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WO1999051270A1 - Nitroreductase nouvelle et usages therapeutiques associes - Google Patents

Nitroreductase nouvelle et usages therapeutiques associes Download PDF

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WO1999051270A1
WO1999051270A1 PCT/US1999/007546 US9907546W WO9951270A1 WO 1999051270 A1 WO1999051270 A1 WO 1999051270A1 US 9907546 W US9907546 W US 9907546W WO 9951270 A1 WO9951270 A1 WO 9951270A1
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nitroreductase
mtz
cells
rdxa
pylori
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PCT/US1999/007546
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Avery Goodwin
Paul S. Hoffman
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Dalhousie University
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Priority to CA002325050A priority Critical patent/CA2325050A1/fr
Priority to EP99917349A priority patent/EP1069911A1/fr
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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to nitroreductases, nucleic acids encoding nitroreductases, microaerophilic bacteria from which such nitroreductases may be isolated, conjugates of targeting compounds and nitroreductases and methods of using same.
  • Metronidazole (Mtz) [l-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] is a key component of combination therapies that are widely used against Helicobacter pylori (Malfertheiner et al, 1997), a microaerophilic, Gram-negative pathogen that is highly specific for the human gastric mucosa.
  • H. pylori tends to establish chronic and often life-long infections that constitute a major cause of peptic ulcer disease and an important risk factor for gastric cancer, one of the most common malignancies worldwide (Correa, 1996). Most residents of developing countries are infected with H.
  • Mtz resistance is an important variable in the treatment of H. pylori infections, indeed its presence markedly reduces the efficiency of Mtz-containing treatment regimens (Chiba et al, 1992; Graham et al; 1992).
  • the incidence of Mtz R also varies geographically with half or more of H. pylori strains from developing countries and approximately 10-30% of strains from the US and Western Europe being Mtz (D ⁇ im et L, 1997) Veldhuyzen van Zanten et al, 1997).
  • the incidence of Mtz among H. pylori isolates generally parallels the level of Mtz usage in a particular society. Thus, it is parsimonious to imagine that many of the H.
  • Mtz H. pylori strains currently resistant to Mtz reflect the frequent use of Mtz and related nitroimidazoles for treatment of anaerobic and protozoan infections, but in dosing c regimens that generally do not eliminate Mtz H. pylori from an infected person. (Grunberg and Titsworth, 1974; Hoff and Sticht-Groh, 1984; Edwards, 1993). Any inhibition of H. pylori growth during such periods of Mtz therapy would enrich or select for Mtz strains.
  • Mtz is relatively rare in anaerobes (Rasmussen et al, 1997), and therefore, one might imagine that the high incidence of Mtz in microaerophiles is due to a mechanism of action that differs from that found in anaerobes.
  • the available evidence from studies of protozoan and anaerobic bacterial species suggests that Mtz toxicity to H. pylori might depend on its reduction to the nitro anion radical and other compounds including hydroxylamine (Moreno et al, 1982; Lindmark and Muller, 1975; Kedderis et al, 1988). Hydroxylamine is particularly damaging to macromolecules such as DNA and proteins (Lindmark and Muller, 1976; Kedderis et al, 1988).
  • pylori merit consideration: decreased Mtz uptake or active efflux; deficiency in Mtz activation or modification; target modification or loss; and increased DNA repair or oxygen scavenging capabilities (Hoffman et al, 1996). Indeed, inactivation of recA, a gene needed for generalized DNA repair and recombination, greatly enhances Mtz susceptibility of wild-type H. pylori (Thompson and Blaser, 1995); and cloned recA gene from a Mtz strain seems to increase the already very high level of resistance that E. coli exhibits (Chang et al, 1997).
  • the gene responsible for metronidazole sensitivity in H. pylori has been identified. Mutational inactivation of the gene, which encodes an oxygen-insensitive NADPH nitroreductase, referred to herein as rdxA (designated HP0954 in the entire genome sequence) (Tomb et al, 1997), is the cause of naturally acquired Mtz m H. pylori.
