Pharmucol. Vol. T/W. 76,Nos. 1-3,~~. 219-242, 1997 Copyright 1997 0 Elsevier Science Inc.

ELSEVIER Associate Editor: D. Shugar

PI1

ISSN0163-7258/97$32.00 SO163-7258(97)00094-6

Active Efflux by Multidrug Transporters as One of the Strategies to Evade Chemotherapy and Novel Practical Implications of Yeast Pleiotropic Drug Resistance
Marcin Kolaczkowski and AndreGoffeau
UNIT~DEBlOCHlMIEPHYSIOLOGlQUE,UNIVERSIT~CATHOLIQUE DELOUVAIN,PLACECROlXDUSUD2/20,
B-1348 LOUVAIN LA NEUVE, BELGIUM

ABSTRACT. Mankind is faced by the increasing emergence of resistant pathogens, including cancer cells. An overview of the different strategies adopted by a variety of cells to evade chemotherapy is presented, with a focus on the mechanisms of multidrug transport. In particular, we analyze the yeast network for pleiotropic drug resistance and assess the potentiality of this system for further understanding of the mechanism of broad specificity and for development of novel practical applications. PHARMACOL. THER. 76:( l-3):219-242, 1997. 0 1997 Elsevier Science Inc. KEY WORDS. CONTENTS 1. INTRODUCTION ............. 2. TRICKSTOSURVIVE ........... .. . . 2.1. ENZYMATICINACTIVATION ... . . . 2.2. TARGETALTERATIONS. 2.3. ALTERATIONSOFACTIVITYOF DRUGTARGET-AND DRUG-METABOLIZINGENZYMES .. .... . 2.4. INCREASEDDNAREPAIR 2.5. FAILURETOUNDERGO APOPTOSIS . . . . . . . . . . . . . 2.6. REDUCEDACCUMULATIONAND SEQUESTRATION . . . . . . . . . . 2.7. AcTIvEEF~ux........... 2.7.1. CANCERCELLS . . . . . . . 2.7.2. OTHERPATHOGENS. . . . . 3. THEDILEMMAOFBROADSUBSTRATE SPECIFICITY . . . . . . . . . . . . . . . 3.1. DRUG-RESISTANCEPROFILES ABBREVIATIONS. 219 220 220 OFMULTIDRUG-RESISTANCE TRANSPORTERS . . . . . . . . . . . 3.2. MECHANISM. . . . . . . . . . . . . 3.3. SUBSTRATERECOGNITION BYMULTIDRUG RESISTANCE-ASSOCIATED PROTEIN............... 4. THEYEASTSACCHAROMYCES CERIWISIAE fiEIOTROPIC DRUG-RESISTANCENETWORK ASATOOLTOUNDERSTANDTHE MECHANISMOFMULTIDRUG RESISTANCEANDTRANSPORT ..... 5. OTHERPRACTICALIMPLICATIONSOF THEYEASTPLEIOTROPIC DRUG-RESISTANCENETWORK ..... ACKNOWLEDGEMENTS............ REFERENCES . . . . . . . . . . . . . . . . . 227 229
Chemotherapy, antibiotic, fungicide, anticancer. substrate specificity.

222

222 223 223 223 224 224 225 227

230

231

232 234 234

ABC, ATP-binding cassette; AIDS, acquired immunodeficiency syndrome; AM, acetoxymethyl; Al, abasic (a purinic or a pyrimidinic); ARA, anthracycline resistance-associated; CFTR, cystic fibrosis transmembrane conductance regulator; DNP-SG, dinitrophenol-S-glutathione; LRP, lung resistancerelated protein; LT, leukotriene; MDR, multidrug resistance; MFS, major facilitator superfamily; MK57 1, 3( [(3(2[7-chloro-2-quinolinyl]ethenyl)phenyl} ((3-dimethylamino-3-oxopropyl) thio] methyl] thio) propanoic acid; MRP, multidrug resistance-associated protein; NBD, nucleotide-binding domain; 4NQO,+nitroquinolineN-oxide; PDR, pleiotropic drug resistance; P-gp, P-glycoprotein; RND, resistance/nodulation/cell division; SMR, small multidrug resistance; Texans, toxin-extruding antiporters; TM, transmembrane; TMA-DPH, l-[+(trimethylamino) phenyll-6-phenylhexa-1,3,5triene.

1. INTRODUCTION An immense variety of small organic molecules was designed in the course of evolution as toxic weapons of chemical warfare carried on by microorganisms survival and elimination of competitors. in their fight for

They are also abundant in plants as a means of killing or repelling herbivores and pathogenic microbes or other plants (Taiz and Zeiger, 1991). Moreover, to protect themselves against in-

*Corresponding

author.

fections, plants, as well as animals and humans, have developed a defense system based on broad-spectrum hydrophobic antimicrobial peptides. In vertebrates, these supplement cell-mediated immunity, protecting mucosal surfaces of the respiratory and gastrointestinal tracts, as well as skin, against invading microorganisms (Nicolas and Mor, 1995). To balance this toxic arsenal, which has been extended during the last 50 years by the development and massive use of antibiotics, evolution has equipped living organisms with a plethora of protecting systems. These systems, to our annoyance, contribute to the large-scale emergence of resis-

220 tant pathogens, as stressed in many recent alarming reports 1997). These include vanfor which there J. G. et al., tuberculosis 1995; Kaye 1996; But(Morris, and agricultural importance. low manipulation and predictable

M. Kolaczkowski and A. Goffeau In the long term, it should alprofile in a controlled

(Neu, 1992; Krause, 1992; Travis, 1994; Gold and Moellering, 1996; Acar and Goldstein, comycin-resistant is no effective (Morris, and enterococcal antibiotic bacteremia,

of the specificity

way to tailor it for our benefit. We believe and new prospects in we

that yeast has much to offer to the study of MDR and will describe below recent developments this field. Before going into more detail on MDR transporters, will present mechanisms, a brief overview of other types of resistance since, in most cases, the resistance is multifacoften link together to cause

treatment

1995; Linden et al., 1996), Frieden, 1996;

multidrug-resistant and Castro, 1996),

S. et al., 1995; Huebner Blanchard,

Plasmodium spp.

(Borst and Ouelette, 1995; Ullman, resistant (Odds, drug-resistant 1996).

199.5; Rubio and Cowman,

ler, 1997) and other parasitic protozoa (Borst and Ouelette, 1995; Rubio and Cowman, (Geerts et al., 1997). fungal clinical The 1996), as well as The number of used in (AIDS) addition1995; us helminths

torial and different mechanisms a poor response to chemotherapy. 2. TRICKS Various TO SURVIVE mechanisms

isolates also tends to increase treatments syndrome

chemotherapeutic immunodeficiency

the global acquired pandemic, nosuppression ally contribute pathogens Although flavonoids,

resistance

found in many microbial are often present in and include: (1) in-

as well as the use of drugs inducing severe immuin transplant and and cancer patients, Castro, 1995; to the incidence of new and drug-resistant Hazen,

and parasitic pathogens different supplementing activation

and cancers combinations

of drugs by enzymes, (2) target alterations resultenzymes, (4)

(Huebner

ing in decreased target affinity, (3) changes in the cellular activity of target enzymes or drug-activating cancer cells, (6) reduced accumulation increased DNA repair, (5) failure to undergo apoptosis by and sequestration, and (7) active efflux (Fig. 1.). 2.1. En-tic

Hazen, 1995; Odds, 1996; Kelly et al., 1997). endogenous alkaloids, defense mechanisms protect from natural toxins, such as the plant pesticides glucosinolates phenols, con-

and saponins,

sumed daily in food, and many others, including, for example, the potent mold carcinogens sometimes cells lining excretory, detoxifying aflatoxins, they can also from turn against us. Thus, tumors originating

Inactivation
inactivation is the major mechanism of resisand and results from hydroly cephalosporins Resistance to newly

Enzymatic

organs, such as the kidresistant to antiduring mechais the acand and et al.,

tance toward P-lactam antibiotics sis of the p-lactam related antibiotics generated 1996). Enzymatic modification tin, kanamycin, mases is common p-lactams (Livermore, (Davies,

ney, gut and liver, are often intrinsically the course of chemotherapy One of the important nisms of drug resistance drug Pastan, resistance 1993, (MDR) (Mattem

ring of penicillins, 1995). 1994; due to point

cancer drugs. Many other tumors acquire resistance and clinically relevant

and Volm, 1993).

mutations Gold

in p-lacta-

and Moellering,

in human malignancies transporters and Volm,

tive extrusion of toxic chemicals 1997; Mattern

by broad specificity multi(Gottesman 1993; Simon 1997).

is also the most clinically signifineomycins) by aminoglycoside

cant mechanism of inactivation of aminoglycosides (streptomy gentamycin, acetyltransferases, phosphotransferases, nucleotidyltransferases and adenylyltransferases, transposons tamases, specificity often associated with plasmids or of modifying enzymes have pathogens are (Shaw et al., 1993). Here, in contrast to p-lacalterations

Schindler,

1994; Skovsgaard et al., 1994; Gottesman

1995; Kane, 1996; Borst and Schinkel, tial danger of this type of mechanism, bacterial, compounds specificity fungal and parasitic from different is in clear contrast

The poten-

widespread among lies in the fact to a plethora of This broad

pathogens,

that a single protein can confer resistance chemical

not been identified in clinical isolates (Davies, 1994). Also widely distributed chloramphenicol Many macrolide-, cleotidyltransferases, among bacterial acetyltransferases lincosamide(Shaw and Leslie, 1989). and streptogramin-inactihydrolases, esterases, nuand glycosyl trans-

classes.

