activity of DNA gyrase, suggesting a balance between the competing activities of topoisomerases that introduce DNA supercoils (i.e., DNA gyrase) and those that remove or relax DNA supercoils (e.g., Topo I).
  • topA mutants that compensate for the loss of topA in a topA mutant have been mapped to a region of the chromosome encoding the pare and parE genes which encode the subunits of the bacterial Type II topoisomerase, Topo IV.
  • compounds that can trap the cleavable complex intermediate formed during the catalytic reaction of the DNA topoisomerases represent important drugs.
  • compounds that trap the covalent intermediates involved in the catalytic reactions of the eukaryotic DNA topoisomerases have been shown to represent important anticancer drugs; e.g., the anticancer drugs Etoposide and Adriamy ⁇ in trap the covalent intermediate of human Type II topoisomerases.
  • camptothecins another important class of anticancer drug, trap the DNA intermediate of human Type I topoisomerase but do not affect the bacterial Type I topoisomerases (Drlica, K. et al., Biochem. 22: 2253- 2259) .
  • quinolones In the area of antibacterial drugs, a major class of antibiotics, the quinolones, have been shown to trap the cleavable complex of the bacterial DNA gyrase enzyme. The quinolones have also been shown to trap the intermediate fo.rmed during the catalytic reaction of Topo IV (Drlica et al., September 1997, Microbiology and Molecular Biology Reviews, 6.1 (No. 3): 377-392).
  • the present invention relates to methods and compositions for the identification of new antibacterial compounds. More specifically, the present invention relates to bacterial assays or screens, wherein the levels of bacterial DNA topoisomerases or target sites within the topoisomerases are varied in bacterial test strains in order to identify compounds that target, i.e., interact with topoisomerase enzymes within the bacterial cell, thereby causing DNA damage which leads to cell growth inhibition and/or cell death.
  • the methods and compositions of the invention therefore identify potent antibacterial compounds that act through their effects on bacterial DNA topoisomerases .
  • screens are performed for identifying test compounds that arrest the reaction catalyzed by the bacterial DNA topoisomerases.
  • Such compounds are referred to as "poisons" and, as herein defined, these poisons interact with the DNA topoisomerase cleavable complex intermediate, trapping the intermediate and thus blocking the DNA rejoining step of the topoisomerase catalytic reaction.
  • these poisons possess an antibacterial therapeutic effect, not through inhibition of the catalytic activity of the enzyme per se, but rather via their ability to cause lethal DNA damage (see, e.g., Liu, supra at pp. 371-372; Wang, supra at p. 9) .
  • one class of poisons acts to trap the bacterial Topo I cleavage complex intermediate.
  • Another class of poisons acts to trap the bacterial Type II DNA topoisomerases, e.g., DNA gyrase and Topo IV.
  • the assays or screens of this invention comprise contacting a test compound to a bacterial test strain A and a bacterial test strain B wherein the test strains are isogenic, except that the two strains exhibit a differential level of test compound topoisomerase "targets", that is, elements or sites within the topoisomerase enzyme that allow interaction with the test compound or poison.
  • a differential level of such targets can be achieved by decreasing or increasing the level of the topoisomerase enzyme itself (e.g. , via deletion or duplication mutations) or by decreasing or increasing the number of functional sites within the enzyme that allow interaction with the test compound or poison.
  • topoisomerase target that enables a topoisomerase poison to exert its antibacterial effect, i.e., DNA damage (due to the interaction between the poison and the target intermediate)
  • elevated levels of the topoisomerase target in one test strain relative to another leads to a differential, e.g., increased sensitivity to the test compound that can be measured as a function of DNA damage. Therefore, if, upon exposure of test strains A and B as defined above to a test compound, a differential sensitivity is detected between the two strains, that test compound is identified as a DNA topoisomerase poison.
  • differential sensitivity refers to a differential amount or measure of DNA damage to the bacterial cells, which can be detected by assays of cell death and/or growth inhibition of the cells (indirect measures of DNA damage) or by standard DNA damage or DNA cleavage assays (direct measures) .
  • a differential sensitivity between the test strains is first detected using an indirect measure of DNA damage such as a growth inhibition assay and the DNA damage ability/activity of the test compound/poison is further confirmed by standard DNA damage or cleavage assays using known substrates (see, e.g.. Sections 6.4 and 6.5, infra) .
  • a screen for identifying Topo I poisons comprises contacting a test compound to two bacterial test strains that are isogenic except that one of the strains has a reduced level of Topo I enzyme targets relative to the other.
  • the differential inhibition of growth and/or cell death ejdiibited by the two strains relative to each other is measured.
  • assays and biochemical tests can be performed to determine the differential DNA damage ejdiibited by the two test strains.
  • a test compound that causes an increased amount of DNA damage, growth inhibition and/or killing of the strain that has the elevated level of Topo I targets relative to the other strain indicates that the test compound works in concert with Topo I to poison the cell; that test compound is therefore identified as an antibacterial Topo I poison.
  • bacterial test strains A and B are isogenic strains that differ only in that one strain contains a topA mutation which decreases the level of Topo I or Topo I targets within the bacterial cell.
  • the topA mutation is a deletion mutation.
  • a test compound is contacted to the test strains and the differential sensitivity of the strains relative to each other is measured.
  • a test compound that demonstrates a differential, in this case, an increased DNA damage, growth inhibition and/or killing of the wild type bacterial strain relative to the topA mutant strain is identified as a Topo I poison.