  • rdxA oxygen-insensitive NADPH nitroreductase
  • Fig. 1 shows the nucleotide sequence and deduced amino acid sequence of rdxA of WT strain 500.
  • the Shine-Dalgarno (SD) ribosome-binding site is underlined on the nucleotide sequence.
  • the underlined amino acid sequence defines a highly conserved region among classic nitroreductase (CNR) proteins. Cysteine residues are highlighted in bold face and the Sphl sites used for insertion of the camR cassette are underlined and noted.
  • Fig. 2. indicates the location of amino acid substitutions in RdxA from
  • H. pylori strain 1107 was created by transforming DNA from Mtz R strain 439 into Mtz s strain 500. Note that the RdxA amino acid sequence is identical, indicating allelic exchange recombination occurred outside the rdxA locus. Other clinical isolates are included for comparison. The five matched pairs of isolates are grouped separately and the amino acid substitutions are listed in Table 3.
  • novel nitroreductases having two or more cysteine residues, an alkaline pi greater than about 6.0, a preference for NADPH as an electron donor, and having the ability to convert a prodrug to one or more cytotoxic compounds.
  • invention nitroreductases have a pi of about 7.99.
  • prodrug refers to compounds of the general structure X-NO 2 , wherein X is an organic radical of structure sufficient to impart to X-NO 2 a low redox potential.
  • X-NO 2 has a redox potential in the range of about -500mV to about -350mV.
  • organic species including, without limitation, pyrroles, furans, thiophenes, imidazoles, oxazoles, thiazoles, pyrazoles, pyridines, pyrimidines, purines, quinolines, isoquinolines, carbazoles, as well as substituted variants thereof.
  • prodrug includes imidazoles, nitrofurazones, furanyls, and derivatives thereof such as nitroimidazoles, and the like.
  • Preferred prodrugs include compounds used to treat Helicobacter infections such as metronidazole, nitazoxanide, and the like.
  • An especially preferred prodrug is metronidazole.
  • a prodrug is characterized by the ability to be converted to one or more hydroxylamines by action of invention nitroreductases.
  • nitroreductases further characterized as being encoded by DNA having greater than about 90% homology with the H. pylori rdxA gene (see SEQ ID NO:l and Fig. 1).
  • invention nitroreductases contain a conserved amino acid motif common to the CNRs (QPWHF) as well as the positioning of a strategic cysteine residue (position 87, see SEQ ID NO:2).
  • QPWHF conserved amino acid motif common to the CNRs
  • invention nitroreductases are isolated from microaerophilic bacterial species such as Helicobacter, Campylobacter, and the like.
  • An especially preferred nitroreductase is the H.
  • pylori nitroreductase and homologues thereof.
  • nitroreductases can be isolated from other Helicobacter species, including, H. acinonyx, H bilis, H. bizzozeronii, H. canis, H. cholecystus, H. cinaedi, H. felis, H. fennelli, H. heilmanni, H. hepaticus, H muridarum, H. mustelae, H. nemestrenae, H. pullorum, H. rodentium, H. salamonis, H suncus, H trogontum, and the like.
  • the presently preferred nitroreductase is the RdxA of H. pylori strain HP950.
  • conjugates comprising a targeting compound and a nitroreductase, as defined herein.
  • conjugates wherein said targeting compound is covalently linked to a nitroreductase.
  • covalently linked refers to a bond between the targeting compound and nitroreductase wherein electrons are donated by one or more atoms of each to form the bond shared between the targeting compound and the nitroreductase.
  • said targeting molecule is an antibody, to include monoclonal antibodies, and the like.
  • Antibodies used in the present invention may be isolated and/or made with specificity cell surface antigens, precancerous cell surface antigens, cell surface antigens characteristic of autoimmune diseases (including for example, arthritis, Lupus, and other autoimmune diseases/conditions), tissue-specific antigens, organ-specific antigens, and the like.