to many other types of resismutations

tance, which are usually limited to a single drug or to a class of structurally closely related drugs. Spontaneous yielding even more effective transporters for selective

vating enzymes (acetyltransferases,

phosphotransferases,

agents or transporters with altered specificity profiles have been reported (Choi et al., 1989; Chen et al., 1997; Klyachko, et al., 1997). Transporters encoding genes can be spread by means of plasmids and transposons (Roberts, 1994), genes as has happened (Davies, 1994). with many other drug-resistance Indeed, some MDR transporters, to disinfecresistance

ferases) have been detected in clinical isolates, although they are less important clinically than p-lactamases (Arthur et al., 1987; Kono et al., 1992). Fosfomycin, encoded used in the treatment S-transferase of sepsis, is inactiby the plasmidvated by its derivatization glutathione with glutathione,

(Suarez and Mendoza,

such as QacA

and Smr, conferring

1991). This is the only example of this type of mechanism in bacteria, which is widespread in other organisms. In fungi, plants, animals, and humans, the intracellular metabolism of endo- and xenobiotics is mediated by cytochrome P450 monooxygenases (Kivisto et al., 1995) and further by glutathione S-transferases (Commandeur et al.,

tants and antiseptics, already have been identified on plasmids from clinical isolates of staphylococci (Littlejohn et al., 1992; Leelapom et al., 1994). To overcome their mechanism MDR transporters, the understanding of of action and regulation is of key clinical

Drug Resistance Mechanisms

221

222 1995; Hayes and Pulford, 1995), UDP-glucuronyl transferases (Bock, 199 1) or sulfotransferases (Falany, 199 1) . These conjugations are key defense mechanisms in the detoxification of endogenous and exogenous electrophilicreactive compounds in eukaryotes. Not only do they eliminate harmful electrophilic moieties, but they provide a molecular flag that signals export of the conjugate from the cell. This mechanism contributes to resistance to chemotherapy of cancer-cells. Various types of other enzymatic inactivation mechanisms have been observed in fungi, plants, animals, and humans. Bleomycin hydrolase, for example, is a cysteine protease involved in tumor resistance to the anticancer drug bleomycin. Increased mRNA levels for this hydrolase have been observed in a variety of tumor cell lines (Bromme et al., 1996).

M. Kolaczkowski and A. Goffeau tance development (Kremsner et al., 1997). Accumulation of mutations in the individual drug targets is the most frequent mechanism of MDR to escape antimycobacterial drugs such as streptomycin, rifampin and isoniazid (Morris, S. et al., 1995; Blanchard, 1996). Multiple resistance to nucleoside analogues and non-nucleoside human immunodeficiency virus reverse transcriptase inhibitors, due to acquisition and accumulation of point mutations in the target enzyme, is also frequent during AIDS chemotherapy, where the high proviral DNA transcription replication rates and the resulting variability contribute to its quick development (Schmit et al., 1996; Brown and Richman, 1997). A similar mechanism of resistance of the human immunodeficiency virus protease to its potent inhibitors, indinavir, ritonavir and others, has also been reported (Molla et al., 1996; Brown and Richman, 1997). Acquisition of new target enzymes (dihydropteroate synthase and dihydrofolate reductase) with reduced affinity for the inhibitor is the major mechanism of resistance to sulfonamides and trimethoprim in bacteria (Huovinen et al., 1995). Acquisition of a transposon-encoded (additional) penicillin-binding protein with low affinity for p-lactam antibiotics, which takes over the function of endogenous enzymes, is the mechanism of methicillin resistance in St&ylococcus aureus (Song ec al., 1987). Until recently, vancomycin was the only reliable drug to treat infections caused by multidrug-resistant enterococci. Vancomycin-resistant microbes have spread recently in hospitals (Morris, J. G. et al., 1995; Linden et al., 1996). Acquired vancomycin resistance results from acquisition of the plasmid-encoded operon mediating the synthesis of altered peptidoglycan precursors of reduced affinity for the antibiotic (Walsh, 1993).

2.2.

Target Alterations

Development of targets with reduced affinity for antibiotics is a major mechanism of resistance when drug-inactivating or -modifying enzymes are absent. It occurs most rapidly and frequently with drugs that inactivate a single target and are not substrate analogs. Most frequently, it results from target modifications, mutations, or acquisition of druginsensitive enzymes replacing the target function. Methylation of ribosomal RNA by rRNA methylases, often carried on plasmids and transposons, is the most often encountered mechanism of bacterial resistance to macrolides, lincosamides and streptogramins in clinical isolates (Leclerq and Courvalin, 1991). Resistance to tetracyclines, often encoded by transposons and conjugative plasmids (Roberts, 1994), can be mediated by protection of the ribosome by TetO or TetM, whose functions at the molecular level have not been fully elucidated yet (Schnappinger and Hillen, 1996). Mutations in the drug targets, resulting in decreased drug binding, occur for all types of drugs. For example, mutations in DNA gyrase confer resistance to fluoroquinolones in bacteria, including Sta@ylococcus aureuS (Ouabdesselam et al., 1996). Therapeutic options for Staphylococcus aurew are quite limited, especially in the case of methicillin-resistant strains. Resistance to p-lactam antibiotics can result from alterations in the penicillin-binding proteins (transpeptidases participating in bacterial cell wall biosynthesis), by creation of hybrid proteins through interspecies recombination (Spratt, 1994). Mutations in topoisomerases I and II are one of the mechanisms of resistance to anticancer drugs targeted to topoisomerase I (camptothecin, topotecan) and II (etoposide, mitoxantrone, doxorubicin, amsacrine, ellipticine, saintopin) (Nitiss and Beck, 1996). Resistance to azole fungicides in clinical isolates can result from their decreased affinity for the target enzyme, the cytochrome P450-dependent sterol 14-cY-demethylase (Marichal and Vanden Bossche, 1995 ) . Combination therapy with drugs affecting different targets strongly diminishes the chance of simultaneous resis-

2.3. Alterations of Activity of Drug Target- and Drug-Metabolizing Enzymes Resistance can also arise by increased target enzyme activity. For example, resistance towards the anticancer drug methotrexate often results from overproduction of dihydrofolate reductase by amplification of its gene, but sometimes from increased mRNA translation (Skovsgaard et al., 1994). The opposite is observed with topoisomerases I and II, which provide the strand breakage-unwinding-ligation activities crucial for DNA replication. Their inhibitors stabilize the DNA-topoisomerase complexes, leading to formation of double-strand breaks and cell death. Resistance to topoisomerase inhibitors, due to decreased enzyme levels, or reduced activity by mutations in the ATP-binding domain, have been observed (Nitiss and Beck, 1996; Houlbrook et al., 1996). Many drugs must be metabolized inside the cell to their active form, and any changes in this process can lead to resistance. The antifungal 5-fluorocytosine exerts its cytotoxic effect upon intracellular conversion and inhibition of DNA synthesis, as well as incorporation into RNA, which is then aberrant. Upon entry to the cell through cytosine

Drug Resistance Mechanisms permease, it is deaminated rouracil, further RNA. which phate pyrophosphorylase phosphorylated 5Fluorouracil synthase. by cytosine deaminase to ~&IOby uridine monophosacid. This is into aberrant into of can is into 5-fluorouridylic and incorporated plasma membrane

223 and have to use specific transporters for in these transporters often lead to resisin the folate transporter common in other

is then converted

this purpose. Alterations tance was related

reduced influx of drugs. In the case of methotrexate, to alterations (Skovsgaard species. Many gram-negative nas spp., are intrinsically to extremely (Nakae, 1995), which tightly microorganisms, et al., 1994), a mechanism

is also converted monophosphate, Resistance

simultaneously

5-fluorodeoxyuridine thymidylate

a potent inhibitor

to 5-fluorocytosine

such as Pseudomodue

result from loss or mutation of any of the enzymes involved in its activation, and is so common that this antifungal no longer recommended Bossche et al., 1994). 2.4. Increased DNA Repair agents that (nitrogen agents nitrogen mustards and nitrosoureas). intraor interstrand mustards). Others bind nonbetween base pairs (anthracy(distamycin groove binding for single-drug therapy (Vanden

resistant to many antibiotics is composed packed

low permeability

of their outer membrane of lipopolysaccharide hydrocarbon unsaturated which,

units of several enter through

chains linked to a single polar head group. The nutrients porin channels, or mutational contribute (Martinez due to their small 1994). of porins Rein DNA is the site of action of many anticancer can bind covalently Some bifunctional (cisplatin, crosslinks covalently, clines) produce pore size, exclude duced production gram-negative many antibiotics (Nikaido,

alterations

bacteria

to their acquired resiset al., 1996). An exresisthe

tance to some antibiotics tant to most antibiotics thick and highly 1995).

