WO1998014784A1 - Identification et utilisation de cellules mutantes caracterisees par la resistance multiple aux anticancereux - Google Patents

Identification et utilisation de cellules mutantes caracterisees par la resistance multiple aux anticancereux Download PDF

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Publication number
WO1998014784A1
WO1998014784A1 PCT/US1997/017999 US9717999W WO9814784A1 WO 1998014784 A1 WO1998014784 A1 WO 1998014784A1 US 9717999 W US9717999 W US 9717999W WO 9814784 A1 WO9814784 A1 WO 9814784A1
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WIPO (PCT)
Prior art keywords
cell
mdr
pump
mutant
test substance
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PCT/US1997/017999
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English (en)
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WO1998014784A9 (fr
Inventor
Kim Lewis
Scott A. Siegel
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Phytera, Inc.
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Priority to JP51694098A priority Critical patent/JP2001503857A/ja
Priority to EP97945517A priority patent/EP0944830A1/fr
Publication of WO1998014784A1 publication Critical patent/WO1998014784A1/fr
Publication of WO1998014784A9 publication Critical patent/WO1998014784A9/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • MDR pumps confer to proteins that function as multi-drug resistance (MDR) pumps.
  • MDR pumps confer to proteins that function as multi-drug resistance (MDR) pumps.
  • MDRs Most bacterial MDRs belong to the large major facilitator (MF) family of
  • Escherichia coli the glucose facilitator of eukaryotic cells, and bacterial
  • QacA and QacB both located on S. aureus plasmids
  • EmrB, EmrD, and Bcr all of E. coli
  • Bmr of B. subtilis
  • NorA of S. aureus
  • a second family of MDR pumps are the smallest known translocases, 100-
  • EmrAB proteins (formerly known as MvrC or EBr).
  • Proteins encoded by acr loci including AcrAB and AcrEF, belong to a
  • An outer membrane protein, TolC is a
  • a fourth family of MDR pumps is a large family referred to as ABC
  • ATP-binding cassette translocases. These proteins use ATP as an energy source and
  • ABC proteins have 12 transmembrane ⁇ -helices. Some of the ABC proteins are uptake translocases,
  • His histidine transporter of E. coli
  • efflux pumps such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the histidine (His) transporter of
  • the P-glycoprotein a eukaryotic MDR pump
  • doxorubicin (adriamycin), a clinically useful anticancer drug.
  • the invention features a method for identifying a substance that inhibits
  • the method includes the steps of: (a) contacting the cell with a test
  • the cell is selected from yeast and, preferably, a mammalian cell, a
  • bacterial cell a saprophyte, a filamentous fungal cell, a parasite cell, and a protozoan.
  • the invention also features a method for identifying a multi-drug
  • the method includes the step of (a) contacting a first cell
  • the first cell includes a wild-type multi-drug
  • the second cell includes a mutated multi-drug resistant pump that cannot
  • the first cell has a wild-type MDR pump
  • the third cell is identical to the second cell, namely, it includes the same
  • Another step in the method is (d) measuring the growth or
  • a "dysfunctional multi-drug resistance pump” is a MDR pump that has
  • antineoplastic agents as efficiently as an otherwise identical cell possessing a
  • an MDR mutant strain may have no expression of the MDR pump protein, it may express a fusion protein, or it may express a truncated protein. All three cases
  • a cell e.g., host, parent, or wild-type cell
  • bacterial cell such as those selected from Staphylococcus aureus, Escherichia
  • Tuberculosis sp. Streptococcus pyogenes, Enterobacter sp., and Pseudomonas
  • a cell can also be a filamentous fungal cell; a cell selected from
  • lactis lactis, and Bacillus subtilis
  • a parasite cell a protozoan selected from Plasmodium
  • the cell can include one or
  • MDR pump mutations e.g., one, two, three, four, five, six, seven, eight, nine,
  • MDR pump mutations in at least one-half, two-thirds, three-fourths, or
  • component generally a protein or enzyme selected from membrane receptors, ion channels, DNA binding proteins, transcriptional regulators, steroid-responsive
  • immunosuppressive target protein cell division control genes (cdc), oncogenes, and
  • Examples include a G-protein linked receptor, a cytoplasmic