  • autoimmune diseases including for example, arthritis, Lupus, and other autoimmune diseases/conditions
  • tissue-specific antigens including for example, arthritis, Lupus, and other autoimmune diseases/conditions
  • tissue-specific antigens including for example, arthritis, Lupus, and other autoimmune diseases/conditions
  • tissue-specific antigens including for example, arthritis, Lupus, and other autoimmune diseases/conditions
  • tissue-specific antigens including for example, arthritis, Lupus, and other autoimmune diseases/conditions
  • organ-specific antigens including for example, arthritis, Lupus, and other autoimmune diseases/conditions
  • antibodies, for use as targeting molecules may be generated with specificity to any cell population with characteristic antigenicity.
  • nucleic acid molecules encoding the invention nitroreductases as defined herein.
  • said nucleic acid is greater than about 90%) homologous to the H. pylori rdxA gene (see SEQ ID NO:l and Fig. 1).
  • the nucleic acid is homologous to the ORF shown in Fig. 1.
  • said nitroreductase-encoding nucleic acid is expressed in a heterotypic cell.
  • heterotypic cell refers to a cell or virus other than that in which said nucleic acid is found in nature.
  • the range of heterotypic cells in which invention nucleic acids can be expressed includes, bacteria, viruses, retroviruses, yeast, eukaryotic cells, and the like. Expression of invention nucleic acids in each of these cell types is contemplated by the present invention, as are the proteins so expressed.
  • methods for selectively killing or inhibiting the growth of target cells comprising administering invention conjugates in conjunction with administration of a prodrug, as defined herein, wherein said nitroreductase converts said prodrug into one or more cytotoxic compounds, resulting in the killing or growth-inhibition of the target cells.
  • target cells are selected from bacterial, (retro)viruses, fungi, yeast, immune system cells such as T-cells, and B-cells, tissue cell, organ cells, diseased cells, tumor cells or neoplastic cells.
  • compositions comprising the nitroreductase, or conjugated nitroreductase as defined herein.
  • pharmaceutical formulations will include a suitable carrier.
  • suitable carriers contemplated for use in the practice of the present invention include carriers suitable for oral, intravenous, subcutaneous, transcutaneous, intramuscular, intracutaneous, inhalation, and the like administration.
  • suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, and the like.
  • suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic 8 esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.
  • Suitable carriers may also include liposomes, microspheres, or latex beads, and the like.
  • Invention compounds can optionally be converted into non-toxic acid addition salts.
  • Such salts are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid.
  • Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, methanesulfonate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate, and the like.
  • Such salts can readily be prepared employing methods well known in the art.
  • methods for detecting plasmid loss by a bacterium comprising transforming a bacterium with a plasmid containing DNA encoding invention nitroreductases as described herein, and assaying for growth of said bacteria on nitroaromatic-containing media, wherein said nitroreductase, as inserted into said plasmid, is expressed in said bacteria, and identifying as having lost the plasmid, any of said transformed bacteria which grow on said nitroaromatic-containing media.
  • Methods for identifying substrates for nitroreductases as defined hereinabove.
  • Methods according to this embodiment comprise transforming a host cell with a plasmid encoding said nitroreductase, and assaying for growth of said host cell on a medium contaimng the putative substrate, wherein said nitroreductase is expressed and converts any substrate present in said medium to one or more cytotoxic compounds such that said transformed cells will be killed or growth-inhibited, and identifying as a substrate any of said putative substrates causing killing or growth-inhibition of said transformed cells.
  • kits for identifying whether a bacterial isolate expresses a nitroreductase as defined herein comprising a substrate for said nitroreductase, wherein said nitroreductase converts said substrate into one or more detectable products, and a means for detecting said product(s).
  • bacteria typically contain several different nitroreductases including flavin and ferredoxin reductases that may exhibit nitroreductase activity (Zenno et al, 1996a,b).