by intercalation

treme case are mycobacteria, ordered

which are intrinsically through outer membrane

or by nonintercalative

unable to penetrate

A). If the extent of DNA damage is high, cells die. Otherwise, small lesions can be repaired by three main mechanisms: (1) direct repair, in which the chemical ing the substituent bonds linkto DNA are cleaved; (2) base excision,

containing

chains of mycolic acid more than 70 carbons long (Brennan and Nikaido, Decreased toxicity can also be caused by changes in the lipid composition in permeability. fectants. The of the membranes, leading to a decrease of bacterial resisThis is the mechanism decreased permeability

in which the modified base is first removed by a glycosylase, followed by removal of the abasic (AP) site by AP lyase/endonuclease, nucleotide (Sancar, with subsequent excision 1995). nucleotide replacement; (3) repair, making nicks on both sides of is then released and filled in anticancer agents (Skovs1995; Sancar, DNA repair is often associated and Lippard,

tance to organic solvents, some of which are used as disinand fluidity of the of their membranes results from cis- to tram-isomerisation antifungals nystatin

the damaged region, which Increased

unsaturated fatty acids (Heipieper to the polyene systemic tericin B, which interact membranes (Cohen,

et al., 1994). Resistance and amphoto form pores in

with resistance to DNA-damaging gaard et al., 1995). 2.5. 1994; Zamble

with ergosterol

1992), results, in most cases, from de(Kelly et

fects in the ergosterol biosynthesis pathway, leading to a decreased ergosterol drugs tance, level in the plasma membrane of other mechanisms et al., 1996). due to changes al., 1997). Existence gested (Josephhorne of polyene resis-

Failure to Undergo Apoptosis by which most anticancer leading to induction effects is the creation of disturbances of apoptosis in

The primary mechanism exert their cytotoxic in cellular metabolism,

not linked to sterol alterations,

has also been sug-

(Seimiya et al., 1997). Apoptosis is the tightly regulated, intrinsic cellular suicide program that assures homeostasis multicellular organisms, the complexity 1997). of which only now beginning son, 1997; McCall controlled to understand and Steller, (Steller, Cancer we are

The reduction of membrane permeability a very efficient by another enzymatic way of resistance, mechanism,

in membrane biophysical properties, or loss of porins, is not unless it is accompanied such as active efflux or resistance inactivation,

1995; Andercells escape ability to unby anticancer (Thompson, Hannun, 1996;

which is often the case (Nikaido,

this natural regulatory mechanism

and proliferate in an un-

1994; Thanassi et al., 1995). As in the case of solid tumors, formation of cell clusters imposes another kind of permeability barrier. These large masses of cells are usually poorly vascularized, their penetration conditions by anticancer ited oxygen and nutrient resistance genes (Simon al., 1995). get is sequestration supply to subpopulations and Schindler, reducing of cells, drugs. This also causes lim-

way. They often show decreased

dergo apoptosis in response to at least some physiological signals; when these include signals elicited drugs, 1995; 1997). 2.6. Reduced Accumulation and Sequestration reduced cellular accumulation of drugs is often a of increased efflux, we will focus first on other resistance Mashima to chemotherapy results et al., 1996; Reed et al.,

that are linked to increased expression of drug1994; Koomagi et

Yet another way of preventing

access of a drug to its tar-

Although

by binding to serum proteins. Drugs can transporters (Yelin and

consequence mechanisms.

also be sequestered in subcellular organelles by active transport, as for vesicular monoamine Schuldiner, 1995; Schuldiner et al., 1995), or by membrane potential (A?) or pH gradient (ApH)-driven diffusion into

Many hydrophilic drugs, for example, the anticancer antimetabolite methotrexate, cannot easily diffuse through the

224

M. Kolaczkowski and A. Goffeau from ATP hydrolysis (Senior et al., 1995). P-gps are apitally localized in epithelial cells lining the intestine and kidney, in the canalicular membrane of liver cells, in endothelial cells of the blood-brain barrier and in pluripotent precursor stem cells of the bone marrow. These observations, and studies with mdrl/mdr2 knockout mice, which did not develop any detectable phenotype apart from marked drug hypersensitivity (Borst and Schinkel, 1997), suggest that these proteins play a key role in excretion and formation of an active permeability barrier for the toxic hydrophobic compounds, which are normal constituents of the environment and which otherwise easily diffuse through lipid bilayers (Eytan et al., 199613). The importance of P-gp in the clinical resistance of several types of cancers to antineoplastic drugs has been well established (Arceci, 1993; Filipits et al., 1996). The clinical relevance of different drug-resistance genes in cancer has also been reviewed recently by Filipits et al. (1996). A second identified human protein involved in clinically relevant broad specificity efflux of anticancer drugs and glutathione conjugates is the MDR-associated protein (MRP), which is a homologue of MDRl and also belongs to the ABC superfamily (Cole et al., 1992; Lautier et al., 1996). The activity of MRP strongly resembles that of the ATPdependent GS-X pump, previously characterized biochemically. The GS-X pump excretes glutathione S-conjugates, cysteinyl leukotrienes (LTs), and certain organic ions from normal and cancer cells (lshikawa, 1992). It is considered to modulate the resistance of cancer cells to cisplatin [cisdiamminedichloroplatinum(ll)] (lshikawa et al., 1994). The functional similarity, and the recent observations of lshikawa et al. (1994, 1996), suggest that the GS-X pump and MRP are identical. It is unclear how many other proteins are involved in transport of the previously discussed glutathione, glucuronide, or sulfate conjugates of endo- and xenobiotics, which are excreted mainly by liver and kidney, and what is their contribution to cancer chemotherapy resistance. Some of these have been characterized biochemically, although the corresponding human genes have not been identified yet (Commandeur et al., 1995). These include the dinitrophenol-S-glutathione (DNP-SG) ATPase, which is distinct from MRP and P-gp. DNP-SG ATPase transports glutathione S-conjugates and the anticancer drugs doxorubicin, daunorubicin, and vinblastine (Awasthi, Y. C. et al., 1992; Awasthi, S. et al., 1994; Saxena et al., 1992). Recently, the gene encoding a rat homologue of MRP, called cMoat, cMrp, or Mrp2, has been cloned (Paulusma et al., 1996). In contrast to MRP, which is expressed in several types of epithelia, muscle cells and macrophages and is located basolaterally in liver cells, cMoat is expressed in the liver and is localized in the canalicular membrane, showing that although functionally related, these are two different proteins (Cole et al., 1992; Paulusma et al., 1996; Lautier et al., 1996). Multidrug-resistant cancer cells often over-express the lung resistance-related protein (LRP), whose gene was cloned recently. The amino acid sequence of human LRP is

acidic organelles such as lysosomes or vacuoles. Daunorubitin, an anticancer drug, has been shown to accumulate in acidic organelles in ho; this occurs to a higher extent in some multidrug-resistant cells (Hindenburg et al., 1989). In many tumor cell lines, the development of MDR has been correlated with an increase in intracellular pH, leading to decreased drug accumulation (Simon and Schindler, 1994). Accumulation of many chemotherapeutic compounds in liposomes, in response to membrane potential (A*) and a pH gradient ( ApH), has been well documented and includes the anticancer drugs vinblastine, vincristine, doxorubicin, daunorubicin, epirubicin, and mitoxanthrone; the antimalarials chloroquine and quinine; and many others (Cullis et al., 1991). Interestingly, the observations of Wei et al. (1995) show that over-expression of the cystic fibrosis transmembrane conductance regulator (CFTR) lowers the membrane potential and intracellular pH and confers resistance to anticancer drugs such as doxorubicin, vincristine, and colchicine.

2.7. Active EffEwr Active efflux by specific or MDR transporters is a strategy to prevent the access of toxic compounds to their intracellular targets. It is also used to excrete products of intracellular metabolism of endo- and xenobiotics, flagged by glutathione, glucuronide, or sulfate. Drug transporters, widespread from bacteria to humans, are found within four families of proteins. These are the ATP-driven ATP-binding cassette (ABC) transporter superfamily (Doige and Ames, 1993; Fath and Kolter, 1993); the proton motive force-driven toxin-extruding antiporters (Texans), a subgroup within the major facilitator superfamily (MFS); the small multidrug resistance (SMR) family, or miniTexans; and the resistance/nodulation/cell division (RND) family (Paulsen et al., 1996a,b; Goffeau et al., 1997; Schuldiner et al., 1997). Other members of the ABC transporter superfamily, not shown to be involved in drug resistance, are involved in secretion, uptake, or intracellular transport of a large variety of different substrates, including proteins, peptides (Fath and Kolter, 1993; Kuchler et al., 1994), lipids (Oude Elferink et al., 1997) and different nutrients (Doige and Ames, 1993). Uptake of nutrients is also mediated by MFS transporters unrelated to drug resistance (Nelissen et al., 1995). 2.7.1. Cancer cells. In cancer cells, resistance to chemotherapy often results from active efflux mediated by the over-expressed MDRl P-glycoprotein (P-gp), a member of the ABC superfamily (Gottesman and Pastan, 1993, 1997; Gottesman et al., 1995; Kane, 1996). Multidrug-transporting P-gps, homologues of human MDRl, have also been identified in rodents. These comprise the mouse Mdrla and Mdrlb, rat Mdrla and Mdrlb, and hamster Pgpl and Pgp2 (Borst and Schinkel, 1997). P-gps extrude with different, but overlapping, specificities a large variety of toxic hydrophobic molecules in an unmodified form, utilizing energy