  • nucleotide phosphodiesterase The nucleotide phosphodiesterase. Cellular growth and metabolism can be measured, for
  • cellular metabolism can be measured by
  • the cell is preferably contacted with a concentration of the antibiotic (or anticancer agent
  • the cell is a mammalian cell that has undergone a neoplastic transformation
  • the second and third cells are also contacted with a test substance.
  • the second and third cells being identical strains.
  • the mutant MDR strain is such that the MIC of a
  • tetracycline, ampicillin, and gentamycin in the wild-type of the (mutant) cell is at least
  • test substance can be a substantially pure compound, or a mixture of
  • test substance can be from a natural source, such as a plant or
  • marine extract or a synthesized or semi-synthesized material, including a mixture of
  • a “substantially pure compound” is a compound separated from the
  • a compound including synthetic reagents or side reaction products.
  • a compound is
  • the compound is at least 75%, more preferably at
  • the pure compound may be chemically synthesized.
  • Purity can be measured by any appropriate method, such as thin layer
  • FIG. 1 is a graph comparing the growth inhibition of wild- type (KLE700;
  • FIG. 2 is a graph comparing the growth inhibition of wild-type (SC5314;
  • knockout mutant were 10.9 ng/ml and 1.2 ng/ml, respectively.
  • FIG. 3 is a plasmid map of pCH23, used to generate the S. aureus ANorA
  • FIG. 4 shows a scheme for disrupting the S. aureus Nor A gene by
  • FIG. 5 is a graph comparing the growth inhibition of wild-type (RN4220;
  • ANorA mutant were 1.5 ⁇ g/ml and 0.23 ⁇ g/ml, respectively.
  • FIG. 6 is a graph comparing the growth inhibition of wild-type (RN4220;
  • mutants were 6.28 ⁇ g/ml and 0.67 ⁇ g/ml, respectively.
  • the invention relates to the use of mutant cells possessing one or more
  • mechanisms e.g., antibiotic, antiprotozaon, antifungal, or antineoplastic agents.
  • the MDR mutant cell is more vulnerable than wild type cells to the
  • mutant cells can be used in a test substance.
  • active compounds may represent novel therapeutic agents.
  • mutant cells of the invention therefore increase efficiency and sensitivity.
  • MDR pump mutants can be used to screen for selective inhibitors
  • a known therapeutic agent that is extruded by the MDR pump e.g., antibacterial,
  • nonpathogenic strain is likely to do so in the pathogenic strain.
  • the invention also features a method of treating cancer or an infection of a
  • microorganism such as a fungus (filamentous, saprophyte, or yeast), bacteria,
  • corresponding therapeutic agents such as one or more anticancer agents or antibiotics.
  • growth or metabolism inhibitor includes administration of one of these two agents before, during, or after administration of the other.
  • metabolism inhibitor can be formulated together or separately.
  • administration is directed toward having the target cell (pathogen or cancerous cell)
  • the mutant cells may be microorganisms, including bacteria, fungi, yeast,
  • filamentous yeast, parasites, and protozoa or mammalian cells, particularly those that
  • MDR knockout strains are generated in
  • pathogenic bacteria yeast, and fungi including: Enterococcus fecalis, Haemophilus
  • influenzae Enterococcus fecium, Pseudomonas aeruginosa, Aspergillus fumigatus,
  • Cryptococcus neoformans and Trichophyton sp., Fusarium sp., as well as prototypic
  • Lactococcus lactis Lactococcus lactis, and Bacillus subtilis. MDR pump inhibitors, growth inhibitors, and
  • MDR pump inhibitor of S. cerevisiae can be assayed for activity against other yeast.
  • An antibacterial agent active against a Gram-positive Lactococcus can be assayed for
  • Example 1 Generation of a Strain of E. coli Deficient in the Expression of a Multi-Drug Resistant Pump Protein.
  • the mutant strain designated KLE701 was
  • the recipient strain was plated on nutrient medium with
  • KLE701 cells contain a mutation in the TolC protein, wild-type KLE700 cells grew
  • the MIC for nalidixic acid was 0.19 ⁇ g/ml for the mutant strain
  • Strain KLE701 is more sensitive to some antibiotics due to the TolC
  • a broth microtiter assay in which bacterial growth is measured by
  • test sample e.g., a crude extract such as an extract of cell cultures, or a purified
  • tissue culture plate ⁇ l of LB medium without E. coli was added to certain wells.