  • a single bacterial test strain containing a topA mutation that results in a loss or decreased level of Topo I targets additionally contains a second DNA sequence encoding a functional topA gene.
  • the strain can contain a plasmid or other extrachromosomal DNA sequence having a functional topA gene under the regulatory control of an inducible promoter for the regulated production of Topo I.
  • a functional topA gene operably linked to an inducible promoter may be inserted directly into the bacterial DNA.
  • the strain is studied under two different conditions: in one, the strain is induced to express Topo I, while in the other, it is not. As with the Topo I poison screen discussed supra.
  • a test compound that causes a differential DNA damage, growth inhibition and/or killing of the bacterial strain induced to produce Topo I relative to the non-induced topA mutant strain is identified as a Topo I poison.
  • the topA mutation is a deletion mutation.
  • a screen for identifying bacterial Type II DNA topoisomerase poisons comprises contacting a test compound to two bacterial test strains that are isogenic except for the fact that one of the strains has a reduced level of bacterial Type II DNA gyrase targets and the second strain has an increased level of Type II Topo IV targets.
  • the levels of the enzymes themselves are varied such that one of the strains has a reduced level of the bacterial gyrase enzyme and the other strain has an increased level of the Topo IV enzyme.
  • the strain with reduced levels of the gyrase enzyme will possess a decreased number of targets for a Type II-specific poison whereas the strain with increased levels of Topo IV will possess an increased level of targets for a Type II-specific poison.
  • a differential, in this case, increased DNA damage, growth inhibition and/or cell death exhibited by the strain with the elevated level of Type II DNA topoisomerase relative to the strain having reduced levels of Type II targets indicates that the test compound works in concert with the Type II topoisomerase enzymes to poison the cell.
  • Such a test compound is identified according to the assays of this invention as an antibacterial Type II DNA topoisomerase poison.
  • the isogenic test strains are topA + and according to another embodiment, they are topA ' .
  • a bacterial strain with reduced gyrase levels and a bacterial strain with increased Topo IV levels are compared respectively, i.e., separately, to isogenic strains that are wild type for each mutation in order to identify Type II poisons that are specific for the gyrase or Topo IV enzymes.
  • topA' mutants the topA mutations must be compensated for by second site mutations in order for the bacterial cells to maintain their viability. All topA ' strains used in the embodiments of this invention therefore further comprise mutations which compensate for topA defects, including but not limited to mutations in the gyrA and/or gyrB genes or in the pare and/or parE genes.
  • FIGS. 1A-1C A schematic diagram showing that various bacterial DNA topoisomerases act in concert to maintain a balance in the level of DNA supercoiling.
  • FIG. B Reducing DNA gyrase activity, and thereby restoring the balance by decreasing DNA supercoiling activity, can compensate for the loss of topA.
  • FIG. C Increasing Topo IV activity, and thereby restoring the balance by increasing DNA relaxing activity, will also act to compensate for the loss of topA.
  • FIG. 2 Series of genetic events involved in the construction of isogenic strains for the identification of bacterial Topo I and Type II topoisomerase poisons.
  • FIGS. 3A-3B Schematic diagrams of the plasmids used for the inducible expression of E.coli topoisomerase I (FIG. 3A) and S . aureus topoisomerase I (FIG. 3B) .
  • FIGS. 4A and 4B Schematic depiction of a Topo I topA deletion screen (FIG. 4A) and a Topo I inducible expression screen (FIG. 4B) .
  • FIG. 5 Schematic depiction of a Type II topoisomerase poison screen comparing strain A containing reduced levels of DNA gyrase with strain B containing elevated levels of Topo IV.
  • poison refers to compounds that interact with the cleavable complex intermediate formed during the catalytic reaction of the bacterial DNA topoisomerases. Such poisons trap the intermediate and block the DNA rejoining step of the catalytic reaction. These types of compounds thus exert their antibacterial effect by damaging the DNA of the bacterial cell (see, e.g., Liu, supra at pp. 371-372; Wang, supra at p. 9).
  • Topic I poison refers to those compounds that act by trapping the cleavable complex intermediate of bacterial Type
  • Topoisomerase I also referred to herein as Topo I.
  • Type II poison refers to those compounds that act by trapping the cleavable complex intermediate of bacterial Type
  • the present invention provides bacterial assays wherein the levels of bacterial DNA topoisomerase enzyme targets are varied in bacterial test strains, thus providing differential levels of targets for topoisomerase poisons.
  • the bacterial test strains are isogenic, except that the strains possess differential levels of DNA topoisomerase targets.
  • the strains possess differential levels of DNA topoisomerase enzymes. The test strains are then exposed to a test compound and the differential sensitivity of the strains to the compound is assessed. As noted supra .
  • a differential sensitivity exhibited by the bacterial cells according to this invention is a differential amount or degree of DNA damage, which can be detected by cell death or growth inhibition assays such as growth sensitivity assays which can be performed in liquid culture media employing icrotiter plates or growth sensitivity assays in agar such as that described in Section 6.2 infra, wherein differential sensitivity to the test compound is measured by assessing zones of growth inhibition.
  • cell death or growth inhibition assays such as growth sensitivity assays which can be performed in liquid culture media employing icrotiter plates or growth sensitivity assays in agar such as that described in Section 6.2 infra, wherein differential sensitivity to the test compound is measured by assessing zones of growth inhibition.