  • flavin and ferredoxin reductases that may exhibit nitroreductase activity
  • isfrxA HP0642
  • isfrxA HP0642
  • FrxA NAD(P)H flavin reductase
  • metronidazole resistance focused on the metabolic enzymes of H pylori; in particular, on pyruvate:ferredoxin/flavodoxin oxidoreductase (POR) and ⁇ -ketoglutarate oxidoreductase (KOR) (Hoffman et al, 1996), in part because studies in anaerobes had shown POR to be responsible for Mtz activation (Moreno et al, 1983; Narikawa, 1986 Lockerby et al, (1991). Our studies showed that POR and KOR activities of Mtz strains of H.
  • POR pyruvate:ferredoxin/flavodoxin oxidoreductase
  • KOR ⁇ -ketoglutarate oxidoreductase
  • Mtz The basis of susceptibility and resistance to the antimicrobial agent metronidazole (Mtz) in H. pylori has been examined.
  • Mtz the antimicrobial agent metronidazole
  • rdxA oxygen-insensitive, chromosomally encoded NADPH nitroreductase
  • rdxA oxygen-insensitive, chromosomally encoded NADPH nitroreductase
  • Mtz strains display no significant changes in metabolic or growth capacity compared with isogenic Mtz strains in culture (Hoffman et al, 1996).
  • the allele ofrdxA that was responsible for transformation of the Mtz S strain to Mtz R in these first experiments contained a nonsense (translational stop) codon 14 codons before the 3' c end of the ORF (as defined by sequences ofrdxA genes from Mtz strains).
  • E. coli which is normally Mtz , was rendered Mtz by cloned rdxA genes from each c _ p to of 8 Mtz H. pylori strains, but not by cloned rdxA genes from any of 8 Mtz strains 11 contain mutant (inactive) rdxA genes. DNA sequencing showed that point mutations (missense and nonsense) at other sites in rdxA were responsible for rdxA inactivation in these strains.
  • Nitroreductases from other organisms are classified as oxygen sensitive or insensitive based on whether the substrates are reduced in one- or two-electron transfer reactions respectively.
  • One-electron transfer reductions of the nitro group of a particular compound produces the nitro-anion radical, which in the presence of oxygen generates superoxide anions and regeneration of the 5 -nitro group (Moreno et al, 1983; Edwards, 1993). It has been suggested that aerobes and facultative anaerobes are resistant to Mtz because under aerobic conditions redox cycling leads to regeneration of Mtz (Smith and Edwards, 1995).
  • Mtz of Actinobacillus actinomycetemcomitans under anaerobic, but not aerobic conditions is consistent with the concept of redox cycling and the nitroreductase activity implicated in Mtz of this species may be of the oxygen-sensitive type (Pavicic et al, 1995).
  • Mtz of H. pylori was not affected by growth under different oxygen tensions; suggesting that one electron transfer is probably not involved in Mtz reduction in this microaerophilic bacterium (Smith and Edwards, 1995). The latter interpretation is supported by the present finding that an oxygen-insensitive nitroreductase is c responsible for the Mtz of H. pylori.
  • Microaerophiles in general are susceptible to Mtz, and display patterns of resistance similar to those noted for H. pylori (Hoff and Stricht-Groh, 1984; Lariviere et al., 1986), suggesting that homologues ofrdxA may be found in these other species.
  • p Naturally occurring Mtz is associated with a Mtz-inducible depression of activity of pyruvate oxidoreductase (POR) and as little as 3-5 ⁇ gml-' of Mtz in the p culture medium is sufficient to abolish POR activity of Mtz strains (Hoffman, et al., 1996).
  • nitroreductases thus far studied are of the oxygen-insensitive type and are capable of reducing nitroaromatic compounds through sequential two- electron reductions, resulting in nitroso intermediates and hydroxylamine end products (Lindmark and Muller, 1976; Bryant and Deluca, 1991).