Drug Resistance Mechanisms 88% identical with the rat major vault protein. As vaults are multisubunit structures involved in nucleocytoplasmic transport, it is likely that LRP mediates resistance by a transport mechanism (Scheffer et al., 1995). Expression of LRP has been observed in many cancer cell lines, often together with P-gp and MRP (Izquierdo et al., 1996a,b), and is associated with a poor response to standard chemotherapy and adverse prognoses in patients with advanced ovarian carcinoma (Izquierdo et al., 1995) and acute myeloid leukemia (List et al., 1996). There are reports suggesting the presence of an altemative, other than the P-gp and MRP, efflux system for daunorubicin in acute myeloid leukemia human cell lines (Hedley et al., 1997). A gene encoding a new ABC, anthracycline resistance-associated (ARA) transporter, overexpressed in the anthracycline-selected human multidrug-resistant leukemia cell line together with MRP, has been cloned recently. ARA is the half-size transporter composed of six transmembrane (TM) spans and one nucleotide-binding domain (NBD) at the C-terminus. This is in contrast to P-gps and MRP, in which this structure is doubled (TM-NBD-TM-NBD), with both halves being homologous. ARA is most similar to the C-terminal part of MRP (Longhurst et al., 1996). It is not known yet whether ARA is involved in anthracycline efflux. Multidrug transport is also a feature of the previously mentioned mammalian vesicular monoamine transporters, which sequester drugs and neurotransmitters within synaptic vesicles and other subcellular organelles and belong to the family of Texans; but their involvement in clinical cancer resistance has not been documented (Yelin and Schuldiner, 1995; Schuldiner et al., 1995). 2.7.2. Other pathogens. Active efflux of antibacterial, antiparasitic, or antifungal agents has not been considered among the most prominent mechanisms of resistance until recently. It began to attract attention following the discovery of the plasmid-encoded energy-dependent efflux system for tetracycline (McMurry et al., 1980). More recently, awareness of the role of efflux mechanisms in microbial resistance has increased significantly (Nikaido, 1994; Poole, 1994; Williams, 1996; Jenkinson, 1996). 2.7.2.1. Bacteria. In bacteria, specific efflux systems contribute to clinical resistance to tetracycline, macrolides, and chloramphenicol. Active efflux of tetracyclines is usually mediated by proton motive force-driven transporters of the MFS, often carried on plasmids and transposons (Roberts, 1994). TetA, encoded by transposon TnlO, is the best known (Schnappinger and Hillen, 1996). Resistance to macrolides in some clinical isolates of St&ylococcus aureus has also been associated with efflux. The identified MsrA transporter belongs to the ABC superfamily (Ross et al., 1990). A plasmid-encoded efflux mechanism, conferring resistance to 14- and 15-membered macrolides, has been isolated from Smphylococcus epidermidis (Goldman and Cappobianco, 1990). Efflux of chloramphenicol can be medi-

225 ated by the transposon Trill 696-encoded CmlA-specific MFS transporter, identified in Pseudomonas ueruginosa (Bissonnette et al., 1991). Other chloramphenicol-specific MFS transporters include the plasmid-encoded CmlB, identified in Rhodococcus fascians (Desomer et al., 1992). High-level resistance to fluoroquinolones usually results from accumulation of several mutations affecting permeability and the target enzyme DNA gyrase. It has been observed among clinical isolates of Smphylococcus aureus, Pseudomonas aeruginosa, and species of Enterobacteriaceae (Poole, 1994; Acar and Goldstein, 1997). In Pseudomonas aeruginosa, the broad specificity multidrug transporter, the MexA-MexB-OprM complex from the RND family, contributes to resistance not only to quinolones, but also to tetracycline, chloramphenicol and p-lactams (Poole, 1994; Li et al., 1994, 1995). In Smphylococcus uureus, the efflux of quinolones, chloramphenicol and puromycin is mediated by the MFS transporter NorA (Kaatz and Seo, 1995). Interestingly, tetracycline resistance is also mediated by other broad-spectrum MDR transporters, including the human P-gp, product of the MDRl gene (Kavallaris et al., 1993) and the yeast Saccharomyces cerevisiae Pdr5p and Yorlp.* PdrSp also mediates resistance to chloramphenicol (Meyers et al., 1992; Leonard et al., 1994) and Yorlp to erythromycin.* Multiple resistance to some antiseptics and disinfectants is mediated by Smr (known also as Ebr/QacC/QacD) (Schuldiner et al., 1997) from the SMR family, which is encoded by a variety of plasmids from clinical isolates of Staphylococcus aureus and other staphylococci (Littlejohn et al., 1992; Leelapom et al., 1994, 1995). Also, the MFS transporter QacA, often found on staphylococcal plasmids, confers resistance to antiseptics and disinfectants (Littlejohn et al., 1992; Leelaporn et al., 1994; Behr et al., 1994). Of many other bacterial MDR transporters now identified, all but one are secondary transporters energized by the proton motive force (Paulsen et al., 1996a,b). The only ATP-driven one is the LmrA of Lactococcus lactis, which shows homology to human P-gp (van Veen et al., 1996). The clinical significance of these transporters isolated from pathogenic strains, however, is not yet clear, due partly to the fact that in most cases, their reported drug-resistance profiles are limited to a few compounds, often not including even the most important groups of clinically used antibiotics. The level of resistance conferred by these transporters is often low. However, the presence of transport systems, even when conferring low levels of resistance to antibiotics, increases the frequency of mutations to higher-level resistance (Takiff et al., 1996; Markham and Neyfakh, 1996). This can result from combination of efflux with other resistance mechanisms, or increased efficiency of efflux due to overproduction or mutation of the transporters. Duplication of Smr, for example, on a transferable pTZ22 plasmid of Staphylococcus uureus doubled the efflux rate and con*Kolaczkowski, yeast multidrug M. et al. In viuo screening of the substrate specificity of the resmance network. Manuscript in preparatim.

226 ferred high-level is analogous resistance to antiseptics. This mechanism cancer fantrine (Wilson

M. Kolaczkowski and A. Goffeau

et al., 1993; Cowman et al., 1994). InterP-gps and MRP the the STE6 deficiency of Sacet al., 1996; Raymond of

to that found in multidrug-resistant P-gp-mediated antibody UIC2 MDR resistance

estingly, Pghl shares with mammalian ability to partially complement

cells, which often overproduce P-gp and MRP. A recent report showing MDRl ment-mediated pamil and anti-P-gp to compleF(ab)* It cytotoxicity, which was reversible by veraand HYB-241 (Weisburg et al., 1996).

charomyces cereoisiae, suggesting that it is able to transport the yeast a-mating factor (Volkman et al., 1992; Ruetz et al., 1996). Recently, chloroquine The the presence Thus, the mechanism

fragments, also merits attention suggests that bacterial tribute to protection lapping substrate specificity

Pghl function seems to be quite complex. of a transport system mediating in several chloroquineuptake was linked to cross (Sanchez responsible uptake and defective rate of chloroquine in a genetic

transporters with MDRl,

that share overmight also concomplement-

of microbes to protection

against

resistant Plasmodium falciparum isolates, has been reported. decreased chloroquine 1997). An Entamoeba histolytica (pathogen showing increased efflux activity, other hydrophobic selected for dysto some has been entery and liver abscesses) mutant resistant to emetine and cross resistance drugs, reversible by P-gp antagonist verof a P-gp homologue, (Samuleson et al., 1990; Descospp. resistance to resistance

mediated lysis by the host immune system. In addition, they may also contribute microbial drophobic tance 2.7.2.2. Ouelette, against the innate antibecause many hyand in the resispeptide-mediated immunity P-gps

et al.,

peptides have also been identified of mammalian

spectrum

(Gottesman

Pastan, 1993). Parasitic protozoa. The resistance of parasitic prochemotherapeutic agents (Borst and 1996) be1995; Ullman, 1995; Rubio and Cowman, tozoa to available

apamil, and over-expression in the laboratory teaux et al., 1992).

is alarming, in particular in Plasmodium fakiparum, the causative sistance Verdier, been agent of malaria responsible tween 1.5 and 2.7 million people yearly (Butler, to chloroquine 1994). Increased in resistant to account 1992), mainly from decreased cellular accumulation been observed proposed

which is

for killing

In Leishmania spp. and Trypanosoma pentavalent treatment antimonials, of espundia,

1997). Reresults has

which are drugs of choice for the kala azar (caused by L.&mania and Papaef-

in Plasmodium fakiparum

(Pussard and

spp.), and sleeping sickness (caused by Trypanosoma spp.), is often observed in clinical dopoulou, 1993; Bacchi, flux was observed in selected somal circles. quences dopoulou, &@A, These circles isolates (Ouelette 1993). Increase in antimonial

active efflux of chloroquine for the resistance genes, homologues

PIasmodium falciparum and has phenotype of mammaof probably resulting from am-

mutants of Leishmania spp., contain genes several repeated (Ouelette segene to

(Krogstad et al., 1987, plification

which often amplify parts of their genome as extrachromoand drug-resistance 1993). One which and Papa-

of the n&-like

lian P-gps (Wilson et al., 1989; Foote et al., 1989) that have been identified in this parasite. Neither the amplification the pfmdrl gene nor efflux of chloroquine, gated with chloroquine resistance The resistance in (Wellems et al., 1990). a genetic 7 (Wellems however, segrecross by et al.,

of these

is the P-gp-related

was shown by transfection

experiments

confer only low-level resistance to oxyanions in the form of pentavalent antimony compounds. The decreased accumuexpression, was of antifungal antibiotics. often lation of 73As 0 2 in the cells, due to @@A not observed however (Papadopoulou 2.7.2.3. Fungi. The number and variety

locus identified

this approach, mapping to chromosome 1991), has not been cloned. Expression parasites conferred hypersensitivity ated with its enhanced increased chloroquine observation, chloroquine acidification, accumulation in contrast

in Chinese hamster associalleles This en-

et al., 1994).