  • the DNA damage inflicted on the bacterial cells by these poisons may be identified by direct DNA damage or DNA cleavage assays or techniques known in the art.
  • an alkali elution profile of cleaved DNA is a well- known method for demonstrating DNA damage (see, e.g., Markovitz et al., 1987, Cancer Research 47: 2050-2055).
  • a DNA damage inducible promoter assay can be used, wherein the level of DNA damage is assessed by measuring the level of a reporter gene whose expression is driven by a regulatory sequence induced by the DNA damage.
  • DNA damage-inducible regulatory sequences can be introduced into the test strains prior to the screen and include, but are not limited to, promoters induced as part of the bacterial SOS response.
  • one such regulatory sequence is the sulA promoter sequence, the sequence of which is well known to those of skill in the art (see, e.g., Mamber et al., 1990, .Antimicrobial Agents and Chemotherapy 3_4.: 1237- 1243 and Kenyon et al., 1982, J. Mol. Biol. 160: 445-457).
  • the DNA damage caused by a potential topoisomerase poison can be detected, for example, by measuring the level of a reporter gene product whose expression is driven by the sulA promoter sequence.
  • the reporter sequence can be any detectable gene product.
  • Reporter sequences can include, but are not limited to, LacZ (betagalactosidase) and CAT (chloramphenicol acetyltransferase) gene products. Methods for detection using reporter sequences including, but not limited to those described above, are well known to those of skill in the art.
  • An E.coli strain, referred to herein for exemplary purposes only, which contains a ⁇ ulA-lac construct as a DNA inducible reporter construct is E.coli strain GR4413 (Huisman, O. et al., 1981, Nature 290:797-799) .
  • the differential DNA damage exhibited by the test strains due to a test compound is detected using a growth inhibition assay, preferably in agar (see Section 6.2, infra). This allows for a more rapid and efficient screening of many compounds in as short a time as possible.
  • the DNA damage activity of a test compound identified as a poison according to this screen can then be further confirmed by various DNA damage or cleavage assays as exemplified infra in Sections 6.4 and 6.5.
  • the DNA damage assay of Section 6.4 can be used to demonstrate actual DNA cleavage that is dependent on the presence of both the specific purified DNA topoisomerase enzyme and the poison compound in question (see, e.g., Anderson et al., 1994, in Topoisomerase Biochemistry and Molecular Biology, Liu (ed.), pp. 83-101). Since, according to this invention, the topoisomerase poison requires the presence of the topoisomerase target for its antibacterial effect, elevated levels of the target in one test strain relative to another leads to a differential, and in such a case, increased sensitivity to a test compound that acts as a topoisomerase poison.
  • topoisomerase target in one test strain e.g., in a topA deletion mutant relative to an otherwise isogenic wild type strain
  • the drug screening approach of this invention is therefore unlike the more traditional drug screening approaches, where the desired cytotoxic effect of a drug is due to a simple inhibition of a target gene product and therefore reduced target levels result in a greater sensitivity to the drug.
  • the drug or antibacterial compound requires the target gene product in order to have its cytotoxic effect and therefore reduced target levels result in a greater resistance to the antibacterial compound, while elevated target levels result in a greater sensitivity to the drug.
  • Test compounds that can be screened for antibacterial activity using the methods and compositions of this invention include, but are not limited to, compounds obtained from any commercial source, including Aldrich (1001 West St. Paul Ave., Milwaukee, WI 53233), Sigma Chemical (P.O. Box 14508, St.
  • any kind of natural products may be screened using the methods of the invention, including microbial, fungal or plant extracts.
  • diversity libraries of test compounds, including small molecule test compounds may be commercially obtained from Specs and BioSpecs B.V.
  • the present invention is divided into the following sections solely for the purpose of description: (a) methods and compositions for screening for bacterial Topo I poisons; and (b) methods and compositions for screening for bacterial Type II topoisomerase poisons.
  • the bacterial test cells utilized are those that are particularly permeable, thereby facilitating entry of the test compounds into the cells.
  • such cells can include, but are not limited to, 5 cells with imp mutations. .
  • the imp mutations that can be utilized in this regard is the imp 4213 mutation (Sampson, B.A.
  • bacterial strains that can be used in the screens of this invention include, but are not limited to, E. coli. 0 Salmonella tvphimurium. S . aureus . s. fecalis. P. aeru ⁇ inosa. M. tuberculosis, and H. pylori.
  • bacterial screens are provided for identifying bacterial Topo I topoisomerase poisons. These bacterial assays comprise contacting a test compound to two bacterial test strains that are isogenic except that one of the strains has a reduced level of bacterial Topo I enzyme targets relative to the other. After an appropriate exposure to the test compound, the differential sensitivity of the test strains to the test compound is determined. A test compound that causes an increased amount of DNA damage, growth inhibition and/or killing of the strain that has the elevated level of Topo I targets relative to the other strain is identified as an antibacterial Topo I poison.
  • isogenic bacterial test strains A and B differ only in that one strain is topA ' .
  • topA ⁇ refers to any topA mutation that decreases the level of Topo I or Topo I targets within the cell, including but not limited to a deletion of the topA gene.
  • isogenic bacterial test strains A and B differ only in that one strain contains a topA deletion mutation or other genetic alteration of the topA gene and thereby exhibits greatly reduced levels of the bacterial Topo I enzyme or is missing the enzyme entirely. See Figure 2 for an illustration of the genetic events involved in the construction of a bacterial strain containing a topA deletion. As noted supra.
