IDENTIFICATION AND USE OF MUTANT MULTI-DRUG RESISTANT CELLS

Abstract: A mutant cell comprising a dysfunctional multi-drug resistance (MDR) pump, and methods for using such cells in the discovery of antibacterial agents, antineoplastic agents, and substances that function as inhibitors of the MDR pump.

Description: IDENTIFICATION AND USE OF MUTANT MULTI-DRUG RESISTANT CELLS The work described herein was supported in part by a grant from the National Science Foundation (MCB-9496291). As a result, the U.S. government may have certain rights to aspects of the inventions disclosed herein. This is a continuation-in-part application of U.S. Serial No. 08/724,540 filed on September 30, 1996. Background of the Invention Most microorganisms and many types of mammalian cells express proteins that function as multi-drug resistance (MDR) pumps. MDR pumps confer resistance to a large variety of chemically unrelated agents by extruding them from the cell (for review, see Lewis, Trends in Biochem. Sci. 19:119-123, 1994). Most bacterial MDRs belong to the large major facilitator (MF) family of membrane translocases, which includes the arabinose/H + symporter of Escherichia coli, the glucose facilitator of eukaryotic cells, and bacterial tetracycline/H + antiporters. Most MF translocases have a transmembrane structure composed of 12 α-helices and use a proton motive force (pmf) as a source of energy. Members of this family include QacA and QacB (both located on S. aureus plasmids), EmrB, EmrD, and Bcr (all of E. coli), Bmr (of B. subtilis), and NorA (of S. aureus). A second family of MDR pumps are the smallest known translocases, 100- mers that span the membrane with four helices. Members of this family include QacC (renamed Smr for Staphylococcus multi-drug resistance) which extrudes a broad range of lipophilic cations from the cell in a pmf-dependent manner, and the related proteins QacE, and EmrE (formerly known as MvrC or EBr). The EmrAB proteins (Lomovskaya et al., Proc. Natl. Acad. Sci. USA 89:8938-8942, 1992) confer resistance to a range of hydrophobic compounds, including the antibiotics thiolactomycin and nalidixic acid. Proteins encoded by acr loci, including AcrAB and AcrEF, belong to a third family, the RND family, and confer resistance to the mutagen acridine and to erythromycin, tetracycline, and β-lactams. An outer membrane protein, TolC, is a component of both the EmrAB and the AcrAB pumps of E. coli. The Mex proteins of the RND family described in Pseudomonas aeruginosa (Poole et al., Mol. Microbiol. 10:529-544, 1993) confer resistance to a broad range of antimicrobial agents and are responsible for the high level of natural resistance of pseudomonads to antibiotics. 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 have 12 transmembrane α-helices. Some of the ABC proteins are uptake translocases, such as the histidine (His) transporter of E. coli, while others are efflux pumps, such as the HlyB translocase of hemolysin. The P-glycoprotein, a eukaryotic MDR pump, extrudes a number of substances from cells in which it is expressed, including doxorubicin (adriamycin), a clinically useful anticancer drug. Summary of the Invention The invention features a method for identifying a substance that inhibits the growth or metabolism of a mutant cell including a dysfunctional multi-drug resistance pump. The method includes the steps of: (a) contacting the cell with a test substance; and (b) determining whether the growth or metabolism of the cell is inhibited. 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 resistance pump inhibitor. The method includes the step of (a) contacting a first cell with a test substance and an antibiotic. The first cell includes a wild-type multi-drug resistant pump that extrudes the antibiotic from the first cell. The method also includes (b) contacting a second cell with the test substance in the absence of the antibiotic. The second cell includes a mutated multi-drug resistant pump that cannot extrude the antibiotic from the second cell, although the second cell is otherwise identical to the first cell. In other words, the first cell has a wild-type MDR pump protein and the second cell has a dysfunctional or mutant MDR pump. The method also includes (c) contacting a third cell with the test substance in the presence of the antibiotic. The third cell is identical to the second cell, namely, it includes the same MDR pump mutation. Another step in the method is (d) measuring the growth or metabolism of the first, second, and third cells. The presence in the test substance of a MDR pump inhibitor is indicated by (i) inhibition of the growth or metabolism of the first cell, and (at the same time) (ii) inhibition of the second cell which is both comparable to the inhibition of the third cell and also lower than the inhibition of the first cell. In other words, if the test substance inhibits the MDR pump, then the growth or metabolism of the wild type cells will be inhibited due to the increased intracellular concentration of the antibiotic and the growth or metabolism of the mutant cell will not be inhibited. This lack of inhibition of the mutant indicates that the inhibition of the wild type cells was caused by blocking the MDR pump, rather than by a different mechanism, such as direct antibacterial, antifungal, or antineoplastic activity of the test substance. Thus, the parallel use of mutant and wild type cells allows the detection of MDR inhibitors and simultaneously demonstrates the mode of action as MDR inhibitors rather than cytotoxic or cytostatic agents which inhibit both the wild type and the hypersensitive mutant cell. Terms used in these two methods are exemplified below. A "dysfunctional multi-drug resistance pump" is a MDR pump that has been altered so that it does not extrude toxic agents (such as antibacterial or antineoplastic agents) as efficiently as an otherwise identical cell possessing a wildtype MDR pump. Any component of the MDR pump may be altered, for example, by an insertional or deletional mutation, or wherein at least one of the genes that encodes a protein within a given MDR pump is not expressed, due to failure of gene transcription or mRNA translation. In addition, a dysfunctional MDR pump may have one or more inappropriately or incompletely modified protein components. In short, 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 result in a functionally defective MDR pump. In the methods of the invention, a cell (e.g., host, parent, or wild-type cell) can be a bacterial cell such as those selected from Staphylococcus aureus, Escherichia coli, Enterococcus fecalis, Haemophilus influenzae, Enterococcus fecium, Tuberculosis sp., Streptococcus pyogenes, Enterobacter sp., and Pseudomonas aeruginosa. A cell can also be a filamentous fungal cell; a cell selected from Aspergillus fumigatus, A. nidulans, and A. flavus; a saprophyte selected from Trichophyton sp. and Fusarium sp.; a nonpathogenic cell selected from Lactococcus lactis, and Bacillus subtilis; a parasite cell; a protozoan selected from Plasmodium falciparum, P. ovale, P. vivax, P. malariae, Trypanosoma brucei, Toxoplasma sp. , and Pneumocystis sp.; or a mammalian cell, such as one that has undergone a neoplastic transformation. Where a cell includes a mutant MDR pump, the cell can include one or more MDR pump mutations (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve up to the total number of MDR pumps in a given cell). As the proportion of dysfunctional MDR pumps increases, the greater the sensitivity and the broader the range of compounds the mutant cell will be capable of detecting. A cell can include MDR pump mutations in at least one-half, two-thirds, three-fourths, or four- fifths of the MDR pump proteins in the cell. "Inhibition of growth or metabolism" includes inhibition of a cell component (generally a protein or enzyme) selected from membrane receptors, ion channels, DNA binding proteins, transcriptional regulators, steroid-responsive elements, protein kinases, protein phosphorylases, viral processing enzymes, an immunosuppressive target protein, cell division control genes (cdc), oncogenes, and signal transducers. Examples include a G-protein linked receptor, a cytoplasmic receptor, a fungal cell wall enzyme, fungal sterol biosynthesis enzymes, and cyclic nucleotide phosphodiesterase. Cellular growth and metabolism can be measured, for example, by assessing the activity of one or more of the enzymes that function in cellular metabolic pathways. Alternatively, cellular metabolism can be measured by any of the surrogate markers that are known to cell biologists. According to the method for identifying an MDR pump inhibitor, the first cell is preferably contacted with a concentration of the antibiotic (or anticancer agent if the cell is a mammalian cell that has undergone a neoplastic transformation) that is less than one-fourth of the MIC 90 for the first cell. This concentration is routinely determined by serial dilution assays for a given cell, antibiotic, and test substance, such that in a wild-type cell, the concentration of antibiotic is not high enough to be cytotoxic, and yet sufficient to inhibit the MDR pump, for example, by competing with the test substance. The second and third cells are also contacted with a concentration of the antibiotic that is less than one-fourth of the MIC 90 for the second cell, the second and third cells being identical strains. Regarding the method of identifying an inhibitor of cell growth or metabolism, in some embodiments, the mutant MDR strain is such that the MIC of a compound selected from chloramphenicol, norfloxacin, acriflavin, nalidixic acid, miconazole, erythromycin, ethidium bromide, fluconazole, levofloxacin, ciprofloxacin, inoxacin, ofloxacin, rhodamine 69, brefeldin A, daunomycin, TPP, tetracycline, ampicillin, and gentamycin in the wild-type of the (mutant) cell is at least two, five, eight, fifteen, twenty, thirty, fifty, one hundred, 150, 250, 300, 400, 800, or 1200 times greater than the MIC in the (mutant) cell. A "test substance" can be a substantially pure compound, or a mixture of compounds. The 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 synthetic compounds such as those resulting from combinatorial or matrix synthesis methods. A "substantially pure compound" is a compound separated from the components with which it is naturally associated or from an artificial combination of compounds, including synthetic reagents or side reaction products. A compound is substantially pure when it is at least 60%, by weight, free from the organic molecules with which it is naturally associated or with which it was combined for experimental testing or synthesis. Preferably, the compound is at least 75%, more preferably at least 90%, and most preferably at least 99% pure, by weight. A substantially pure compound may be obtained, for example, by extraction from a natural source such as a mammalian, plant, or microbial cell, or the culture medium in which these cells were grown. Alternatively, the pure compound may be chemically synthesized. Purity can be measured by any appropriate method, such as thin layer chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. This description is meant to illustrate, not limit, the invention. Other features and advantages of the invention will be apparent from the following detailed description