  • the tissue culture plate ⁇ l of LB medium without E. coli was added to certain wells.
  • the IC 50 of erythromycin using the wild-type strain (KLE700) was determined.
  • knockout KLE701 and validate the assay described above in terms of its ability to
  • microbiological sources were prepared. The crude extracts were added to microbroth
  • Example 3 Generation of a C. albicans Strain Deficient in the Expression of Multi-Drug Resistance Pump Proteins.
  • a quadruple knockout mutant designated DSY1024 Acdrl, Acdr2, Aben, and
  • test sample i.e., crude
  • phenazine methosulfate was added to each well, and plates were incubated at
  • the mutant strain within such an assay the sensitivity of the mutant, DSY1024, to
  • miconazole was compared to that of the wild type, SC5314.
  • Balzi selected from Balzi and Goffeau, Biochim. Biophys. Acta 1187:152-162 (1994); Balzi
  • Example 4 Generation of a Strain of Staphylococcus aureus Deficient in the Expression of Multi-Drag Resistance Pump Proteins.
  • the mutant strain, KLE819 was constructed by transforming
  • CAT acetyltransferase
  • selection medium containing chloramphenicol to select a stable isolate with a
  • KLE820 a disrupted NorA gene, generating strain KLE820 ANorA (hereinafter "KLE820").
  • KLE820 was tested for a ANorA phenotype using microbroth dilution
  • growth medium was soy broth supplemented with chloramphenicol (34 ⁇ g/ml).
  • mutant strain was more sensitive to bactericidal compounds that are known MDR
  • bromide was 0.23 ⁇ g/ml for the mutant and 1.5 ⁇ g/ml for the wild-type strain
  • IC 50 for acriflavin was 0.67 ⁇ g/ml for the mutant and 6.28 ⁇ g/ml for the wild-type
  • mutant strain was 0.25 ⁇ g/ml versus 1 ⁇ g/ml for the wild-type strain, representing a
  • KLE820 genotype examples include two pump genes, LmrA and LmrP, described in the gram positive bacterium Lactococcus lactis (van Veen et al.
  • Saccharomyces cerevisiae and Schizosaccharomyces pombe are the most abundant Saccharomyces cerevisiae and Schizosaccharomyces pombe.
  • MDR pumps have been disabled by insertional mutagenesis and exhibit increased
  • G-protein linked receptors cytoplasmic receptors, ion (e.g., potassium, calcium, or
  • antiviral targets e.g., host proteins or enzymes that are necessary for the virus life
  • immunosuppressive targets wherein the target proteins are inhibited to cause
  • the two hybrid system uses a galactose
  • Such screens employ an easily measured parameter, such as changes in cell growth or reporter gene expression (e.g. ⁇ -galactosidase-based
  • HTTP high throughput screening
  • genetically manipulated bacterial hosts have been used for antiviral and
  • MDR-deficient cells e.g., as screening strains or as host strains
  • S. cerevisiae would be ⁇ pdrl, Apdr3, ⁇ pdr5, and
  • a sensitive bacterial strain can be used to detect antibiotic activity in a mixture of compounds when such activity would
  • the invention allows detection of new antimicrobial and antineoplastic
  • efflux pumps can be readily identified by skilled artisans and targeted for mutation.
  • the genes include those described by Lewis (1994) hereby incorporated by reference.
  • novel MDR pumps may be identified by screening cDNA
  • novel genes can be expressed by
  • novel MDR pumps may be identified in cells that exhibit
  • NorA is more resistant to ethidium bromide, acridine, and norfloxacin
  • one or more components of the newly identified MDR pump can be isolated by
  • the plasmid can then be isolated from any recombinant clone that exhibits
  • a gene of interest such as an MDR (NorA) gene of S. aureus, is cloned
  • the gene may be cloned by
  • PCR chain reaction
  • the primers used to amplify the DNA may include one or more restriction
  • a fragment (cassette) that will be used to disrupt the gene is then prepared
  • a suitable vector such as a Tn5 -containing vector that confers
  • An excised cassette usually contains a gene that confers
  • the excised cassette is ligated into the gene, disrupting its sequence, and rendering the
  • the recombinant vector, carrying the disrupted gene is
  • the cells are plated onto nutrient medium containing
  • antibiotic resistant are those in which homologous recombination produced a
  • the gene may be amplified again by PCR and the presence of an insert
  • gene encoding a protein that functions within an MDR pump may be achieved by
  • the gene can be characterized by testing the protein it encodes for the ability