  • topA mutant strains must possess a second site mutation, such as in the gyrA and/or gyrB genes, or in the pare and/or parE genes, in which the second site mutation compensates for the loss of the relaxing activity of the Topo I enzyme.
  • a second site mutation such as in the gyrA and/or gyrB genes, or in the pare and/or parE genes, in which the second site mutation compensates for the loss of the relaxing activity of the Topo I enzyme.
  • mutations in the gyrA or gyrB genes such mutations reduce the endogenous supercoiling activity of the gyrase enzyme and thus compensate for the loss of the relaxing activity normally provided by the Topo I enzyme.
  • the second site mutation involves duplications in the par and/or parE genes, such mutations provide increased relaxing activities to the cell, thus compensating for the relaxing activity lost via the topA mutation (see FIG. 1) .
  • strains used in this embodiment of the invention must be isogenic except for the absence or presence of the topA mutation, the non- topA' test strain must also contain the same compensatory second site mutations contained in the topA mutant strain.
  • Strains that can be used according to this invention include E.coli RFM475 (gyrB 1 * topAJ (Drolet, 1995, Proc. Natl. Acad. Sci.
  • Novel sets of isogenic strains can be constructed in accordance with this invention as described in Section 6.1 and Table I, infra and as depicted in FIG. 2.
  • Novel strains for use in the Topo I screen of this invention include topA-wild type strain SAD15 and its isogenic topA-deleted companion strain S.AD16; topA-wild type strain RFM431 and its isogenic topA-deleted companion strain SAD17; and topA-wild type strain SAD19 and its isogenic topA- deleted companion strain SAD18 (see FIG. 4A and Table I, infra). See also. Table II, in Section 6.2 infra. which lists pairs of isogenic test strains that can be used in the Topo I and Type II poison screens of this invention.
  • a test compound is contacted to the test strains and the differential sensitivity of the strains relative to each other is measured.
  • a test compound that acts by trapping the cleavable complex intermediate of the Topo I enzyme will only inhibit or kill a strain that possesses the target enzyme, but will have no effect on a strain that is lacking Topo I.
  • a test compound that demonstrates a differential (i.e., increased) DNA damage, growth inhibition and/or killing of the wild type bacterial strain relative to the topA mutant strain is identified as a Topo I poison. See Figure 4A for a depiction of this screening embodiment of the invention.
  • a single bacterial test strain contains a topA deletion and therefore 5 lacks the Topo I target enzyme, but it additionally contains a second DNA sequence encoding a functional topA gene under the regulatory control of an inducible promoter for the regulated production of Topo I.
  • the strain can contain a second DNA sequence inserted into its bacterial DNA
  • topo I can contain a plasmid or other extrachromosomal DNA sequence that carries a functional bacterial topA gene operably linked to an inducible promoter.
  • inducible promoters that can be used for the regulated expression of Topo I according to this invention include the
  • FIGS. 3A and 3B represent schematic depictions of plasmids that can be used according to this embodiment of the invention for the inducible e.xpression of
  • Novel test strains containing inducible topA genes that can be used according to this embodiment of the invention include S.AD22 and SAD23, which strains can be constructed as described infra in Section 6.1.
  • topA 35 is studied under two different conditions; it is either induced to ej ress Topo I or it is not (see FIG. 4B) .
  • the regulated expression of the topA gene according to this embodiment provides the ability, in a single strain, to examine conditions of no or low expression versus conditions of high level expression. Since the general viability of topA' strains (without compensatory mutations) is weak, it is preferred to carry out this screen using low versus high induction conditions.
  • basal (non-induced) Topo I levels are less than 10% of wild type levels (as assayed by Western analysis) and induction produces a greater than 5-fold increase over basal levels (as assayed by Western analysis) .
  • Topo I poison a test compound that causes an increased sensitivity, i.e., DNA damage, growth inhibition and/or killing of the bacterial strain, when induced to produce Topo I relative to the non- or low-induced condition is identified as a Topo I poison.
  • this embodiment of the invention provides a drug screening assay that can be set up to evaluate levels of Topo I expression in excess of those that would be tolerated under normal conditions.
  • a topA-deleted test strain containing a plasmid bearing the topA gene can be induced to produce higher than normal levels of Topo I. That strain can then be compared with an otherwise isogenic strain that is wild type for topA and the differential sensitivity to test compounds determined.
  • isogenic topA + strains with inducible topA genes such as S.AD20 or SAD21 (which can be constructed as described infra in Section 6.1) can be used to compare induced versus uninduced conditions.
  • SAD20 could be induced to produce higher than normal levels of Topo I and compared to an SAD20 culture that is uninduced and produces wild-type levels of Topo I.
  • E.coli can be used as the host for the e.xpression of topA gene products from other bacteria such as S. aureus (see FIG. 3B and Section 6.1, infra) .
  • the topA gene from other pathogens such as Salmonella typhimurium. S. fecalis. P. aeru ⁇ inosa. M. tuberculosis . and H. pylori could also be used.
  • the use of a pathogen-specific target, e.g., Topo I of S . aureus . in the screens of this invention may be desirable in cases where pathogen-specific antibacterial agents are sought.
  • the Topo I poison identified by the screen causes DNA damage within the bacterial cell.
  • such confirmation can be achieved using techniques known in the art such as DNA damage assays and/or DNA cleavage assays.
