WO1998014784A1 - Identification and use of mutant multi-drug resistant cells - Google Patents

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 MIC₉₀ 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 MIC₉₀ 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 IC₅₀'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 IC₅₀'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 IC₅₀'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 IC₅₀'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 10⁵ 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. A₆₅₀),

and growth inhibition was calculated as the percent decrease in A₆₅₀ 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 IC₅₀ of erythromycin using the wild-type strain (KLE700) was

16.6 μg/ml, while the IC₅₀ 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 A₂₆₀), 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 IC₅₀ 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 IC₅₀ 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

IC₅₀ 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

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 MIC₉₀ 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

MIC₉₀ 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.