ABC Multidrug Transporters

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    10.2217/14622416.9.1. 105 2008 Future M edicine Ltd ISSN 1462-2416 Pharmacogenomics (2008) 9(1), 105127 105

    part of

    ABC multidrug transporters: structure,function and role in chemoresistanceFrances J Sharom

    University of Guelph,Department of Molecular& Cell ular Biology, GuelphOntari o, N 1G 2W1, Canada Tel. : +1 519 824 4120ext. 52247; Fax: +1 519 837 1802; E-mail : fsharom @uoguelph.ca

    Keywords: ABCG2 (BCRP),ABC superfamily, ATPhydrolysis, drug efflux,drug therapy, MRP1/ABCC1,mult idrug resistance,P-glycoprotein/MDR1/ABCB1, polymorphisms,transport mechanism

    Three ATP-bind ing ca sset te (ABC)-superfa mily multidrug eff lux pumps are kno w n t o b erespon sible f or chemo resista nce; P -glycop rot ein (ABCB1), MRP1 (ABCC1) an d ABCG2(BCRP). These tra nsporters play a n import an t ro le in no rmal physiology b y prot ectingt issues from to xic xenobiot ics and endo geno us meta boli tes. Hydrophobic amph ipathiccompo und s, including m an y clinically used drug s, intera ct w ith th e substra te-bindingpocket o f t hese prot eins via f lexible hydroph ob ic an d H-bo nding intera ctions. These effluxpumps are expressed in many huma n t umors, whe re they l ikely cont r ibute to resista nce tochemo th erapy trea tm ent . How ever, the use of ef flux-pump mo dula to rs in clinical cancertrea tm ent ha s proved d isap point ing. Sing le nucleo tide po lymorphisms in ABC drug -effluxpumps ma y play a ro le in responses to drug th erapy a nd d isea se susceptibility. The eff ect of

    various geno types and haplot ypes on the expression an d fun ct ion of these proteins is notyet clear, and th eir true impa ct rema ins cont roversial.

    The ATP-binding cassette (ABC) superfamily of proteins is one of the largest protein families inbiology [1]. It consists largely of membrane pro-teins that transport a diverse array of substrates,including sugars, amino acids, drugs, antibiotics,toxins, lipids, sterols, bile salts, peptides, nucleo-tides, endogenous metabolites and ions. ABCproteins are present in the cytoplasmic (inner)

    membrane of bacteria, and in both the plasmamembrane and organelle membranes in eukary-otes. The human genome encodes 49 ABC pro-teins [2,3], only a fraction of which have beencharacterized in terms of their biochemistry andfunction. They have been classified into sevensubfamilies based on phylogenetic analysis [2].ABC proteins in their functional form comprizea minimum of four core domains; two mem-brane-bound domains that form the permeationpathway for transport of substrates, and twonucleotide binding domains (NBDs) that hydro-lyze ATP to power this process. In bacteria, thesefour domains exist as two or four separatepolypeptides, whereas in eukaryotes, the fourdomains are often fused into a single large pro-tein with an internal duplication. Proteins in theABCC subfamily possess an extra N-terminaltransmembrane (TM) domain of unknownfunction. While prokaryotic ABC proteins canbe either importers or exporters, eukaryotic fam-ily members are exclusively exporters. ABC pro-teins are active transporters, pumping theirsubstrates up a concentration gradient using theenergy of ATP hydrolysis.

    Mammalian ABC proteins have gained promi-nence as their involvement in maintaininghuman health became evident; a total of 14 human ABC transporters have now been asso-ciated with a specific disease state [4]. In somecases, the physiological substrate is known(e.g., ABCB11 transports bile salts; and loss-of-function mutations produce progressive familial

    intrahepatic cholestasis type2 [PFIC-2]), whereasfor some proteins it remains to be determined(e.g., the disease Pseudoxanthoma elasticum iscaused by loss-of-function mutations in ABCC6 ,whose physiological substrate has not yet beenidentified). Several ABC proteins are multidrugefflux pumps that not only protect the body fromexogenous toxins, but also play an important rolein the uptake and distribution of therapeuticdrugs [5]. They can, therefore, profoundly affectdrug therapy, and resistance to treatment by mul-tiple drugs has been associated with their expres-sion in the target tissue. For example, multidrugresistance (MDR) to chemotherapeutic drugs is aserious barrier to successful treatment of manyhuman cancers. Polymorphisms in ABC drugtransporters have been increasingly studied overthe past few years, since it seems likely that theywill be responsible for varying responses to drugtherapy in the population. This review focuses onwhat we currently know of the molecular struc-ture, substrates, transport mechanism and poly-morphisms of the three most important ABCmultidrug transporters, and discusses their role indrug resistance in clinical therapy.