  • topA bacterial Type II topoisomerase enzyme
  • DNA gyrase which aids in supercoiling of the DNA
  • an increase in the levels of Topo IV which possesses its own DNA relaxing activity, can compensate for the loss of the Topo I relaxing activity.
  • TopA ' strains that compensate for the topA defect via reduced gyrase levels will have reduced levels of Type II topoisomerase targets (reduced gyrase plus normal Topo IV)
  • topA-deleted strains that compensate for the topA defect via increased Topo IV levels will have increased Type II topoisomerase targets (normal gyrase plus elevated Topo IV)
  • the cells with increased levels of Type II topoisomerase targets will therefore display an increased sensitivity to Type II topoisomerase poisons that trap the cleavable complex intermediates of the target enzymes.
  • the strategy of varying the levels of the target for the desired antibacterial compound, wherein increased target levels translates into increased sensitivity to the antibacterial compound parallels the approach described above with respect to the Topo I poisons.
  • bacterial screens are provided for identifying Type II poisons, i.e., those compounds that trap the cleavable complex intermediate of the bacterial Type II DNA topoisomerases.
  • bacterial assays are performed comprising contacting a test compound to bacterial test strains that are isogenic except for the fact that one of the strains has a reduced level of bacterial Type II DNA gyrase targets, while the second strain has an increased level of Type II Topo IV targets.
  • one strain has a reduced level of the gyrase enzyme while the other strain has an increased level of the Topo IV enzyme.
  • the isogenic strains used can be either topA ' or topA + as long as they are isogenic for the topA gene.
  • the strain with reduced levels of the gyrase enzyme will possess a decreased number of targets for a Type II-specific poison whereas the strain with increased levels of Topo IV will possess an increased level of targets for a Type II-specific poison.
  • the strains are contacted with a test compound and the differential sensitivity of the cells are measured.
  • topA-deleted test strain A contains a compensatory mutation in the gyrA and/or gyrB genes that reduces the level of DNA gyrase in the cell and topA-deleted test strain B contains a compensatory mutation (i.e., a duplication) in the pare and/or parE genes that increases the levels of Topo IV in the cell.
  • the two strains are exposed to a test compound and the differential sensitivity of the cells to the compound is measured. Exposure of the two strains to a test compound that is a Type II topoisomerase poison that traps the cleavable complex intermediate of both the gyrase and Topo IV Type II topoisomerases will result in a significant differential sensitivity between the two strains since strain A contains Type II target levels below wild type while strain B contains Type II target levels above wild type. See Figure 5 for a schematic depiction of this screen.
  • test strains A and B containing a gyrA and/or gyrB mutation and a pare and/or parE mutation, respectively can be topA + .
  • Such strains can be constructed as described in Section 6.1, infra . e.g., RFM431 or SAD19 (gyrB mutants) versus SAD15 (parC:E duplication mutant) .
  • the screens of this embodiment involve two different Type II topoisomerase targets, additional tests can be performed to determine whether the poison is in fact interacting with both targets (as in the case of the quinolone antibiotics) or either target alone. Further characterization of the Type II poison can involve comparing the differential sensitivities displayed by the test strains with the differential sensitivities displayed by those test strains with respect to known antibiotic and other antibacterial drugs.
  • the quinolone antibiotics are known to be Type II poisons that trap the intermediates of both gyrase and Topo IV; therefore, comparison of the differential sensitivity displayed by the test strains to a test compound according to this invention with the differential sensitivities those same strains display to a known quinolone antibiotic can confirm the discovery of novel compounds with a mechanism of action similar to that of the quinolones.
  • DNA damage assays and/or DNA cleavage assays for the detection of DNA damage caused by the test compound allows for a further confirmation of the compound as a Type II poison.
  • the sensitivity of strains with reduced gyrase levels or increased Topo IV levels can be separately tested against an isogenic wild type strain.
  • This embodiment of the invention allows for the detection of antibacterial compounds that interact with one or the other of the Type II topoisomerases. Please note that since this screen involves a comparison with a strain that is wild type for the Type II topoisomerases and the test strains must be isogenic and viable, both strains in this embodiment should be topA + .
  • bacterial strains A and B are isogenic except that strain A contains a mutation in a gyr gene which results in reduced gyrase levels within the mutant strain while strain B is wild type for the gyr gene.
  • Test compounds are then contacted to the test strains and the differential sensitivity of the strains relative to each other is measured.
  • a test compound that causes a decreased sensitivity on the part of strain A containing a gyr mutation relative to the wild type strain B, is identified as a Type II DNA gyrase poison.
  • Novel strains that can be used in this embodiment include R.FM431 or SAD19, which are gyrB mutants, versus wild type RFM430; see Section 6.1, infra .
  • bacterial strains A and B can be isogenic except that strain A contains a mutation in a pare and/or parE gene which results in increased Topo IV levels within the mutant strain while strain B is wild type for those genes.
  • Test compounds are then contacted to the test strains and the differential sensitivity of the strains relative to each other is measured.
  • a test compound that causes an increased sensitivity on the part of strain A containing a pare and/or parE mutation relative to the wild type strain B, is identified as a Type II Topo IV poison.
  • Novel strains that can be used in this embodiment include SAD15, which is a parC:E duplication mutant versus wild type RFM430 (see Section 6.1 and Table I , infra) .
  • Table I lists various strains that can be used in accordance with the present invention.