and from the claims. Brief Description of the Drawings FIG. 1 is a graph comparing the growth inhibition of wild- type (KLE700; squares) and mutant (KLE701; triangles) strains of E. coli by various concentrations of erythromycin. The calculated IC 50 's for the wild-type and Tol C mutant were 16.6 μg/ml and 1.0 μg/ml, respectively. FIG. 2 is a graph comparing the growth inhibition of wild-type (SC5314; squares) and mutant (DSY1024; triangles) strains of C. albicans by various concentrations of miconazole. The calculated IC 50 's for the wild- type and quadruple 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 mutant strain. FIG. 4 shows a scheme for disrupting the S. aureus Nor A gene by homologous recombination of plasmid pCH23 into the S. aureus chromosome. FIG. 5 is a graph comparing the growth inhibition of wild-type (RN4220; squares) and mutant (KLE820; triangles) strains of S. aureus by various concentrations of ethidium bromide. The calculated IC 50 's for the wild-type and the ANorA mutant were 1.5 μg/ml and 0.23 μg/ml, respectively. FIG. 6 is a graph comparing the growth inhibition of wild-type (RN4220; squares) and mutant (KLE820; triangles) strains of S. aureus by various concentrations of acriflavin. The calculated IC 50 's for the wild-type and the ANorA mutant were 6.28 μg/ml and 0.67 μg/ml, respectively. Detailed Description of the Invention The invention relates to the use of mutant cells possessing one or more dysfunctional MDR pumps in drug discovery. For example, such mutant bacteria are more susceptible to antibacterial agents and therefore provide a more sensitive screening tool. The mutant cells used to identify compounds which are MDR pump inhibitors and biologically active (cytotoxic or cytostatic) compounds in a test substance mixture. Clinically, co-administration of an MDR pump inhibitor may enhance the efficacy of new or existing agents that are subject to MDR pumping mechanisms (e.g., antibiotic, antiprotozaon, antifungal, or antineoplastic agents). The MDR mutant cell is more vulnerable than wild type cells to the inhibitory influence of a test substance. First, the mutant cells can be used in a hypersensitive assay to rapidly screen test substance mixtures. The dysfunctional pump increases the intracellular concentration of pharmacophores that would otherwise be present at intracellular levels that are ineffective against wild type cells because the pharmacophore is extruded from the cell. Thus the pharmacophore would remain undetected by screening mixtures in assays using wild-type cells. Yet these low abundance, active compounds may represent novel therapeutic agents. The mutant cells of the invention therefore increase efficiency and sensitivity. Second, MDR pump mutants can be used to screen for selective inhibitors of these pumps. Both mutant and wild type cells are tested; the latter in the presence of a known therapeutic agent that is extruded by the MDR pump (e.g., antibacterial, antifungal, antineoplastic, or antiprotozoan agent). As discussed in the Summary section, a compound that (a) does not by itself inhibit the growth of a mutant strain when incubated in the absence of a known inhibitor, but that (b) inhibits wild-type cell growth in the presence of an ineffective or weakly effective concentration of a known inhibitor, is likely to be an effective MDR pump inhibitor. According to the invention, where there is a high degree of homo logy between a pathogenic strain and a nonpathogenic strain, a test substance that inhibits an MDR pump in the 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, parasite, or protozoan, by co-administering an MDR pump inhibitor with the corresponding therapeutic agents, such as one or more anticancer agents or antibiotics. Co-administration of an MDR pump inhibitor and a therapeutic agent, for example, a growth or metabolism inhibitor, includes administration of one of these two agents before, during, or after administration of the other. The pump inhibitor and growth or metabolism inhibitor can be formulated together or separately. The formulation and administration is directed toward having the target cell (pathogen or cancerous cell) exposed to both the MDR pump inhibitor and the therapeutic agent. The mutant cells may be microorganisms, including bacteria, fungi, yeast, filamentous yeast, parasites, and protozoa, or mammalian cells, particularly those that have undergone neoplastic transformation. MDR knockout strains are generated in pathogenic bacteria, yeast, and fungi including: Enterococcus fecalis, Haemophilus influenzae, Enterococcus fecium, Pseudomonas aeruginosa, Aspergillus fumigatus, A. nidulans, A. βavus, Tuberculosis sp. , Streptococcus pyogenes, Enterobacter sp. , Cryptococcus neoformans, and Trichophyton sp., Fusarium sp., as well as prototypic organisms not known for pathogenicity such as Saccharomyces cerevisiae, Lactococcus lactis, and Bacillus subtilis. MDR pump inhibitors, growth inhibitors, and metabolism inhibitors of the knockout strains of prototypic microorganisms can be further assayed for similar activity against pathogenic organisms. For example, an 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 activity against the closely-related Enterococcus. Examples Example 1 : Generation of a Strain of E. coli Deficient in the Expression of a Multi-Drug Resistant Pump Protein. A mutant E. coli that bears a dysfunctional MDR pump was generated from a wild-type strain of E. coli. The mutant strain, designated KLE701, was constructed by transducing a transposon insertion in the tolC gene from a strain carrying multiple mutations into wild type E. coli. Transducing phage PI was used to lyse strain