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Abstract

Cellule mutante comprenant une pompe dysfonctionnelle de résistance multiple aux anticancéreux (MDR) et méthodes d'utilisation de ces cellules dans la découverte d'agents antibactériens, d'agents antinéoplasiques et de substances fonctionnant comme inhibiteurs de la pompe de MDR.
PCT/US1997/017999 1996-09-30 1997-09-30 Identification et utilisation de cellules mutantes caracterisees par la resistance multiple aux anticancereux WO1998014784A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP51694098A JP2001503857A (ja) 1996-09-30 1997-09-30 変異型多剤耐性細胞の同定および使用
EP97945517A EP0944830A1 (fr) 1996-09-30 1997-09-30 Identification et utilisation de cellules mutantes caracterisees par la resistance multiple aux anticancereux

Applications Claiming Priority (2)

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US72454096A 1996-09-30 1996-09-30
US08/724,540 1996-09-30

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WO1998014784A1 true WO1998014784A1 (fr) 1998-04-09
WO1998014784A9 WO1998014784A9 (fr) 1998-08-13

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079257A2 (fr) * 2000-04-14 2001-10-25 Phytera, Inc. Pompes d'ecoulement mdr
WO2002055732A3 (fr) * 2001-01-11 2003-11-06 Trustees Of Tufts College Mdt(a), nouvelle proteine d'efflux creant une resistance antibiotique multiple, et utilisations correspondantes
US7011957B2 (en) 2001-09-26 2006-03-14 Northeastern University Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon
CN113621547A (zh) * 2021-07-26 2021-11-09 中南民族大学 一种豌豆根瘤菌rl3841菌株突变株的构建方法及应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CYTOMETRY, 1994, Vol. 17, WEIDER et al., "Isolation and Characterization of a Chinese Hamster Ovary Cell Mutant with Improved Staining for Indo-I", pages 33-38. *
JOURNAL OF BACTERIOLOGY, May 1995, Vol. 177, No. 9, LOMOVSKAYA et al., "EmrR is a Negative Regulator of the Escherichia Coli Multidrug Resistance Pump EmrAB", pages 2328-2334. *
PROC. NATL. ACAD. SCI. U.S.A., April 1996, Vol. 93, PAULSEN et al., "Multidrug Resistance Proteins QacA and QacB from Staphylococcus Aureus: Membrane Topology and Identification of Residues Involved in Substrate Specificity", pages 3630-3635. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079257A2 (fr) * 2000-04-14 2001-10-25 Phytera, Inc. Pompes d'ecoulement mdr
WO2001079257A3 (fr) * 2000-04-14 2002-03-21 Phytera Inc Pompes d'ecoulement mdr
WO2002055732A3 (fr) * 2001-01-11 2003-11-06 Trustees Of Tufts College Mdt(a), nouvelle proteine d'efflux creant une resistance antibiotique multiple, et utilisations correspondantes
US7011957B2 (en) 2001-09-26 2006-03-14 Northeastern University Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon
CN113621547A (zh) * 2021-07-26 2021-11-09 中南民族大学 一种豌豆根瘤菌rl3841菌株突变株的构建方法及应用
CN113621547B (zh) * 2021-07-26 2023-10-03 中南民族大学 一种豌豆根瘤菌rl3841菌株突变株的构建方法及应用

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JP2001503857A (ja) 2001-03-21

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