  • the table indicates the relevant genotype of each strain as well as the type of mutation possessed by the strain which is relevant to this invention.
  • strain N99 also known as W3102, see Bachmann, in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 1st ed. , 5 Neidhardt (ed.), p. 1201, ASM Press 1987.
  • the gyrB temperature-sensitive/coumermycin-resistant allele, gyrB221 (cou R ) gyrB203 (Ts) was isolated in this genetic background as previously described (Menzel and Gellert, 1983, Cell 3_4: 105-113) . To ensure an isogenic derivative, this
  • a trpE deletion was placed in the N99 and N4177 genetic backgrounds by the sequential phage Pl-mediated transduction of pyrB AtrpE zci::TnlO (with phage grown on PLK831) by the sequential phage Pl-mediated transduction of pyrB AtrpE zci::TnlO (with phage grown on PLK831) by the sequential phage Pl-mediated transduction of pyrB AtrpE zci::TnlO (with phage grown on PLK831) by the
  • Strain SAD17 ' has the unusual property of being both temperature-sensitive (due to the gyrB allele) and cold- sensitive (due to the combination of the AcysBtopA mutation and the gyrBTM allele which fails to compensate for the topA deletion at low temperature, e.g., 25°C) . Strain SAD17 ' was therefore used to select for the transposon-mediated duplication (Anderson and Roth, 1979, Cold Spring Harb. Symp. Quant. Biol. 43. Pt. 2: 1083-1087) of the parC,E genes following transposon mutagenesis at 18°C, using the transposon TnlOdkan (Eliot and Roth, 1988, Mol. Gen. Genet.
  • the irap+ precursors of these strains are designated herein with a prime symbol (e.g., precursor strain SAD 1 ; actual test strain for screening SAD).
  • strain RFM430 is transformed with plasmids, PECOTOP or PSTPTOP to form strains, S.AD20 and SAD21, respectively.
  • PECOTOP contains a functional E. coli topA gene operably linked to a tac promoter and can be formed as follows: PCR primers, GGTGAATATGGGTAAAGCTC and GCTCTTCCACCAGTGGC are used to amplify DNA corresponding to the E. coli topA gene from an E.
  • Strain SAD22 can then be derived from SAD20 by transduction to Trp + and retention of the cys" ( ⁇ cystop) co- transductant using bacteriophage PI as described above for the creation of SAD17 from RFM431.
  • PSTPTOP which contains a functional S ⁇ . aureus topA gene operably linked to a tac promoter
  • PSTPTOP which contains a functional S ⁇ . aureus topA gene operably linked to a tac promoter
  • PCT primers ATGGGCAGCAGCCATCATCA and TTATTTCTGCGCTGCCTCTT for the amplification of DNA corresponding to the gene for S . aureus Topo I from an S . aureus genomic library (Clontech) .
  • the PCR fragments can be cloned into the PG.EM-T vector and transformed into JM109 and subsequently excised therefrom to replace the human Topo I gene of PMSTOPl.
  • the resultant PSTPTOP plasmid (FIG. 3B) is transformed into strain R.FM430 to create strain SAD21.
  • Strain SAD23 is then constructed from SAD21 by transduction to Trp + and retention of the cys" ( ⁇ cystop) co-transductant using bacteriophage PI as described above.
  • Strain SAD24 is formed by the transformation of strain GR4413 with PECOTOP.
  • 6.2 DIFFERE.NTIAL SENSITIVITY ASSAY IN AGAR The following procedure was used for the identification of bacterial Topo I poisons as well as bacterial Type II topoisomerase poisons.
  • paired test strains were grown in culture and then transferred to agar plates. If bacterial strains with the imp mutation are used, i.e., to increase permeability to the test compound, the strains should not be kept on agar (either on a plate or on a slant) for more than a day or two.
  • the cultures which were the source of the cells used for screening can be stored either at -80°C or preferably, in 20% glycerol at -20°C. Such a working frozen glycerol stock can be used for up to a year. Small aliquots of the frozen culture were used to inoculate liquid broth prior to each e * xperiment.
  • a Topo I screen compared strains SAD15 and S.AD16
  • a Type II topoisomerase screen compared S.AD16 and SAD17
  • Table II infra . screens similar to those exemplified herein can be readily performed using the paired test strains listed in that Table.
  • Test compounds (dissolved in 100% DMSO at a nominal concentration of 2.5 Mm) arrayed in microtiter plates were delivered (1 ⁇ l) to the agar surface using a 96-pin device (VP Scientific, San Diego CA) .
  • a 96-pin device VP Scientific, San Diego CA
  • dilutions of the DNA gyrase poison, Norfloxicin were included in control wells. Plates were inverted and incubated overnight at 37°C.
  • test compound is a Topo I or Type II topoisomerase poison, depending upon the particular screen performed.
  • Figure 4A depicts a typical Topo I screen of the invention, wherein an otherwise isogenic test strain pair, i.e., strains that are isogenic except that one strain is topA' and the other is topA + , are exposed to a test compound.
  • a test compound that acts as a general poison will create a zone of growth inhibition on both the topA ' and topA + plates.
  • a test compound that is non-poisonous will create no zone of inhibition on either plate.
  • a test compound that is a Topo I poison i.e., acting through its effect on Topo I targets, can only inhibit the topA + test strain which will show a zone of inhibition; since the topA-deleted test strain does not contain a Topo I target, it will demonstrate no zone (or a dramatically reduced zone) of inhibition.