MC4100, araD139, de(argF-lac)U1698, rpsL150, relA, flbB5301, ptsF25, deoCl, TolC::Tnl0. The lysate, which carried DNA fragments packaged into defective PI , was then used to infect the recipient wild-type E. coli strain K12 (KLE700) according to standard methods (see, e.g., J.H. Miller "A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria" CSH Laboratory Press, Cold Spring Harbor, N.Y., 1992). Following infection, the recipient strain was plated on nutrient medium with tetracycline. Transductants that received a disrupted tolC gene, with a transposon (TnlO) insertion conferring tetracycline resistance, grew on the tetracycline- containing medium. The resulting strain, designated KLE701 tolC::TnlO (hereinafter "KLE701"), was analyzed for the display of a mutant TolC phenotype. Colonies of KLE700 and KLE701 were streaked on plates containing McConkey medium, which included bile salts that kill tolC mutant cells. Consistent with the hypothesis that KLE701 cells contain a mutation in the TolC protein, wild-type KLE700 cells grew on McConkey medium, but KLE701 cells did not. In a second test of the mutant strain, disks impregnated with neomycin and erythromycin (30 and 15 μg, respectively; Difco Laboratories, Detroit, MI) were placed on lawns of KLE700 or KLE701 cells. Strain KLE700 appeared to be resistant to these antibiotics: the wild- type E. coli strain, bearing a functional MDR pump, apparently extruded the antibiotics and grew normally. In contrast, a clear appeared around the disks that were placed on KLE701 lawns, indicating inhibition, i.e., little or no growth of the mutant cells. In a third test of the mutant strain, strips (from AB BioDisk), each impregnated with a gradient of a known antimicrobial agent, were placed on a lawn of KLE700 or KLE701 cells. MICs were determined based upon the concentration of the antimicrobial agent at the position of the strip at which a clear zone was first evident. Mutant strain KLE701 was more sensitive to some of the drugs tested, as evidenced by clear zones that were larger than those observed with the wild-type strain. For example, the MIC for nalidixic acid was 0.19 μg/ml for the mutant strain versus 6 μg/ml for the wild-type strain, representing a 31.6-fold increase in sensitivity. Similarly, the MIC for chloramphenicol was 0.38 μg/ml for the mutant strain versus 6 μg/ml for the wild-type strain, representing a 15.8-fold increase in sensitivity. Strain KLE701 is more sensitive to some antibiotics due to the TolC knockout mutation (FIG. 1). In further examples, other deletional mutations of MDR pumps are superimposed upon the KLE701 genetic background. For example, MDR pump bcr (Nichols and Guay, Antim. Agents Chemotherapy 33:2042-2048, 1989; Bently et al. Gene 127:117-120, 1993), can be mutated to generate a double-knockout strain useful in an assay screening for antibiotics. Example 2: Determination of Wild-type and Mutant E. coli Growth in the Presence of Potential Pharmacophores A broth microtiter assay, in which bacterial growth is measured by turbidity, was performed for E. coli strains KLE700 and KLE701. Approximately 2 μl of a test sample, e.g., a crude extract such as an extract of cell cultures, or a purified compound, and 98 μl of LB containing approximately 5 X 10 5 CFU/ml of either strain (based on a visual comparison to a MacFarland standard) were added to the wells of a 96-well tissue culture plate. Typically, the assay was performed in triplicate. The LB used to dilute the recombinant strain (KLE701) was supplemented with 5 μg/ml tetracycline to maintain selective pressure on the mutation. As a negative control, 98 μl of LB medium without E. coli was added to certain wells. The tissue culture plate was then placed in a 36 °C incubator for 18-20 hours. At the conclusion of that period, turbidity was assessed by measuring the optical density at 650 nm (i.e. A 650 ), and growth inhibition was calculated as the percent decrease in A 650 compared to control cell cultures. To validate both the assay described above and the mutant strain, the sensitivity of mutant (KLE701) and wild-type (KLE700) E. coli cells to erythromycin was determined. The IC 50 of erythromycin using the wild-type strain (KLE700) was 16.6 μg/ml, while the IC 50 of erythromycin using the mutant strain (KLE701) was 1 μg/ml (FIG. 1), based on fitting the data to a four parameter fit (Graph Pad™ Prism Software). Since erythromycin is known to be extruded from wild-type E. coli via the AcrAB/TolC MDR pump, these results demonstrate the effectiveness of the MDR knockout KLE701 and validate the assay described above in terms of its ability to detect the greater potency of pumped antibiotics in the mutant, MDR knockout bacterial strain. A pilot screen was performed in order to establish the utility of MDR mutant bacteria in screens for novel antibiotics. Two thousand five hundred crude extracts, aqueous and organic extracts from plant cell cultures and marine microbiological sources, were prepared. The crude extracts were added to microbroth cultures of the mutant and wild-type strains, and cell growth was measured by optical density (at A 260 ), as described above. An extract was designated "active" if, in two consecutive tests, growth was inhibited by at least 50% at the standard test concentration. The standard test concentration for plant culture extracts and marine samples, respectively, was 100 μg/ml and 125 μg/ml, respectively. None of the 2500 extracts tested was active against the wild-type strain, whereas 10 samples (0.39%) were active against the mutant strain. After repeated rounds of bioassay-guided fractionation, one novel compound was identified using the mutant strain. Example 