  • This interpretation is supported by the direct demonstration that the enteric homologues of RdxA (CNRs NfsB of E. coli, Cnr of Salmonella typhimurium, and NfsB of E. Cloacae) reduce 4- and 5 -nitro compounds by two-electron transfer reactions (Bryant and Deluca, 1991; Zenno et al, 1996a;, Yamada et al, 1997).
  • the substrate specificity of the CNRs is often a function of the redox potential of the 5-nitro group (Bryant and Deluca, 1991), and in this regard the intrinsic resistance of enteric bacteria to Mtz is due to the very low redox potential of Mtz (Narikawa, 1986).
  • reduction of Mtz and other nitroaromatic compounds to mutagenic end products by S. typhimurium has been demonstrated in the Ames test (Lindmark and Muller, 1976). Null mutations in the S. typhimurium gene for Cnr, an rdxA homologue, renders S. typhimurium resistant to the mutagenic effects of nitro-containing compounds (Yamada et al, 1997).
  • Example 1 Identification of a nitroreductase that confers Mtz sensitivity in H. pylori
  • Mtz strains could transform Mtz strains to Mtz .
  • Mtz determinant independent of whether naturally occurring Mtz is caused by a particular type of allele of a normal chromosomal gene, or by an added gene that is absent from the genomes of Mtz strains, a cosmid cloning approach was employed.
  • the H. Pylori isolates used in this study were isolated from human gastric biopsy samples and were obtained from the Victoria General Hospital, Why, Nova Scotia, Canada, and have been previously described (Hoffman et al, 1996). Paired Mtz R and Mtz s from the same patient that were found to be closely matched in overall genotype had been isolated from biopsies from Peruvian and Lithuanian patients, which were kindly provided by Drs. R. H. Gilman and H. Chalkauskas respectively. Bacterial strains were grown at 37°C on Brucella agar plates supplemented with 10% fetal calf serum (FCS) in a microaerobic incubator maintained at 7%O 2 , 5% CO 2 .
  • FCS fetal calf serum
  • Liquid cultures were grown in Brucella broth with 10% FCS in 125ml screw-capped flasks; the medium was equilibrated with 7%O 2 , 5% CO 2 in the microaerobic incubator for 1 h before inoculation, and then the flasks were sealed and placed on a rotary shaker at 150r.p.m. Unless otherwise indicated, metronidazole-resistant strains were grown with 18 ⁇ gml- of Mtz, which is one half the minimal inhibitory concentration. Bacteria were harvested by centrifugation after 3-4 days of growth, 15 and either used immediately or stored as a pellet at -70°C. E. coli strains DH5 ⁇ (BRL) and ER1793 (New England Biolabs) were grown on Luria-Bertani (LB) agar plates supplemented with the appropriate antibiotics.
  • Cosmid DNAs were prepared in batches from the growth of 96 clones per microtitre plate in 50ml of LB broth and the cosmid
  • Mtz mutants were isolated by spreading 0.10ml of turbid cultures (5x.l0 cells) on Brucella agar containing between 8 and 18 ⁇ gml- Mtz.
  • ORFs Two open reading frames (ORFs) were found in the 2.3kbp fragment.
  • One ORF (corresponding to HPO955 in the entire H. pylori genome sequence, (Tomb et al, 1997)) had strong protein-level homology to the gene for prolipoprotein p diacyglycerol transferase Igt and seemed unlikely to be involved in Mtz .
  • the second ORF had protein-level homology to classical oxygen-insensitive NAD(P)H nitroreductases (CNRs) of several other Gram-negative bacteria (see Table 1) and was a good candidate because some of its homologues are known to reduce metronidazole or related compounds (Lindmark and Muller, 1976; Yamada et al, 1997).