ovary cells of the pfmdrl gene from chloroquine-sensitive to chloroquine, to the mutant in lysosomes and their

agents is inferior to the plethora of antibacterial The therapeutic caused by the emerging nosuppressive new pathogens

options for treating fungal infections,

whose incidence are

from chloroquine-resistant hypersensitivity along with analogues

isolates, which failed to confer (Van Es et al., 1994a,b). (the protein the failure of photoactivatable

has increased due to the AIDS pandemic and use of immudrugs in transplant and cancer patients, antibiotics) limited by the relatively low number and structural variety (as compared with antibacterial developed of antifungals limitations are in the last decades. Additional

to bind to Pghl

coded by the pfmdrl gene) (Foley et al., 1994), and the fact that chloroquine is a weak base accumulating in acidified liposomes (Cullis et al., 1991) suggest that Pghl might affect its intralysosomal ously observed concentration indirectly by pH alterin Pghl, previresistance ations. These data confirm (Foote et al., 1990), that mutations

imposed by their selectivity, pharmacokinetic profiles, and side effects (Como and Dismukes, 1994; Georgopapadakou and Walsh, 1994; Vanden Bossche, 1995; Hazen, 1995; Tuite, 1996). The antifungal agents effective against lifeand threatening infections of deep tissues comprise the three

to be linked

to chloroquine

indeed can contribute

to chloroquine

azoles fluconazole,

itraconazole,

and ketoconazole,

resistance. In addition, chloroquine selection of Plasmodium fakiparum lines with amplified pfmdrl leads to its deamplification and reduced production of Pghl, but unexpectedly increases sensitivity to mefloquine (Barnes et al., 1992). Indeed, amplification of the pfmdrf gene has been correlated quinine, and halowith increased resistance to mefloquine,

5-fluorocytosine and amphotericin B (Como and Dismukes, 1994). For some infections, however, there is no effective therapy. It is worrying that the efficacy of existing antifungals pathogenic is threatened by shifting of the population of fungi towards the intrinsically resistant species of resistance in the species usually re-

and development

Drug Resistance Mechanisms garded as sensitive (Price et al., 1994; Vanden Bossche et al., 1994; Marichal and Vanden Bossche, 1995; Hazen, 1995; Odds, 1996). Although active efflux as a mechanism of fungal pathogen resistance has been appreciated only recently, it has been suggested as an important mechanism of resistance to fluconazole (DuPont et al., 1996). Multidrug transporters of the MFS-type such as CaMDRl/Benr (Fling et al., 1991) and ABC-type such as CDRl (Prasad et al., 1995) and CDR2 (Sanglard et al., 1997) conferring a broad spectrum drug resistance (see Table l), have been identified in the principal human yeast pathogen Candida &cans. CDRl and CDR2 turned out to be homologues of the previously identified Snq2p and Pdr5p of the yeast Saccharomyces cerevisiae (Servos et al., 1993; Balzi et al., 1994; Bissinger and Kuchler, 1994; Hirata et al., 1994) (56% identity between CDRl and PdrSp and 42% identity between CDRI and Snq2). Snq2p and Pdr5p are the first identified members of a distinct subgroup of ABC transporters, characterized by their inverted domain organization in the primary sequence. These resemble the mirror image of the mammalian P-gps in that they contain a tandem repeat of an ATP-binding domain (NBD) followed by six TM helices (NBD-TMNBD-TM). In contrast, the primary structure of mammalian P-gps can be abbreviated TM-NBD-TM-NBD (Decottignies and Goffeau, 1997). ABC transporters of this type have only been reported so far in fungi and plants, and also include AtrA and AtrB of the model filamentous fungus Aspergillus nidulans (Del Sorbo et al., 1997). AtrB also confers MDR in the heterologous host Sacchuromyces cereplisiae. Importantly, various filamentous fungi, including Aspergillus species, are also responsible for a variety of human infections (Vanden Bossche, 1995), including pulmonary aspergillosis, which is a leading cause of mortality in bone marrow transplant recipients. In Candida al&cans CaMDRl/Benr, CDRl or CDR2 deletions lead to hypersensitivity to many drugs, including the most important fungicides in clinical use (Goldway et al., 1995; Sanglard et al., 1996, 1997). Their over-expression has been observed in resistant clinical isolates (Sanglard et al., 1995, 1997; Albertson et al., 1996). Activity of MDR transporters, at least in part, may account for the general low permeability of many fungal pathogens, such as different Candida species, to a variety of inhibitors of potential antifungal targets. Many such inhibitors show poor intracellular accumulation and cannot be developed as antifungal drugs. This permeability barrier is reminiscent of the long-known MDR phenomenon called pleiotropic drug resistance (PDR) observed in the nonpathogenic yeast Saccharomyces cerevisiae (Balzi and Goffeau, 1995; Goffeau et al., 1997).

227 ingly broad substrate specificity. The resistance mechanisms discussed in Section 2 are usually limited to sets of closely structurally related molecules with a similar mode of action. Even though a few specific mechanisms can accumulate together and confer an MDR phenotype, it is not comparable with the wide resistance profile of a single MDR transporter.

3.1. Drug-Resistance Profiles of Mu&drug-Resistance Transporters

3. THE DILEMMA
BROAD

OF SPECIFICITY

SUBSTRATE

The common and intriguing feature of MDR transporters in contrast to their non-MDR counterparts is their surpris-

The most intensively investigated MDR transporters in this respect are the mammalian P-gps. Their substrate specificity profile includes a huge variety of hydrophobic compounds, ranging from peptides and steroid hormones to anticancer drugs, such as daunorubicin, doxorubicin, vinblastine, vincristine, taxol, dactinomycin, etoposide, teniposide, and others (Gottesman and Pastan, 1993). Understanding of this profile comes mainly from comparisons of drug-resistance profiles of multidrug-resistant cell lines that overexpress P-gp, selected on increasing concentrations of different cytotoxic agents, to their sensitive parental lines. Only a few reports, however, deal with P-gp transfectants not exposed to cytotoxic drug selection. It is known that such selection can favor mutant transporters with altered specificity profiles (Choi et al., 1989), and can also lead to over-expression of other transporters with overlapping specificity and other resistance mechanisms not necessarily associated with efflux. Different research groups use cell lines from different tissues, and even species (human, mice, hamster), usually selected by different procedures, while each study reports on the effects of relatively few metabolic inhibitors. This is why it is difficult to qualitatively and quantitatively estimate the full contribution of human MDRl P-gp to resistance to these compounds. The only large-scale screen reported so far is that carried out by the National Cancer Institute (Bethesda, MD, USA), but only a small part of their results have been published (Lee et al., 1994; Alvarez et al., 1995). This attempt was made to predict P-gp substrates by correlation of mdr-1 expression and cyclosporin A (P-gp antagonist) reversible rhodamine 123 efflux with cytotoxicity data of more than 30,000 compounds on 60 human tumor cell lines from the National Cancer lnstitute drug screen database. The information is then applied to identification and characterization of P-gp antagonists of potential clinical application as MDR-reversing agents. There are surprisingly few transporters reported for which a large number of compounds was used to generate the drug-resistance profiles directly for instance, by comparison of cells transformed with the transporter expressing vectors with ones transformed with control vectors or by comparison of transporter expressing cells with cells in which the transporter-encoding gene has been deleted. These are the MFS-type CaMDRl/Benr of Candida al&cans, MdfA of Escherichia cob, as well as the ABC-type CDRl, CDR2 of Candida a&cans and their Saccharomyces cerevisiae Pddp homologue (see Table 1).

228 TABLE 1. Overlapping MDR Tranmorters

M. Kolaczkowski

and A. Goffeau

Drug Resistance Profiles of Candidu albicans, Sacchmomyces cerevisiue, and Escherichia coli candida albicuns CaMDR 11 MFS CDRl CDR2 PdrSp2 ABC Saccharomyces cerwisiae Snq2p Yorlp Atrlp4 Sgelp MFS Escherichia coli MdfA

3-Amino-1,2,4,-triazole Amphotericin B Amorolfine Antimycin Benomyl Brefeldin A Camptothecin CCCP Cerulenin Compactin Chloramphenicol Crystal violet Cycloheximide Daunorubicin Dinitrophenol Erythromycin Ethidium bromide Filipin Fluconazole Fluphenazine Itraconazole Ketoconazole Miconazole Nitrogen mustard 4-Nitroquinoline-N-oxide Nystatin Oligomycin l,lO-Phenantroline Rhodamine 6G Staurosporine Sulfometuron methyl Terbinafine Triaziquone