3: Generation of a C. albicans Strain Deficient in the Expression of Multi-Drug Resistance Pump Proteins. A mutant C. albicans strain with four disrupted multi-drug resistance pump genes was generated from a wild-type C. albicans strain by methods described in Fonzi and Irwin (Genetics 134: 717-728, 1993). The wild-type strain, designated SC5314, possesses a Ura " phenotype due to a deletion of the Ura3 locus, and was used as a parent strain for targeted mutagenesis. Sequential disruption of the pump genes was done by Sanglard, Ischer, Monod, and Bille (Microbiology 143: 405-416, 1997), according to methods described by Alani, Cao, and Kleckner (Genetics 116: 541-545, 1987). A quadruple knockout mutant, designated DSY1024 Acdrl, Acdr2, Aben, and Aflu (hereinafter "DSY1024") was generated. To test the drag sensitivity of mutant strain DSY1024, cell metabolic activity (and hence growth) was measured by a colorimetric assay in a high-throughput broth microtiter assay. Two microliters of a test sample, i.e., crude extract or purified compound and 98 μl of RPMI, 0.1 M MOPS, pH 7, containing approximately 500 CFU/ml of either SC5314 or DSY1024 (based on hemacytometer counts of a liquid suspension) were added to the wells of a 96-well microtiter plate. Ninety-eight microliters of RPMI/MOPS (minus cells) was used as a negative control, and 98 μl of RPMI/MOPS containing 500 CFU/ml and 2 μl of drug diluent (DMSO) were used as a positive control for cell growth. Assays were done either in duplicate or in triplicate. Microtiter plates were incubated at 35°C for 18-22 hours, after which 25 μl of a solution containing the metabolic dye 2,3-bis(2'-methoxy-4- nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H tetrazolium hydroxide (XTT) and phenazine methosulfate (PMS) was added to each well, and plates were incubated at 35°C (with agitation) for two hours more. The reduction of the tetrazolium salt XTT results in the formation of a colored product, the amount of which is proportional to the metabolic activity of the culture (Tellier et al., Antimicrobial Agents and Chemotherapy 36:1619-1625, 1992). Growth and metabolic activity respectively were assessed by measuring the optical density of each microculture sample at 650 nm and 450 nm. Growth inhibition was calculated as the percent decrease in absorbance, compared to the positive control cells. In order to prove the validity of the assay described above, and the use of the mutant strain within such an assay, the sensitivity of the mutant, DSY1024, to miconazole was compared to that of the wild type, SC5314. The IC 50 for miconazole was 1.2 ng /ml for the mutant, and 10.9 ng/ml for the wild-type, after fitting the data to a four parameter fit. This represented a 9-fold increase in sensitivity to miconazole for the mutant versus the wild-type strain (FIG. 2). Since miconazole is known to be extruded by MDR pump proteins, these results demonstrate the utility of the knockout strain in enhancing the detection and identification of antifungal compounds that are MDR pump substrates. To extend this observation, 1760 crude extracts from plant cell cultures and marine microbiological sources, similar to those described in Example 1, were tested. An extract was designated "active" if, in two consecutive tests, growth was inhibited by at least 50% at the standard test concentrations, namely, 25 μg/ml for plant extracts and 2 μg/ml for marine extracts, respectively. None of the 1760 extracts tested was active against the wild-type strain, whereas 2 samples (0.11%) inhibited growth of the mutant strain. After repeated rounds of bioassay-guided fractionation, one active compound was isolated. This was identified as brefeldin A, which has previously been shown to be a pump substrate for bfrl+ of Schizosaccharomyces pombe (Nagao et al., J. Bacteriol. Ill: 1536-1543, 1995). Although a known antimicrobial agent, failure to detect inhibition in the wild-type is attributed to the low concentration in the extract. The product of this gene is structurally related to the ATP-binding casette superfamily, which includes the cdrl and cdr2 gene products of C. albicans. Identification of this compound supports the utility of using MDR-deficient strains as screening tools for antimicrobial compounds. C. albicans and other Candida species that have genes homologous to those described in S. cerevisiae. As a result, examples of other MDR mutants are selected from Balzi and Goffeau, Biochim. Biophys. Acta 1187:152-162 (1994); Balzi and Goffeau, J. Bioenerg. Biomem. 27:7176 (1995); and Goffeau, et al. Yeast 13:43- 54, 1997. Example 4: Generation of a Strain of Staphylococcus aureus Deficient in the Expression of Multi-Drag Resistance Pump Proteins. A mutant S. aureus strain with a disrupted MDR pump gene was generated from a wild-type strain. The mutant strain, KLE819, was constructed by transforming wild-type S. aureus RN4220 with a plasmid (pCH23; FIG. 3) containing a non- functional portion of the Nor A gene preceded by a functional chloramphenicol acetyltransferase (CAT) gene. Because pCH23 lacks the S. aureus replicon, it must be integrated into the host chromosome to stably confer chloramphenicol resistance. Homologous recombination of pCH23 into the host chromosome disrupted the NorA coding region (FIG. 4). A single colony of KLE819 was serially passaged on selection medium containing chloramphenicol to select a stable isolate with a disrupted NorA gene, generating strain KLE820 ANorA (hereinafter "KLE820"). KLE820 was tested for a ANorA phenotype using microbroth dilution assays similar to those described in Example 2. Colonies of RN4220 and KLE820 were inoculated into microbroth cultures as