  • This H. pylori gene corresponds to the ORF designated HP0954 in the full genome sequence (Tomb et al, 1997) and, interestingly, exhibits 54% similarity with another 17
  • ORF that encodes a NAD(P)H flavin nitroreductase (f ' rxa ' herein), also a CNR homologue.
  • the sequences have been deposited in GenBank (AFO12552, AFO12553).
  • Salmonella Cnr 30 50 typhimurium
  • the inferred RdxA product from Mtz H. pylori strain 439 is 196 amino acids long. PCR amplification and sequencing of the corresponding segment from the Mtz strain 500 revealed an ORF that is 14 codons longer at the 3' end (210 codons, see Fig. 1).
  • the rdxA gene from a Mtz R transformant of strain 500 (strain HP 1107) that was made with genomic DNA from strain 439 was identical in DNA sequence to that p of the 439 parent strain (Fig. 2).
  • the WT rdxA gene was 630bp in length and had a Shine-Dalgarno sequence 5bp upstream of the start codon.
  • One of the cysteine residues (position 87) is conserved in the CNR 18 proteins of the enterics.
  • the cysteine located at position 159 is in a motif (L/IDSCI/PI) shared with the inferred product of fix A.
  • Another motif common to all of the CNRs is QPWHF (PW is absolutely conserved) located within a highly conserved region between positions 43-59 in RdxA.
  • Example 2 Nitroreductase activity and rdxA expression in E. coli c p
  • H pylori independent of whether NADPH or NADH were used as electron donors; this is consistent with earlier observations (Hoffman et al, 1996).
  • the inability to detect Mtz reductase activity in cell-free extracts of H pylori might be attributable to oxidation of key components during the preparation, or to an inability of the assays used to detect very low levels of Mtz reductase activity.
  • E. coli DH5 containing pBluescriptSK ⁇ /4 clones from all H pylori strains c used in this study were screened for Mtz on Luria Bertani medium containing a range of Mtz concentrations from 0 to 60 ⁇ gml- .
  • the plates were streaked for isolation of colonies or a 1 : 1 :00 dilution of a 0.40D 660 broth culture was spread onto the medium. The plates were incubated under aerobic conditions at 37°C and then scored for growth at 16-24h.
  • the cloned rdxA gene from the H. pylori strain that rendered E. coli most susceptible to Mtz was tested for nitroreductase activity by spectrophotometric assay.
  • Cell-free extracts from E. coli harboring rdxA from this strain exhibited 40-fold higher than background NADPH-dependent nitroreductase activity using metronidazole as the electron acceptor, and assayed by following either Mtz reduction or oxidation of NADPH (Table 2). No detectable reductase activity was found using NADH instead of NADPH as the electron donor, nor was any detected using extracts of.E.
  • Metronidazole reduction was measured in crude extracts of E. coli strain JF626 grown aerobically in LB broth.
  • pBSK is pBluescript vector control;
  • pBS950 is WT rdxA cloned into pBSK and
  • pBS1043 is MtzR rdxA cloned in pBSK.
  • the assay contained NADPH and Mtz.
  • the enzymatic reaction was followed at 320 nm to measure Mtz reduction and at 340 nm to measure NADPH oxidation. The values are corrected for NADPH oxidase activity. No activity was found when NADH was used as substrate.
  • Cell-free extracts were prepared from bacteria that had been grown to mid to late log phase in the appropriate medium and where indicated, either in the presence or absence of 18 ⁇ gml " Mtz.
  • the reaction mixture contained Tris/acetate (lOOmM Tris-HCl, 50mM acetate) pH 7.0, 0.05mM Mtz and 0.3mM NADPH or HADH, POR (EC 1.2.7.1) was assayed under anaerobic conditions with 74mM potassium phosphate (pH 7.3), lOmM sodium pyruvate, 5mM benzyl viologen, 0.18mM coenzyme A (CoA), and 5 ⁇ M thiamine PP as described previously (Hoffman et al, 1996). Reduction of benzyl viologen was followed at 546nm and specific activity was determined for the reaction using an extinction coefficient of 9.2mM " cm . Specific activities were reported as nmoles per min per mg of protein. Protein determinations were performed using the Bradford procedure (Bio-Rad) with bovine serum albumin as the standard.