X6
x7

x6
R7 XO R7 -

R7 x7 R7 -

R* X9

R X3
X3 -

R6 RO -

Xl

R5 X5 X5
x5 x5

R X2
R3

RO x7

R7 RS

R7

Xl4
X4 -

R7 -

X7
RS G z XS RO RO RO RO
R5
XO X5

R7 R7 -

R R R9

x4 R6 Rs R6
R6

Rs RO RO XO XO X6
Rl8

X3 RO
R3

R2 Rs X8

R9 -

R7 R7 R7 R7 -

6 Rs RO RO G x9 R2 Rj R

RO RO X20 R*O

Xl4 -

R2 x5 -

X6
RO
x7

x7 -

5
RI4

x7 R
; R7 -

x7 R7 x7 R7

R22 R3 R22 R20

-

R20 R7 -

R6 -

R, confers resistance; X, not shown to confer resistance; -, not tested; CCCP, carbonyl cyanide-m-chlorophenylhydrazone. CaMDRl/Benr also confers resistance to benzimidazole-2-yl-carbamate, l-benzoyl benztriazole, I-(2sfluoryl)-5-trifluormethyl benztriazole, and methotrexate, but not to l-deaza-7,8-dihydropteridine, echinocandin, 5-fluorocytosine, hygromycin, indole propyl carbamate, papulocandin, or parafluorophenylalaline (Fling et nl., 1991). Tddp also confers resistance to doxorubicin, deoxycorticosterone, ionophore A23187, monensin, nigericin, progesterone, rhoadmine 123, tamoxifen, trifluoperazine (Kolaczkowski et al., 1996), sporidesmin (Bissinger and Kuchler, 1994), 1 mcomycin, and venturicidin (Meyers et al., 1992), but not to FCCP (Kolaczkowski et al., 1996). Pdr5p modifies intracellular accumulation of corticosterone, dexamethasone, triamcinolone acetonide (Kralli ec al., 1995), and, together with SnqZp, estradiol (Mahe et al., 1996a). 3Yorlp also confers resistance to acetic acid, benzoic acid, cadmium chloride, leptomycin B, propionic acid, reveromycin A, and tautomycin (Cui er al., 1996). 4Atrlp has not been shown to confer resistance to canavanine, ethionine, p-fluorophenylalanine, triazolealanine, or vinblastine (Kanazawa et al., 1988). MdfA also confers resistance to benzalkonium, ciprofloxacin, kanamycin, neomycin, norfloxacin, puromycin, rifampin, tetracycline, and tetraphenylphosphonium, but not to nalidixic acid, methyl viologen, or spectinomycin (Edgar and Bibi, 1997). 5Kanazawa et al. (1988). 6Fling et nl. (1991). 7Sanglard et al. (1997). RMeyers etal. (1992). 9Leppert et al. (1990). OSanglard etal. (1995). Reid et al. (1997). l*Kolaczkowski et al. (1996). Hirata et al. (1994). 4Cui et al. (1996). Trasad et al. (1995). 6Edgar and Bibi (1997). Ehrenhofer-Murray et nl. (1994). Ben-Yaacov et al. (1994). Amakasu et ai. (1993). 2Haase et al. (1992). 21Gompel-Klein and Brendel (1990). **Servos et al. (1993).

Drug Resistance Mechanisms 3.2. Mechanism mediate resistance available to so many difhas in-

229

the inside positive membrane and transports colchicine (Ruetz and Gros, 1994a,b). by purified reconstituted on this matter P-gps. Additional changes 1995). in the Comparable

potential

of almost 90 mV gradient by

A still unresolved question can be asked at this point: How can a single transporter ferent compounds? Most of the information been obtained formation with the mammalian

against its concentration P-gp was not pH

The transport of Hoechst 33342 accompanied (Shapiro and Ling,

intraliposomal rates of ATP

hydrolysis and substrate in proand

comes from the analysis of other

transporters,

(valinomycin teoliposomes Another transport (Gottesman

s6Rb+ complex)

transport has also been ob-

since their drug-resistance

profiles often overlap with that similarity. Several lines of evcan directly recognize to this the variety of comAlternative (Fig. 2), attempts to assuming indirect changes pH, have also been P-gp expo-

served recently with the hamster P-gp reconstituted (Eytan et al., 1996a).

of P-gp, suggesting functional and export

idence suggest that P-gp somehow from the cell membrane

argument favoring direct drug interaction is the fact that many point mutations

in P-gps proto imwhich

pounds to which it mediates resistance. or the classical pump model hypothesis explain the huge spectrum of resistance, drug redistribution in membrane (1994a), proposed (Roepe, however, potential or intracellular

et al., 1995) and other MDR transporters (Klythe level of resistance resistance of some structural labeling of mutant mediated

achko et al., 1997) specifically alter the drug-resistance files, often increasing certain suggests the existence Altered photoaffinity (Kajiji drugs and decreasing to others,

in response to P-gp-dependent

preferences

1997). A recent report by Ruetz and Gros shows that the mouse Mdrla

posed by the mutant transporters been observed photolabeling

(Gottesman

et al., 1995). P-gps has also and

pressed in yeast secretory vesicles can mediate vinblastine transport and is not affected by changes in membrane tential and pH gradient. In addition, Mdrla I-gp also transports the lipophilic cation tetraphenylphosphonium against

et al., 1993).

The mutagenesis

studies pointed out the importance of TM re(Pawagi et al., 1994; Gottes-

gions in substrate recognition man et al., 1995).

embrane insertion

flip-flop (slow)

membrane release

CYTOPLASM
FIGURE 2. Possible routes of multidrug transport. According to the vacuum cleaner hypothesis, drugs may be extruded directly from the membrane (inner or outer leaflet) into the extracellular space or flipped from the inner to the outer leaflet (flippase model). Direct extrusion from the cytoplasm into the extracellular space cannot be excluded (classical pump model). Transport can be energized by ATP hydrolysis (ABC transporters) or proton motive force (MPS, RND, and SMR transporters).

230 Support for the idea that P-gp recognizes the drugs directly from the lipid phase came from energy transfer experiments with daunorubicin (a fluorescent P-gp substrate) azide (Raviv et access of aceAM ester) upon and the photoaffinity label iodonaphthalene the intracellular potentially

M. Kolaczkowski and A. Goffeau may gain access to the central pore and extraet al., 1997). Since the substrates are hydrophobic, It seems that by diin membranes.

cellular space (Rosenberg they tend to accumulate

of P-gps and many related transporters

al., 1990). This view was further supported by the observations that P-gp prevents toxymethyl (AM) esters of BCECF become (BCECF-AM-2,7-bis-

rect extrusion of these compounds from the lipid phase, the transporters not only prevent drugs from accessing their intracellular targets, but in parallel, also may protect against What determines the broad specificity of the adverse effects of these lipophilic compounds on membrane integrity. Yet another regulate Gunther, 1997). transport is still an open question. intriguing aspect of MDRl (Higgins, and Tsui, is its ability to Jentsch ABC and transchloride channels Interestingly, (Zielenski 1995; 1995),

(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and other dyes, which

brightly fluorescent

reaching the cytoplasm and upon hydrolysis by intracellular esterases (Homolya et al., 1993; Ho110 et al., 1994). Similar observations other have been made with multidrug-resistant fluorescent probe mutant cells of Luctococcus lactis. These cells also extrude anlipophilic I-[4-( trimethylamino) (TMA-DPH), and the conphenyl]e6-phenylhexa-1,3,5triene efflux rate has been correlated centration in the cytoplasmic

a homologous

porter, the CFTR rectifying 1997). lurea chloride

itself func-

tions as a chloride channel Similarly, receptor, another regulates and Gunther,

and regulator of the outwardly (Jilling and Kirk, the sulfony(Higgins, of this ABC transporter,

with the TMA-DPH

and sodium channels potassium

leaflet of the plasma memmay recognize et al., were also

brane, suggesting that an MDR transporter 199613). The same observations

channels

its substrates localized in the inner leaflet (Bolhuis with TMA-DPH

1995; Jentsch regulation ate. ATP

1997). The mechanism

is not known.

It might involve direct proteinbut its

made with Luctococcus lactis cells specifically over-expressing the LmrP MDR transporter, and were confirmed with plasma membrane vesicles (Bolhuis et al., 1996a). The removal of Hoechst 33342 from the membranes by purified and reconsti, tuted P-gp has been reported by Shapiro and Ling ( 1995). These studies, however, do not fully resolve whether the drugs are extruded directly from the membrane tracellular model medium, (Raviv et al., 1990; Gottesman into the ex1994), or as proposed by the vacuum cleaner et al., leaflet, from (Hig

protein interactions

or the outflow of a soluble intermedi-

was suggested as a possible intermediate, 1995; Abraham

transport by the above ABC transporters is a matter of controversial debate (Al-Awqati, this context, it is interesting et al., 1997; 1997a,b). In Reddy et al., 1997; Grygorczyk and Hanrahan,

to note that the yeast MDR

transporter genes PDR.5 and SNQ.2 have been shown to be induced by ionic stress imposed by Na+, Li+, and Mn++ cations. Deletion of these genes leads to hypersensitivity intracellular accumulation of these and slightly increased

flipped from the inner to the outer membrane

which they can diffuse outside (the flippase model)

cations (Miyahara et al., 199613).

gins, 1994). They also leave a possibility that at least some drugs are recognized directly in the cytoplasm and extruded outside (classical mechanism ble for maintaining cal membranes MDRl Helvoort translocate translocate pump model) (see Fig. 2). The flippase asymmetry in biologiIndeed, the human (van have been shown to not inElfwas also proposed earlier for proteins responsithe phospholipid 1993). recently (Zachowski, 3.3. Substrate Recognition by Protein is also complex. charged LTD4, and transport

Multidrug Resistance-Associated MRP substrate recognition In membrane endogenous LTE,, 1994), glutathione

vesicles, MRP transports negatively conjugates, conjugates such as LTC,,

and mouse Mdrla et al., 1996),

a range of short acyl chain phospholipids and their close homologues MDRZ, (human

and glutathione ethacrynic

of lipophilic compounds, monochloro

such as 2,4-dinitrophenol melphalan of LTC,, glutathione (anticancer)

(Leier et al., 1994; Muller et al., (Jedlitschky

volved in MDR erink et al., 1997).

as well as mouse Mdr2) into the bile (Oude

acid (Zaman et al., 1996), (Leier et al., 1996).