described in Example 2, except that the growth medium was soy broth supplemented with chloramphenicol (34 μg/ml). The mutant strain was more sensitive to bactericidal compounds that are known MDR pump substrates than the wild-type strain. For example, the IC 50 for ethidium bromide was 0.23 μg/ml for the mutant and 1.5 μg/ml for the wild-type strain, representing a 6.5-fold increase in sensitivity for the mutant (FIG. 5). Similarly, the IC 50 for acriflavin was 0.67 μg/ml for the mutant and 6.28 μg/ml for the wild-type strain, representing a 9.3-fold increase in sensitivity. These data demonstrated the increasing sensitivity of the representative drug screening assay for antibacterial compounds that are also MDR substrates. In a second test of the mutant strain, strips (from AB BioDisk), impregnated with a gradient of known antimicrobial agents, were placed on lawns of RN4220 and KLE820 cells, as described in Example 1. The mutant strain was more sensitive to some of the drags tested, as evidenced by clear zones that were larger than those observed with the wild-type strain. For example, the MIC for norfloxacin in the mutant strain was 0.25 μg/ml versus 1 μg/ml for the wild-type strain, representing a 4-fold increase in sensitivity. Two-fold increases in sensitivities of the wild-type versus mutant strains were observed for various classes of antimicrobial compounds including nalidixic acid, chloramphenicol and ceftazidime. The NorA knockout mutation rendered KLE820 more sensitive to some antibiotics. In other examples, other deletional mutations are superimposed on the KLE820 genotype. Examples of such mutations include two pump genes, LmrA and LmrP, described in the gram positive bacterium Lactococcus lactis (van Veen et al. Biochemistry 93:10668-10672, 1996); Bolhuis et al. J. Biol. Chem. 270:26092-26098, 1995). Genes which have homologues in S. aureus are targets for mutagenesis. Example 5: Optimization of Bioassays Via The Use of Host Cells Deficient in MDR Pump Proteins. Saccharomyces cerevisiae and Schizosaccharomyces pombe are experimental organisms that are genetically well-characterized, and for which there are well-developed molecular biology methods. A large number of MDR pump homologues have been identified in the completely sequenced genome of S. cerevisiae (Balzi and Goffeau, 1994, 1995, and Goffeau et al, 1997). Some of these MDR pumps have been disabled by insertional mutagenesis and exhibit increased sensitivity to antimicrobial compounds. There are numerous yeast-based screens that identify compounds that affect the biological activities of therapeutic targets, such as G-protein linked receptors, cytoplasmic receptors, ion (e.g., potassium, calcium, or sodium) channels, fungal cell wall enzymes, fungal sterol biosynthesis enzymes, antiviral targets (e.g., host proteins or enzymes that are necessary for the virus life cycle), immunosuppressive targets wherein the target proteins are inhibited to cause immunosuppression, cyclic nucleotide phosphodiesterase, and oncogenes (reviewed in Kirsch, Current Opinion in Biotechnology 4:543-552, 1993). Yeast also have been used to study the function of oncogenes, to study protein-protein interactions, and to screen for potential antitumor agents. The two hybrid system uses a galactose selection to identify proteins that interact with a known protein (Fields and Song, Nature 340:245-246, 1989). Such screens employ an easily measured parameter, such as changes in cell growth or reporter gene expression (e.g. β-galactosidase-based blue-white discrimination; Dhundale and Goddard, J. Biomol. Screen. 1 :115, 1996), and hence are well suited for high throughput screening (HTPS) format. Other recombinant microorganisms can also be used for HTPS, as reviewed in Klein and Geary, J. Biomolecular Screening 2:41-49, 1997. For example, genetically manipulated bacterial hosts have been used for antiviral and antiparasite target screening, Kadam et al. J. Antibiot. 47:492, 1994, and Eakin et al. Antimicrob. Agents Chemotherapy 39:620-625, 1996. Mammalian cell lines are also suitable for HTPS and possess MDR genes that have similar structure and function to homologous gene families in yeast (McGrath and Varshavsky, Nature 340:400-404, 1989). The use of MDR-deficient cells (e.g., as screening strains or as host strains providing a platform for other mutations) increases the sensitivity of assays that screen for biological activity when the biologically active agent must enter the cell to exert its activity. An example of S. cerevisiae would be Δpdrl, Apdr3, Δpdr5, and Asnq2 with further deletions sequentially introduced from the MDR-related genes listed in Balzi and Goffeau (1994), (1995) and Goffeau et al., 1997. Advantages and Use Many cell types, including bacterial and mammalian cells, harbor MDR pumps that extrude a variety of chemically unrelated substances from the cell. A number of anticancer drags and natural antibiotics, as well as synthetic antimicrobial agents such as quinolones are also extruded by MDR pumps. In the disclosed process of identifying antibiotic agents, for example, a sensitive bacterial strain can be used to detect antibiotic activity in a mixture of compounds when such activity would otherwise remain undetected. As demonstrated, bacterial strains that possess MDR pumps, and therefore extrude antimicrobial agents from the cell, are significantly less effective in detecting antimicrobial agents than are MDR mutant strains. The invention allows detection of new antimicrobial and antineoplastic agents using mutant cells that possess dysfunctional efflux pumps and will therefore not prevent these agents from exerting their full effect. The genes