  • rdxA sequences from various strains of H pylori were amplified and cloned into pBluescript using primer pairs Mtz6EF (forward) 5'- TGAATTCGAGCATGGGGCAG and reverse primer Mtz R Bgl 5'- AGCAGGAGCATCAGATAGATCTGADNA.
  • pDH26 a chimeric shuttle vector
  • H pylori strain 500 sequences spanning the rdxA ORF were excised from pBluescript by EcoRY and Sail digestion and subcloned into similarly restricted pDH26.
  • H pylori strain 1061 was made MtzR by natural transformation of pBluescriptSKnbC4 originating from Mtz strain 439. The pDH26rdxA plasmid was introduced into strain
  • CM colonies were subsequently screened for Mtz s phenotype on Brucelia agar containing CM and 18 ⁇ gml "1 Mtz to demonstrate dominance of wild-type rdxA through loss of the Mtz phenotype.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Selon la présente invention, le gène responsable de la sensibilité au métronidazole chez H. pylori a été identifié. L'inactivation mutationnelle du gène, qui code pour une NADPH nitroréductase insensible à l'oxygène, appelée ici RdxA (désignée par HP0954 dans la séquence du génome entier) (Tomb et al.,1997), est la cause de MtzR naturellement acquis dans H. pylori. Selon une des réalisations, la présente invention concerne un procédé d'utilisation de RdxA et de composés associés, éventuellement conjointement avec des composés de ciblage, destiné à convertir des composés aromatiques nitrés en cytotoxines pour les utiliser dans la destruction sélective ou l'inhibition de croissance de populations de cellules cibles. Selon une autre réalisation, la présente invention concerne un procédé d'utilisation de RdxA et de composés associés destiné à convertir des composés aromatiques nitrés en cytotoxines pour les utiliser dans l'élimination, par sélection, des cellules qui expriment RdxA.
PCT/US1999/007546 1998-04-06 1999-04-06 Nitroreductase nouvelle et usages therapeutiques associes WO1999051270A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057237A3 (fr) * 2000-02-02 2002-05-16 Amersham Biosciences Uk Ltd Procede et reactif de detection par fluorescence
US7906106B2 (en) 2007-06-27 2011-03-15 General Electric Company In vivo cell trafficking
US8021647B2 (en) 2007-06-29 2011-09-20 General Electric Company In vivo optical imaging
EP2793871A4 (fr) * 2011-12-23 2015-07-22 Auckland Uniservices Ltd Composés et procédés pour l'imagerie et/ou l'ablation sélectives
CN109593842A (zh) * 2018-12-21 2019-04-09 上海芯超医学检验所有限公司 一种检测和判定幽门螺旋杆菌耐药性的试剂盒和方法
US10357577B2 (en) 2010-07-16 2019-07-23 Auckland Uniservices Limited Bacterial nitroreductase enzymes and methods relating thereto
CN110613687A (zh) * 2019-10-30 2019-12-27 北京利普松生物科技有限公司 一种复方呋喃西林纳米脂质体及其制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5387598A (en) * 1994-04-13 1995-02-07 Rossignol; Jean-Francois Composition and galenic formulation suitable for combatting affections of the lower abdomen
WO1997002020A1 (fr) * 1995-07-05 1997-01-23 Byk Gulden Lomberg Chemische Fabrik Gmbh Composition pharmaceutique administrable par voie orale contenant des agents actifs antimicrobiens et du pantoprazole a liberation differee