phosphatidylcholine

et al., 1996), and also

In oiao transport by alkylated inhibited

oxidized glutathione P-gps and the

It is not understood how the mammalian

which was competitively

Plasmodium falcipurum Pghl transport the relatively big, as compared with drugs, a mating factor, which was proposed as a mechanism of complementation of the defect in the yeast Succharomyces cerevisiae STM cific transporter Volkman Recent (Raymond a-mating factor-speet al., 1992; Ruetz et al., 1996; three-dimensional struc-

derivatives, was also inhibited by taxol, VP-16,

vincristine, and vinblastine, but surprisingly, this inhibition was efficient only in the presence of glutathione. Vincristine itself was transported by MRP in membrane vesicles in the presence of glutathione ated in vitro transport inhibited competitively (Loe et al., 199613). MRP mediwas DNP-SC, of azidophenacylglutathione by oxidized glutathione,

et al., 1996).
data on low-resolution

tures of hamster P-gp suggest that it forms a central membrane-spanning chamber closed at the cytoplasmic side and open to the extracellular space but large enough to allow passage of known P-gp substrates. This chamber within the membrane has an opening to the lipid phase, which might be the putative substrate-binding site through which drugs

the LTD, receptor nyl]ethenyl)phenyl}

antagonist 3([(3(2[7-chloro-2-quinoli{(3-dimethylamino-3-oxopropyl) thio)

methyl] thio) propanoic acid (MK571), arsenate, daunorubicin, vincristine, and etoposide (Shen et al., 1996). In addition to glutathione conjugates, in membrane vesicles, MRP transports glucuronides, such as 17+estradiol-

Drug Resistance Mechanisms 17-P-D-glucuronide (Loe et al., 1996a), glucuronosylhyodeoxycholate, and glucuronosyletoposide, and sulfates such as sulfatolithocholyltaurine (Jedlitschky et al., 1996). Transport of 17-P-estradiol-17-P-D-glucuronide was competitively inhibited by other cholestatic-conjugated steroids (Loe et al., 1996a). The transport of calcein, calcein acetomethoxymethyl ester, and pyrenemaleimide glutathione conjugate by MRP in whole cells has also been suggested (Feller et al., 1995; Ho110 et al., 1996). In addition to the negatively charged compounds, in membrane vesicles, MRP was shown to transport neutral or mildly cationic cytotoxic lipophilic drugs such as daunorubicin, vincristine, and etoposide. Daunorubicin transport was competitively inhibited by reduced and oxidized glutathione, azidophenacylglutathione, dinitrophenyl glutathione, arsenate, genistein and MK571 (Paul et al., 1996a,b,c). In another study, reduced glutathione did not inhibit LTC, transport (Loe et al., 1996b), and no MRP-mediated transport of doxorubitin, daunorubicin and vinblastine was observed (Jedlitschky et al., 1996). Direct interaction of MRP with vincristine, VP16, reduced and oxidized glutathione, however, was suggested by their stimulation of vanadate-induced trapping of MgATP (Taguchi et al., 1997). Finally, expression of MRP in the yeast Saccharomyces cerevisiae ste6 null mutant partially restored mating, suggesting that MRP can transport the a mating pheromone (Ruetz et al., 1996).

231 has been implicated additionally in the red pigment formation of Succharomyces cerevisiae a&l and ade2 mutants, presumably by transporting the glutathione conjugates of the endogenous metabolites in the adenine biosynthetic pathway (phosphoribosylaminoimidazole and phosphoribosylaminoimidazole carboxylate) into the vacuoles (Chaudhuri etal., 1996). Yaplp and Yap2p also influence the expression of PDRS and SNQ2 genes encoding ABC transporters, as their heat shock-induced expression becomes very low in the yap], yap2 double disruptant (Miyahara et al., 1996a). The second network, called the PDR network (Balzi and Goffeau, 1995), is regulated by the Pdrlp and Pdr3p transcription factors and the unidentified PDR4, PDR7, and PDR9 loci (Dexter et al., 1994). Many spontaneously isolated mutations in Pdrlp and Pdr3p result in over-expression of the ABC transporter genes SNQ2, PDRS, and YORI , which initially were cloned as genes conferring resistance to cycloheximide (Leppert et al., 1990; Balzi et al., 1994), 4NQ0 (Haase et al., 1992; Servos et al., 1993), and oligomycin (Katzmann et al., 1995; Cui et al., 1996), respectively. On the other hand, the double deletion of both PDRI and PDR3 genes results in drug hypersensitivity and strongly reduced expression of SNQ2, PDRS, and YORl (Decottignies et al., 1995; Mahe et al., 1996b; Katzmann et al., 1995). Two other MDR transporter-encoding genes belonging to the MFS, energized by proton motive force, have been identified in yeast. These are ATRl, conferring aminotriazole and 4NQO resistance (Kanazawa et al., 1988; Gompel-Klein and Brendel, 1990), and SGE1, conferring resistance to crystal violet and ethidium bromide (Amakasu et al., 1993; Ehrenhofer-Murray et al., 1994). The transcript of /&iR1 has not been affected in multidrug-resistant mutants of PDR I, suggesting that it probably is not under the control of PDRl and PDR3 (Balzi et al., 1994). Finally, sequencing of the yeast genome unraveled 13 other ABC homologues of PDRS , SNQ2, and YORl (Decottignies and Goffeau, 1997). This inventory revealed that Pdr5p and Snq2p belong to a new family of ABC transporters, which so far contained no human homologues. In contrast, the family comprising Yorlp has several human homologues, including MRP, which shows 33% amino acid identity to Yorlp (Katzmann et al., 1995). Also, 26 other major facilitators homologous to ATRI and SGEf have been identified in the yeast genome (Goffeau et al., 1997). Some of these are likely to be involved in multidrug transport. The resistance profiles of the identified Saccharomyces cerevisiae MDR transporters reported to date are summarized in Table 1. We have observed, however, that the drug-resistance profile of Pddp is much larger than previously believed. It comprises many other steroids, fungicides of different chemical classes, herbicides, detergents and other toxic compounds. Its substrate specificity largely overlaps with that of Snq2p and Yorlp. These data, ob*Kolaczkowski, yeasr multidrug M. et al. In uivo screennxg of the substrate specificity resistance network. Manuscript in preparatmn. of the

4. THE YEAST SACCHAROMYCES CEREVlSlAE PLEIOTROPIC DRUG-RESISTANCE NETWORK AS A TOOL TO UNDERSTAND THE MECHANISM OF MULTIDRUG RESISTANCE AND TRANSPORT In the yeast Saccharomyces cerevisiae, two networks of genes involved in MDR have been identified. The first one is reg ulated by Yaplp, a bZIP-containing transcription factor (Moye-Rowley et al., 1989) protecting yeast against oxidative stress (Schnell et al., 1992; Kuge and Jones, 1994; Kuge et al., 1997). Together with Cadlp, a Yaplp homologue, this network also protects yeast against toxic effects of cadmium, zinc, l,lO-phenanthroline, and cycloheximide (Wu et al., 1993). Yaplp also confers resistance to sulfomethuron methyl (Leppert et al., 1990), 4-nitroquinoline-Noxide (4NQO), N-methyl-N-nitro-N-nitrosoguanidine, trenimon, and triaziquone (Hertle et al., 1991; Haase et ai., 1992). Overproduction of YapZp, the homologue of Yaplp that also specifically binds the AP-1 recognition DNA sequence, mediates resistance to l,lO-phenanthroline. In addition, yap2 null mutants show increased thermotolerance under iron and zinc starvation conditions induced by the chelator l,lO-phenanthroline (Bossier et al., 1993). Cadmium resistance mediated by Yaplp requires the presence of an ABC transporter Ycflp (a homologue of MRP) (Wemmie et al., 1994), which sequesters bis(glutathionato)-cadmium in the vacuoles (Li et al., 1997). Ycfl

232

M. Kolaczkowski and A. Goffeau Yeast has been used for analysis of the mechanism of action of certain anticancer drugs (Nitiss and Wang, 1988; Abe et al., 1994; Fox et al., 1994; Nitiss, 1994; Kauh and Bjornsti, 1995; Ishida et al., 1995I/Karavokyros and Delitheos, 1997) and other drugs (Wdoden et al., 1997). In this context, we have shown that PdrSp confers resistance to several anticancer agents (Kolaczkowski et al., 1996), apparently by reducing their accumulation in the cells. Recently, resistance to the anticancer, topoisomerase-targeted drug camptothecin has been associated with the PDR network. In particular, SnqZp, but also PdrSp, when overproduced, conferred some resistance to this compound (Reid et al., 1997). Yeast has also been used for functional and mutational analysis of cloned steroid hormone receptors (Lind et al., 1996). The influence of the PDR network on intracellular hormone availability interfering with the transcriptional activity of steroid hormone receptors has been indicated by Gilbert et al. (1993). As mentioned in Section 4, the transporters Pdr5p and Snq2p are involved in the efflux of a large series of steroids (Kralli ec al., 1995; Mahe et al., 1996a; Kolaczkowski et al., 1996; Kolaczkowski et al.*). Use of more permeable yeast strains deleted in MDR transporters or their regulators would eliminate their interference with these applications. The mammalian hepatobiliary metabolism and excretion of endo- and xenobiotics responsible for the clearance of many chemotherapeutic drugs are used to determine the pharmacokinetic behavior of compounds important in drug development (Kling, 1996). In this respect, yeast Saccharomyces cerewisiae serves as an alternative to mammalian cell cultures to study the metabolism of a variety of drugs and xenobiotics by the heterologously expressed human enzymes involved in detoxification, including cytochrome P450 (Pompon et al., 1995). Again, the active permeability barrier of the PDR network interferes with these investigations. One of the important transport systems of the hepatic canalicular membrane, the understanding of which would be highly facilitated by cloning its gene, is the bile acid transporter. Our data* suggest that the PDR network is involved in resistance to certain bile acids, likely to be mediated by the Bat1 ABC transporter, mediating taurocholate uptake into the secretory vesicles (St-Pierre et al., 1994; Oritz et al., 1997). Therefore, the yeast system should offer an easy screen for expression cloning of the human canalicular bile acid transporter. Development, testing, and release of new antibiotics is a costly and time-consuming process. An alternative strategy, of which the P-lactamase inhibitors (clavulanic acid, sulbactam, tazobactam) co-administered with p-lactam antibiotics, are the first successful examples, is to overcome resistance by its inhibition (Coleman et al., 1994). Inhibition of bacterial efflux pumps has also been pursued to revitalize old antibiotics, such as tetracycline, that lost their efficacy due to widespread resistance (Service, 1995). Inhibition of
*Kolaczkowski, yeast multidrug M. et nl. In viva screening of the substrate specificity resistance network. Manuscript in preparation. of the