  • Topo I poison screen exemplified herein, wild type, topA + test strain SAD15 and its isogenic, topA ' companion strain SAD16 were tested with various test compounds and in numerous instances, a zone differential of growth inhibition was observed, i.e., SAD15 exhibited a zone of inhibition whereas SAD16 did not, or exhibited a very small zone of inhibition. Those test compounds that gave these results were considered Topo I poisons and were arrayed to new plates and retested to confirm activity (see Results Section 6.3, infra) . As indicated in Figure 4A and in Table II below, similar screens can be run with paired, isogenic strains RFM431/SAD17 and SAD19/SAD18.
  • the test strain containing the inducible gene is treated so as to induce the production of Topo I prior to exposure to the test compound.
  • 10 cm petri dishes containing 40 ml of LB-agar with 50 ⁇ g ampicillin/ml with (+) or without (-) IPTG are overlaid with 10* midlog topA-deleted test strain cells in 4 ml of soft (0.7% agar) LB-agar at 48°C. when the soft-agar overlays have solidified, 4 mm cylindrical holes or wells are cored to the bottom of each petri dish using a small probe attached to a vacuum.
  • test compound is then added to the wells and the zones of inhibition around the wells are measured after 14-18 h of growth at 30°C.
  • either of the topA-deleted test strains SAD22 and SAD23 which contain, respectively, the PECOTOP and PSTPTOP plasmids described in Section 6.1 above and depicted in Figures 3A and 3B, can be grown with or without IPTG (an inducer of topA expression through its effect on the lad gene of the plasmid) .
  • IPTG an inducer of topA expression through its effect on the lad gene of the plasmid
  • the inducer When the inducer is present, the topA gene will be expressed and produce Topo I targets in the form of the Topo I enzyme; without the presence of the inducer, the strain will lack or have reduced Topo I targets.
  • Test compound to the plates containing the test strain in an induced vs. non-induced or low-induced condition, will indicate whether that test compound is a Topo I poison as follows: A general poison will produce an essentially identical zone of inhibition on both plates, while a compound that is not a poison will demonstrate no zone of inhibition on either plate. However, a Topo I poison will produce a larger zone of growth inhibition on the induced plate than on the non- or low level-induced plate since the induced test strain will contain a greater number of Topo I targets for interaction with the poison than the non- or low-induced strain.
  • test strains SAD20 and 21, which contain the PECOTOP and PSTPTOP plasmids, respectively, can be used in this inducible Topo I screen wherein either strain can be grown in the presence of IPTG for high level expression of Topo I and compared with the strain in an uninduced condition, i.e., having a wild type level of Topo I expression.
  • a differential zone of growth inhibition displayed by the test pair upon exposure to a test compound indicates that the test compound is a Topo I poison.
  • Type II topoisomerase poison screen of the invention the procedure was the same as described above utilizing the appropriate isogenic test strains, e.g., one strain that contained a reduced level of Type II topoisomerase targets and the other strain that contained an elevated level of such targets.
  • isogenic strains one of which contains a mutation in the gyrA and/or gyrB genes and one of which contains a duplication of the pare and/or parE genes, were utilized.
  • a test compound that is a Type II topoisomerase poison will create a larger zone of growth inhibition on the plate containing the strain having the greater level of Type II targets for the poison.
  • strains SAD16 and SAD17 were tested using this screen. Numerous test compounds gave a differential zone of growth inhibition in this screen, i.e., SAD16 exhibited a greater zone of inhibition than SAD17. See, e.g.. Section 6.3, infra for results.
  • other isogenic test strain pairs that can be used in this Type II screen include RFM431 and SAD15, SAD19 and SAD15, and SAD18 and SAD16.
  • test strain pairs RFM431/RFM430, SAD19/RFM430 or SAD15/RFM430 could also be used. Table II below sets forth various pairs of isogenic strains that can be used in the Topo I or Type II poison screens of this invention.
  • Type II poison screen Using the Type II poison screen exemplified above, 56 test compounds were scored as Type II poisons and of these, seven have been confirmed to date, using the DNA damage and DNA cleavage assays described infra. These compounds have also been shown to inhibit DNA gyrase using techniques as described by Reece and Maxwell, 1989, J. Biol. Chem. 266: 3540-3546. Two of these poisons are quinolones, an expected compound class as discussed supra . thus confirming the screening procedure. Five of the seven confirmed poisons represent novel structures not previously reported to have Type II inhibitory activity.
  • Type II poisons because quinolones were known to be Type II poisons, known quinolones were run as controls in the Type II screen of the invention to verify its validity. Furthermore, Table III below shows the results of a Type II poison screen of the invention using two quinolone compounds, which are known to be Type II topoisomerase poisons.
  • Ciprofloxicin (io ug/ml) 11.5 mm 8.0 mm
  • This table indicates the behavior of the type II topoisomerase test strains SAD16 and SAD17 with two known type II poisons (norfloxicin and ciprofloxicin) and two control antibacterial compounds.
  • the assay was performed as described in Section 6.2, supra.
  • the zone size is the measured diameter of the area around the well where bacterial growth was inhibited.
  • the two quinolones created a larger zone of growth inhibition on S.AD16 than on SAD17, a result indicative of a Type II poison (whereas the expected differential sensitivity was not observed with the control antibacterial compounds) .
  • This control experiment indicates that the Type II screen of the invention operates as disclosed herein.