encoding these 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. Additional genes may be subsequently identified. Other Embodiments In addition to the MDR pumps described herein and presently known to skilled artisans, the invention may be practiced with MDR pumps discovered as follows. In one approach, novel MDR pumps may be identified by screening cDNA or genomic libraries with all or part of the sequence encoding a protein that functions within a known MDR pump. Once isolated, the novel genes can be expressed by standard methods and tested for their ability to participate in drug extrusion from a cell. In a second approach, novel MDR pumps may be identified in cells that exhibit an elevated resistance to one or more toxic substances (such as antimicrobial or antineoplastic agents). Among the cells that are resistant to toxic substances will be those that overexpress an MDR pump. These cells can be identified, for example, by a cross-resistance test. Typically, cells that overexpress an active MDR pump are resistant to several chemically unrelated drags. For example, a mutant cell that overexpresses NorA is more resistant to ethidium bromide, acridine, and norfloxacin than are wild type cells. The presence of an active drug efflux process can be documented by preloading cultured cells with a drag they are capable of extruding via an MDR pump, supplying the cells with an energy substrate, and measuring the extrusion of the drag into the culture medium. The locus containing genes encoding one or more components of the newly identified MDR pump can be isolated by placing a chromosomal DNA library on a multicopy vector, which is then used to transfect appropriate cells. The presence of the locus in multiple copies will lead to increased expression of the MDR pump, which will in turn cause an increased resistance to toxic, e.g., antibacterial, agents. Due to this increased resistance, when wild type cells that have been transfected with the chromosomal DNA library are plated on a medium containing an agent to which cells expressing the MDR pump in question are resistant, only cells containing a vector bearing the MDR genes will survive. The plasmid can then be isolated from any recombinant clone that exhibits increased resistance, and the locus characterized by restriction mapping and sequencing. Genes encoding proteins that participate in drag extrusion can be disrupted according to the invention in Gram-negative bacteria, Gram-positive bacteria, mammalian cells, protozoans, parasite cells, filamentous yeast, and fungi. The extent of success of a particular method will depend on the combination of the cell type and vector employed, a determination that is well within the abilities of a skilled artisan. In general, a gene of interest, such as an MDR (NorA) gene of S. aureus, is cloned into a suitable vector by standard techniques. The gene may be cloned by conventional methods from a genomic library or may be amplified by the polymerase chain reaction (PCR) from chromosomal DNA. In the latter case, to facilitate cloning the gene, the primers used to amplify the DNA may include one or more restriction sites. Once the DNA is amplified and purified, it is digested with an appropriate restriction endonuclease and ligated into the corresponding cloning site of a vector that has been digested with restriction endonucleases to create, preferably, staggered, cohesive ends. If this is not possible, the ligation may take place using blunt ended fragments and a similarly prepared linear vector. The recombinant vector is then propagated, for example in E. coli, purified, and digested at a unique site within the cloned gene. A fragment (cassette) that will be used to disrupt the gene is then prepared by excision from a suitable vector such as a Tn5 -containing vector that confers resistance to kanamycin. An excised cassette usually contains a gene that confers antibiotic resistance; instead of conferring resistance to kanamycin, the gene may confer resistance to, for example, tetracycline, chloramphenicol, or erythromycin. The excised cassette is ligated into the gene, disrupting its sequence, and rendering the target non-functional. The recombinant vector, carrying the disrupted gene, is transformed into the target cell, e.g., S. aureus. The vectors used for gene disraption do not propagate in the target cell. After transformation, the cells are plated onto nutrient medium containing an antibiotic, resistance to which is conferred by the cassette disrupting the gene. Since the plasmid cannot propagate within the target cells, the only cells that become antibiotic resistant are those in which homologous recombination produced a replacement of a native gene (such as NorA) with a disrupted gene (NorA with an inserted kan-resistance gene, for example). Testing according to the invention for the lack of resistance to substrates of the MDR pump verifies the disruption. For example, increased sensitivity to quinolone compounds which are NorA substrates would result. The gene may be amplified again by PCR and the presence of an insert verified by restriction mapping. Methods for disrupting gene expression within mammalian cells are also well known to skilled molecular biologists. For example, targeted disruption of a gene encoding a protein that functions within an MDR pump may be achieved by homologous recombination. Many of the genes encoding such proteins have been identified, and additional genes can be easily isolated as described above. Once isolated, the gene can be characterized by testing the protein it encodes for the ability to participate in extrusion of toxic compounds when expressed in a cell system. As described above, mammalian cells that have undergone a neoplastic transformation may be especially useful in methods of the invention. Generating a mutation of an MDR pump in these cells, many types of which are available from the American Type Culture Collection (Rockville, MD), can provide the means for identifying antineoplastic agents. Other embodiments are within the following claims. What is claimed is:

Claims: CLAIMS 1. A method for identifying a substance that inhibits the growth or metabolism of a mutant cell comprising a dysfunctional multi-drug resistance pump, said method comprising the steps of: (a) contacting said cell with a test substance; and (b) determining whether the growth or metabolism of said cell is inhibited, said cell being selected from a mammalian cell, a bacterial cell, a saprophyte, a filamentous fungal cell, a parasite cell, and a protozoan. 2. A method for identifying a multi-drag resistance pump inhibitor, said method comprising the steps of: (a) contacting a first cell with a test substance and an antibiotic, said first cell comprising a wild-type multi-drug resistant pump that extrudes said antibiotic from said first cell; (b) contacting a second cell with said test substance in the absence of said antibiotic, said second cell comprising a mutated multi-drag resistant pump that cannot extrude said antibiotic from said second cell, said second cell being otherwise identical to said first cell; (c) contacting a third cell with said test substance in the presence of said antibiotic, said third cell being identical to said second cell; (d) measuring the growth or metabolism of said first, second, and third cells, wherein the presence in the test substance of a multi-drag resistance pump inhibitor is indicated by (i) inhibition of the growth or metabolism of said first cell, and (ii) inhibition of said second cell is both comparable to said third cell and also lower than the inhibition of said first cell. 3. The method of claim 1 or 2, wherein said test substance is a substantially pure compound. 4. The method of claim 1 or 2, wherein said test substance is a mixture of compounds. 5. The method of claim 4, wherein said test substance is from a natural source. 6. The method of claim 4, wherein said test substance is a mixture of synthetic compounds. 7. The method of claim 1 or 2, wherein said cell is a bacterial cell. 8. The method of claim 7, wherein said bacterial cell is selected from Staphylococcus aureus, Escherichia coli, Enterococcus fecalis, Haemophilus influenzae, Enterococcus fecium, Tuberculosis sp., Streptococcus pyogenes, Enterobacter sp. , and Pseudomonas aeruginosa. 9. The method of claim 1 or 2, wherein said cell is a filamentous fungal cell. 10. The method of claim 9, wherein said cell is selected from Aspergillus fumigatus, A. nidulans, and A. flavus. 11. The method of claim 1 or 2, wherein said cell is a saprophyte selected from Trichophyton sp. and Fusarium sp.. 12. The method of claim 1 or 2, wherein said cell is a nonpathogenic cell selected from Lactococcus lactis and Bacillus subtilis. 13. The method of claim 1 or 2, wherein said cell is a parasite cell. 14. The method of claim 1 or 2, wherein said cell is a protozoan selected from Plasmodium falciparum, P. ovale, P. vivax, P. malariae, Trypanosoma brucei, Toxoplasma sp. , and Pneumocystis sp.. 15. The method of claim 1 or 2, wherein said cell is a mammalian cell that has undergone a neoplastic transformation. 16. The method of claim 1 or 2, wherein said cell comprises a single MDR pump mutation. 17. The method of claim 16, wherein said cell comprises two MDR pump mutations. 18. The method of claim 17, wherein said cell comprises four MDR pump mutations. 19. The method of claim 1 or 2, wherein said cell has a single MDR pump mutation. 20. The method of claim 18, wherein said cell comprises MDR pump mutations in at least three- fourths of the MDR pump proteins in said cell. 21. The method of claim 1, wherein said inhibition of growth or metabolism is inhibition of a cell component selected from membrane receptors, ion channels, DNA binding proteins, transcriptional regulators, steroid-responsive elements, protein kinases, protein phosphorylases, viral processing enzymes, an immunosuppressive target protein, cell division control genes (cdc), oncogenes, and signal transducers. 22. The method of claim 21, wherein said cell component is selected from G-protein linked receptor, a cytoplasmic receptor, a fungal cell wall enzyme, fungal sterol biosynthesis enzymes, and cyclic nucleotide phosphodiesterase. 23. The method of claim 2, wherein said first cell is contacted with a concentration of said antibiotic that is less than one-fourth of the MIC 90 for said first cell. 24. The method of claim 2, wherein said second and third cells are contacted with a concentration of said antibiotic that is less than one- fourth of the MIC 90 for said second cell. 25. The method of claim 1, wherein the MIC of a compound selected from chloramphenicol, norfloxacin, acriflavin, nalidixic acid, miconazole, erythromycin, ethidium bromide, fluconazole, levofloxacin, ciprofloxacin, inoxacin, ofloxacin, rhodamine 69, brefeldin A, daunomycin, TPP, tetracycline, ampicillin, and gentamycin in said the wild-type of said cell is at least five times greater than the MIC in said cell. 26. The method of claim 25, wherein said MIC in the wild-type of said cell is at least fifteen times greater than the MIC in said cell.

Inventors: LEWIS KIM, SIEGEL SCOTT A, LEWIS, KIM, SIEGEL, SCOTT, A.
Applicants: LEWIS KIM, PHYTERA INC, SIEGEL SCOTT A, LEWIS, KIM, PHYTERA, INC., SIEGEL, SCOTT, A.
Application year: 1997
Main classification: G01N33
IPC codes: C12Q1, G01N33