    For reprin t o rders, please cont act: [email protected]

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    ABC multidrug tra nsportersThe role of ABC proteins in resistance to anti-cancer drugs has been known for over30 years [6]. A total of 15 family members canfunction as drug-efflux pumps, and have beenimplicated in potentially conferring resistance tochemotherapeutic agents (see [5,7] for reviews).However, three ABC proteins appear to accountfor most observed MDR in humans androdents; P-glycoprotein (Pgp/MDR1/ABCB1),MDR-associated protein (MRP)1 (ABCC1)and breast cancer resistance protein ABCG2(variously known as BCRP, ABCP or MXR)[8].The year 2006 represented the 30th anniversaryof the discovery of Pgp. These drug transportersare located in the plasma membrane, where theyengage in active efflux of drugs and drug conju-gates. Pgp and MRP1 are 170190 kDa singlepolypeptides, while ABCG2 is a 72 kDa half-transporter, and likely functions as ahomodimeric complex [9]. Pgp is the mamma-lian ABC protein that we know most about interms of its structure and mechanism. It hasbeen described as a double-edged sword, inthat it protects sensitive tissues from potentiallytoxic xenobiotics and yet also causes MDR intumors, thus preventing effective chemothera-peutic treatment. ABCG2 and MRP1 also sharemany of these features with Pgp.

    Pgp and ABCG2 can export both unmodified

    drugs and drug conjugates, whereas MRP1exports glutathione and other drug conjugates,and unconjugated drugs together with free glu-tathione. All three demonstrate overlapping drugspecificity. This redundancy indicates that acomplex network of efflux pumps is involved inprotecting the body from toxic xenobiotics. Pgptransports a wide range of structurally dissimilarcompounds, many of which are clinically impor-tant, including anticancer drugs, HIV-proteaseinhibitors, analgesics, antihistamines, H 2-recep-tor antagonists, immunosuppressive agents,cardiac glycosides, calcium-channel blockers,calmodulin antagonists, antiemetics, anti-helminthics, antibiotics, steroids (see Box 1 for alist of selected compounds that interact withPgp). All are amphipathic, lipid-soluble com-pounds, with molecular weights in the range of 300 to 1000, often with aromatic rings and apositive charge at physiological pH. Physiologi-cal substrates for Pgp potentially include steroidhormones, lipids, peptides and small cytokines.To date, endogenous biomolecules that havebeen identified as likely Pgp substrates includeseveral phospholipids (phosphatidylcholine,

    phosphatidylethanolamine, phosphatidylserineand sphingomyelin) [1012], simple glycosphin-golipids (glucosylceramide, galactosylceramideand lactosylceramide) [12], platelet-activating fac-tors [13,14], aldosterone [15], -estradiol-17-D-glucuronide [16], -amyloid peptides [17] and sev-eral interleukins[18]. The ABCB4 gene product isa very close relative, with 78% sequence homol-ogy to ABCB1. This Pgp isoform functions toexport phosphatidylcholine from the liver canal-icular cells into the bile, and is believed to be alipid flippase [19], although it can also transportdrugs with low efficiency[20].

    MRP1 transports a variety of endogenous mol-ecules of physiological significance [21], includingfree glutathione, glutathione-conjugated leukot-rienes and prostaglandins (LTC 4, LTD4 andLTE4, prostaglandin A2-SG, hydroxynonenal-SG), glucuronide conjugates ( -estradiol--D-glucuronide and glucuronosyl-bilirubin) and sul-fate conjugates (dehydroepiandrosterone-3-sul-fate and sulfatolithocholyl-taurine) (Box2).Similarly to Pgp, MRP1 also confers resistance toa variety of anti-cancer agents, although not tax-ols. MRP1 prefers anionic substrates, and drugsare either exported as anionic glutathione, glucur-onate or sulfate conjugates, or cotransported withfree glutathione. MRP1 also transports heavymetal oxyanions such as arsenite and trivalentantimonite [22].