tained with isogenic strains in which the PDRS, ShJQ2, and YORl genes have been deleted in different combinations, allowed not only for qualitative determination of their drug-resistance profiles, but also gave gross quantitative estimation on the contribution of particular transporters to the resistance toward a few hundred compounds. These observations and that of others (Mahe et al., 1996a; Sanglard et al., 1997) establish that the presence of several transporters with overlapping specificity often prevents the observation of phenotypes associated with their single deletions. Cautious interpretation of results from drug-resistance assays, aiming at determination of drug-resistance profiles of particular transporters, is thus required, especially for assays performed with cell lines obtained by stepwise drug selection, which may involve several overlapping resistance determinants. In nature, the presence of such a flexible system assures very efficient protection of cells against a variety of toxic insults. To better understand Pdr5p-mediated transport of cytotoxic compounds, we have developed new sensitive fluorescence-based assays, allowing for the in viva and in vitro characterization of drug transport. In particular, the sensitive in vitro assay for ATP-dependent Pdr5p-mediated rhodamine 6G fluorescence quenching in plasma membrane preparations is the first reported assay allowing for a detailed largescale kinetic characterization of multidrug transporter inhibitors (Kolaczkowski et al., 1996). This assay, combined with in viva toxicity screening, provides a wealth of information for rational molecular modeling of new MDR transporter antagonists. The highly overlapping specificity profiles of the yeast MDR transporters with other prokaryotic and eukaryotic ones (Table 1) imply some functional and mechanistic similarity. It is likely, therefore, that our understanding oI the PdrSp mechanism of multidrug transport will contribute to an understanding of clinically relevant transporters such as, for example, MDRl P-gp or the PdrSp homologues CDRl and CDR2 involved in MDR of the pathogenic yeast Cundida albicans. It is likely that our transport assay could be also functional with heterologous MDR genes expressed in yeast plasma membranes. Heterologous expression of ABC proteins has been achieved in Saccharomyces cerevisiae (Kuchler and Thorner, 1992; Ruetz and Gros, 1994a), but the level of over-expression is often limiting. The huge over-expression of PDR5 in the regulatory mutants of PDRl and PDR3, and the availability of strains deleted in the endogenous yeast transporters, makes it an attractive system for overproduction, under the control of the PDR5 promoter, of other clinically relevant ABC transporters, some involved in MDR, such as P-gp, but also others such as CFTR and sulfonylurea receptor. 5. OTHER PRACTICAL IMPLICATIONS OF THE YEAST PLEIOTROPIC DRUG-RESISTANCE

NETWORK

Information on the PDR network modifying transport of a variety of compounds can be exploited for several practical applications.

Drug Resistance Mechanisms P-gp is one of the strategies to increase the efficiency of chemotherapy of certain tumors, particularly of the hematopoietic system. Although some improvement has been observed in several cases by co-administration of P-gp antagonists with anticancer drugs, it usually suffers from many side effects. These side effects result from the toxicity of the modulators, as well as changes in the pharmacokinetic profile of anticancer drugs, which gain easier access to the key organs normally protected by P-gp, such as brain. Several new P-gp modulators are at the stage of clinical trial (Hegewisch-Becker, 1996). The biochemical characterization of multidrug transport, necessary for rational design of inhibitors, has been hindered by lack of convenient in vitro assays of drug transport. The assays based on uptake of radioactive substrates require high amounts of precious biological samples, suffer from high noise, and are expensive, laborious and time-consuming; hence, they are not suitable for large-scale analysis. The only three fluorescence-based in vitro assays reported to date, allowing monitoring of transport in real time, lack sufficient sensitivity for the kinetic characterization of inhibitors (Guiral et al., 1994; Shapiro and Ling, 1995; Bolhuis et al., 1996a). Development of in vitro screening assays, such as the one described for the yeast Pdr5p (Kolaczkowski et al., 1996), allowing rapid characterization of MDR transporter inhibitors, combined with the crystallization and generation of high-resolution three-dimensional structures, may contribute significantly to the rational design of new MDR modulators. The hypersensitivity of yeast deleted in the regulators of the PDR network to many chemotherapeutic drugs points to the possibility of overcoming MDR by inhibition of regulators of MDR pumps. This is an interesting alternative to inhibition of transporters. Yeast and other MDR transporters can also be used as in uiuo selectable markers in expression vectors. This has implications not only for industrial fermentations, but also for gene therapy, which is hampered by inefficient DNA transfer and unstable expression of transgenes (Blau et al., 1997). Among other chemoresistance genes, MDR transporters offer the possibility of positive selection of transfected cells, not only in cell culture, but also in piivo by means of drugs that have been well characterized pharmacokinetically. This approach may improve the efficiency of gene therapy, particularly of hematopoietic disorders (Kane, 1996; Bank, 1996; Licht et al., 1997; Gottesman and Pastan, 1997; Moritz and Williams, 1997). For example, the efficiency of expression of the nonselectable glucocerebrosidase gene has been increased by its translational fusion with MDRI, which resulted in elevation of glucocerebrosidase expression upon drug selection (Aran et al., 1996). Anticancer chemotherapy suffers from severe, dose-limiting side effects, often resulting from the toxicity to cells of the hematopoietic system. Transfusions and administration of hematopoietic growth factors are applied in clinics to reduce morbidity after chemotherapy. One of the strategies to circumvent this problem is the protection of normal hematopoietic

233 cells by introduction of vectors expressing drug-resistance genes such as MDRJ (Gottesman and Pastan, 1997; Moritz and Williams, 1997). The possibility of biotransformation of compounds by combined expression of different biosynthetic genes (Roessner and Scott, 1996) has received a recent impetus by the development of directed DNA shuffling. It is based on generation of a large variety of different combinations of mutations by several rounds of DNA fragmentation and primerless polymerase chain reaction, combined with specific selection for the desired traits. This technology has been applied successfully for the improvement of single gene products, but also whole operons (Crameri et al., 1997; Wackett, 1997). It generates a large diversity of different combinations of desirable mutations, allowing, for example, the isolation of novel antibiotics not previously encountered in nature by manipulation of different genes involved in their biosynthesis. The diversity of generated new combinations of enzymes and resulting metabolites is limited, however, by their toxicity to the producing host. One of the strategies to overcome this problem, widely used in nature by antibiotic-producing organisms, would be to employ MDR transporters to actively extrude such toxic metabolites outside the cells. Recently, the production by yeast and other microorganisms of diverse surface active molecules has received considerable attention as biodegradable alternatives to traditional chemically synthesized surfactants. Many compounds of medicinal importance, steroids in particular, are produced by microbial biotransformations, including yeast (Ward and Young, 1990; Voishvillo et al., 1994; Mahato and Garai, 1997). Our observations, which indicate the involvement of the yeast MDR transporters in resistance to several detergents,* as well as steroids, might be exploited to better control their production. Better knowledge of the specificity of MDR transporters and the ability to manipulate them in a predictable way would be an obvious advantage in the design of particular biotransformations. Expression of transporters not affecting substrates, but extruding end products of biotransformations or unwanted intermediates to culture media, would increase product yield and facilitate purification. There is no reason to believe that this technology cannot be developed in yeast, using Pdr5p or other yeast MDR pumps. The yeast Saccharomyces cerevisiae can be used for presentation of peptide or antibody libraries, production of recombinant vaccines and of immobilized whole cell biocatalysts. These applications require the surface presentation of proteins or smaller peptide epitopes. Presentation of different small peptide epitopes in bacteria has been successfully achieved by fusion with the surface-exposed loops of outermembrane proteins (Stahl and Uhlen, 1997; Georgiou et al., 1997). Due to the high overproduction and membrane localization of Pdr5p in the PDRl mutants, this protein
*Kolaczkowski, M. et al. In viva screening of the substrate specificity yeast multidrug resistance network. Manuscript in preparation. of the

234

M. Kolaczkowski

and A. Goffeau

might offer attractive attachment points for such applications. In brief, due to homology with mammalian, parasite, and other microbial resistance systems, the yeast Succhomyces cereoisiae PDR network proves to be a good model to investigate MDR mechanisms. The understanding of the mechanism of broad specificity of MDR transporters may contribute significantly to the improvement of not only clinical, therapeutic applications, but also of biotechnological biotransformations of pharmaceutical interest. These can profit from the remarkable genetic properties of Saccharomyces cerewisti and its generally regarded as safe status, which allows its use for food and pharmaceutical production.
Acknowkdgements-We wish to thank Anna Kolaczkowska, Susan Cronin, Elisabecta Balzi, and Bart van den Hazel for general support and helpful comments and discussion. This work was supported in part by grants from the Service de la Politique Scientifique: Action Sciences de la Vie and by Fonds National de la Recherche Scientifique, Belgium.

Singh,

S. V., He, N. G. and Awasthi, transport

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