US5633158A (en) * 1991-10-23 1997-05-27 Cancer Research Campaign Technology Limited Bacterial nitroreductase for the reduction of CB 1954 and analogues thereof to a cytotoxic form

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5633158A (en) * 1991-10-23 1997-05-27 Cancer Research Campaign Technology Limited Bacterial nitroreductase for the reduction of CB 1954 and analogues thereof to a cytotoxic form
US5387598A (en) * 1994-04-13 1995-02-07 Rossignol; Jean-Francois Composition and galenic formulation suitable for combatting affections of the lower abdomen
WO1997002020A1 (fr) * 1995-07-05 1997-01-23 Byk Gulden Lomberg Chemische Fabrik Gmbh Composition pharmaceutique administrable par voie orale contenant des agents actifs antimicrobiens et du pantoprazole a liberation differee

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE MPSRCH GENBANK 1 January 1900 (1900-01-01), XP002921454, Database accession no. AE000604 *
GOODWIN A, ET AL.: "METRONIDAZOLE RESISTANCE IN HELICOBACTER PYLORI IS DUE TO NULL MUTATIONS IN A GENE (RDXA) THAT ENCODES AN OXYGEN-INSENSITIVE NAPDH NITROREDUCTASE", MOLECULAR MICROBIOLOGY., WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 28, no. 02, 1 January 1998 (1998-01-01), GB, pages 383 - 393, XP002921452, ISSN: 0950-382X, DOI: 10.1046/j.1365-2958.1998.00806.x *
LEVINSON, ET AL.: "PATHOGENESIS & EPIDEMIOLOGY", MEDICAL MICROBIOLOGY AND IMMUNOLOGY, BERLIN, DE, 1 January 1994 (1994-01-01), DE, pages 98/99, XP002921455 *
TOMB J.-F., ET AL.: "THE COMPLETE GENOME SEQUENCE OF THE GASTRIC PATHOGEN HELICOBACTER PYLORI.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 388., no. 6642., 7 August 1997 (1997-08-07), United Kingdom, pages 539 - 547 + TABEL., XP002062106, ISSN: 0028-0836, DOI: 10.1038/41483 *
TOWNSON S M, ET AL.: "RESISTANCE TO THE NITROHETEROCYCLIC DRUGS", ACTA TROPICA., ELSEVIER SCIENCE BV., AMSTERDAM., NL, vol. 56, no. 02/03, 1 January 1994 (1994-01-01), NL, pages 173 - 194, XP002921453, ISSN: 0001-706X, DOI: 10.1016/0001-706X(94)90062-0 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057237A3 (fr) * 2000-02-02 2002-05-16 Amersham Biosciences Uk Ltd Procede et reactif de detection par fluorescence
JP2003522333A (ja) * 2000-02-02 2003-07-22 アマシャム バイオサイエンス ユーケイ リミテッド 蛍光検出法および試薬
AU782381B2 (en) * 2000-02-02 2005-07-21 Ge Healthcare Uk Limited Methods employing increased dye-fluorescence upon nitroreduction
US7906106B2 (en) 2007-06-27 2011-03-15 General Electric Company In vivo cell trafficking
US8021647B2 (en) 2007-06-29 2011-09-20 General Electric Company In vivo optical imaging
US10357577B2 (en) 2010-07-16 2019-07-23 Auckland Uniservices Limited Bacterial nitroreductase enzymes and methods relating thereto
EP2793871A4 (fr) * 2011-12-23 2015-07-22 Auckland Uniservices Ltd Composés et procédés pour l'imagerie et/ou l'ablation sélectives
CN109593842A (zh) * 2018-12-21 2019-04-09 上海芯超医学检验所有限公司 一种检测和判定幽门螺旋杆菌耐药性的试剂盒和方法
CN109593842B (zh) * 2018-12-21 2021-01-01 上海芯超生物科技有限公司 一种检测和判定幽门螺旋杆菌耐药性的试剂盒和方法
CN110613687A (zh) * 2019-10-30 2019-12-27 北京利普松生物科技有限公司 一种复方呋喃西林纳米脂质体及其制备方法

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