  • DNA Damage ASSAY it may be desirable to confirm the DNA- da aging effect of the Topo I or Type II poisons identified by the screens of this invention.
  • An example of one such confirmatory test is a DNA damage assay wherein the ability of a test compound to cause DNA damage can be assessed by measuring 3-galactosidase activity in a culture of E.coli cells containing a DIN (DNA damage inducible) promoter fused to S-gala ⁇ tosidase (Huisman, 0. et al., supra: and Kenyon C. et al., 1982, J. Mol. Biol. 16£: 445-457) following the ejposure of such a culture to the compound in question.
  • DIN DNA damage inducible
  • strain GR4413 Human et al., supra
  • 190 ⁇ l aliquots were added to duplicate 10 ⁇ l samples of test compound (in 100% DMSO) distributed into the wells of a flat bottom polystyrene microtiter plate.
  • the microtiter plate was incubated overnight (12-16 hours) at 30°C without shaking.
  • a /3-galactosidase assay was initiated by using an automated pipetting device to carefully (so as to not disturb the CHC1 3 in the bottom of the microtiter plate well) transfer 20 ⁇ l of the CHCl 3 -treated culture to 180 ⁇ l of prewarmed (28 °C) Z-buffer (60 mM Na 2 HP0 4 , 40 M NaH 2 P0 4 , 10 mM KC1, 1 mM MgS0 4 , and 50 mM /3-mercaptoethanol) with 1 mg/ml chlorophenol-red 3-D-galactopyranoside (CPRG) present in each of the wells of a clear flat bottom polystyrene microtiter plate.
  • CPRG chlorophenol-red 3-D-galactopyranoside
  • a plasmid substrate such as pBR322
  • a restriction endonuclease e.g., EcoRl
  • This linear substrate was radioactively end-labeled employing standard methods well known to those practiced in the art (see Ausubel, supra or Chen et al., 1984, Proc. Natl. Acad. Sci. 259: 13560-13566) .
  • the actual cleavage reaction (20 ⁇ l) was performed by mixing the test compound, the end-labeled DNA fragment (50 ng) and the appropriate purified topoisomerase enzyme, e.g., either E.
  • DNA was freed from the topoisomerase-DNA cleavage product by protease treatment (1 ⁇ l of 1.5 mg/ml proteinase K per 20 ⁇ l, 1 hour at 42 °C) .
  • protease treatment (1 ⁇ l of 1.5 mg/ml proteinase K per 20 ⁇ l, 1 hour at 42 °C) .
  • the samples were then deproteinized with CHC1 3 - phenol as described by Ausubel, supra . and electrophoresis was used to resolved the end-labeled cleavage products on either agarose gels (1%, low resolution) or acrylamide gels (8%, 29:1 acrylamide to bis, for high resolution) according to standard procedures. Following electrophoretic resolution, visualization of the products was accomplished by autoradiography. Cleavage was indicated by the appearance of discrete low molecular weight species.

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Abstract

La présente invention a trait à des procédés et à des compositions utiles pour une modulation d'enzymes de topoisomérase bactérienne dans des cellules bactériennes. Plus spécifiquement, la présente invention a trait à des dosages bactériens dans lesquels on fait varier les taux d'enzymes de topoisomérase bactérienne ou les taux de sites cibles au sein des enzymes dans des souches d'essai bactériennes afin de rechercher des composés qui ciblent, c.-à-d. qui interagissent avec les enzymes de topoisomérase, provoquant des dommages à l'ADN, et par conséquent, une inhibition de la croissance bactérienne et/ou la mort de cellules. Les procédés et les compositions sont utiles en vue d'une identification et d'une caractérisation de nouveaux composés antibactériens thérapeutiques.
PCT/US1998/023384 1997-11-03 1998-11-03 Procedes et compositions utiles pour une modulation d'enzymes de topoisomerase bacterienne WO1999023244A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6589738B1 (en) 1999-11-09 2003-07-08 Elitra Pharmaceuticals, Inc. Genes essential for microbial proliferation and antisense thereto
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
US6783985B1 (en) 2000-02-18 2004-08-31 Elitra Pharmaceuticals Inc. Gene disruption methodologies for drug target discovery
US7838230B2 (en) 2004-11-11 2010-11-23 Plant Bioscience Limited Assay for measuring an enzyme's capability to modify supercoil topology of nucleic acids and modulators

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FLAMM R. K., ET AL.: "IN VITRO EVALUATION OF ABT-719, A NOVEL DNA GYRASE INHIBITOR.", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 39., no. 04., 1 April 1995 (1995-04-01), US, pages 964 - 970., XP002916319, ISSN: 0066-4804 *
NAKANE T., ET AL.: "IN VITRO ANTIBACTERIAL ACTIVITY OF DU-6859A, A NEW FLUOROQUINOLONE.", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 39., no. 12., 1 December 1995 (1995-12-01), US, pages 2822 - 2826., XP002916320, ISSN: 0066-4804 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
US6589738B1 (en) 1999-11-09 2003-07-08 Elitra Pharmaceuticals, Inc. Genes essential for microbial proliferation and antisense thereto
US6783985B1 (en) 2000-02-18 2004-08-31 Elitra Pharmaceuticals Inc. Gene disruption methodologies for drug target discovery
US7838230B2 (en) 2004-11-11 2010-11-23 Plant Bioscience Limited Assay for measuring an enzyme's capability to modify supercoil topology of nucleic acids and modulators

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