    ABCG2 is a broad specificity drug transporterlike Pgp, however it appears to transport bothpositively and negatively charged drugs, includ-ing sulfate conjugates (Box 3) [23,24]. It was firstdiscovered based on its ability to transportmitoxantrone, which is a poor substrate for Pgpand MRP1. ABCG2 cannot transport taxols, cis-platin and verapamil (Pgp substrates), calcein (anMRP1 substrate) or Vinca alkaloids and anthra-cyclines (substrates for both Pgp and MRP1),indicating that its substrate specificity partiallyoverlaps with that of the other two transporters.ABCG2 transports both Gleevec (imatinib)and Iressa (gefitinib), two recently introducedanticancer drugs that are tyrosine kinase inhibi-tors; these compounds also interact with Pgpand ABCC1, but substantially higher concen-trations are required. The list of ABCG2 sub-strates is rapidly expanding, highlighting theimportance of this protein.

    All three ABC drug-efflux pumps are able totransport fluorescent compounds (Boxes 1, 2 and 3) .These have proved very useful as a tool forexploring the transport activity of the ABC pro-teins in intact cells. For example, quantitation of

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    the uptake of these dyes into cells by flowcytometry, and its inhibition by other substratesand modulators, is an excellent indicator of

    transporter function. Fluorescent transportassays can also be used to distinguish betweendrug pumps. For example, calcein is a specificsubstrate for MRP1 and is not transported byeither Pgp or ABCG2. Calcein-AM, on theother hand, is an excellent substrate for Pgp andforms the basis for commercially available kitsfor screening Pgp substrates.

    MDR mod ulat orsDrug resistance resulting from ABC multidrugtransporters can be prevented by another group of

    compounds, known variously as MDR modula-tors, reversers, inhibitors or chemosensitizers [25].Cells do not display resistance to modulators. Inintact cells, the observation is that a modulator,when combined with a drug to which cells areresistant, will restore its cytotoxicity by shiftingthe LD50 to a much lower value. Modulatorsshow the same diversity of chemical structure assubstrates, and appear to act in several differentways. The mechanism of action of modulatorshas been explored in detail for Pgp. Some Pgpmodulators compete with transport substrates forthe drug-binding pocket of the transporter. Many

    Box 1. Clinically relevant drugs andother compounds that interact withP-glycoprotein (ABCB1).Ant icancer drug s

    Vinca alkaloids (vinblastine and vincristine)

    A nthracyclines (doxorubicin and daunorubicin) Taxanes (paclitaxel and docetaxel) Epipodophyllotoxins (etoposide and teniposide) Camptothecins (topotecan) A nthracenes (bisantrene and mitoxantrone)

    HIV prot ease inhibito rs

    Ritonavir Saquinavir Nelfinavir

    Analgesics

    M orphine

    Ant ih is tamines

    Terfenadine Fexofenadine

    H 2 -recept or antagon ists

    Cimetidine

    Immu nosuppressive agents

    Cyclosporine A Tacrolimus (FK506)

    Ant iar rhythmics

    Quinidine Amiodarone Propafenone

    Ant iepi lep t ics

    Felbamate Topiramate

    Fluorescent compou nds

    Calcein-AM Hoechst 33342 Rhodamine 123

    HM G-CoA reductase inhibit ors

    Lovastatin Simvastatin

    Ant iemet ics

    O ndansetron

    Tyrosine kinase inhibit ors Imatinib mesylate Gef itinib

    Cardiac gl ycosides

    D igoxin

    Ant ihe lminth ics

    Ivermectin

    Calcium -channel blockers

    Verapamil Nifedipine Azidopine Diltiazem

    Calmod ulin ant agonists

    Trifluoperazine Chlorpromazine Trans -flupentixol

    Ant ihyper tens ives

    Reserpine Propanolol

    Ant ib io t ics

    Erythromycin G ramicidin A

    Steroids

    Corticosterone Dexamethasone A ldosterone Cortisol

    Pesticides

    M ethylparathion

    Endosulfan Cypermethrin Fenvalerate

    Natura l products

    Curcuminoids Colchicine

    Ant ia lcohol i sm drug

    Disulfiram

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    of these compounds are transported by the effluxpump (e.g., cyclosporin A or verapamil) and canbe viewed as competitive inhibitors. On the otherhand, several hydrophobic steroid modulators arenot transported by Pgp, but bind to a high-affin-ity site close to the ATP-binding site [26]. Thehigh-affinity modulator XR9576 appears tointeract with Pgp at a location distinct from thesite of interaction of transport substrates, whichmay serve a modulatory function [27]. Themodulator disulfiram (used to treat alcoholism)interacts with Pgp and M RP1 in a unique man-ner, binding to the drug-binding pocket andalso modifying cysteine residues at the catalyticsite [28]. Overall, the different mechanisms bywhich modulators exert their action at themolecular level are not well understood, mak-ing the rational design of new templates achallenging task.

    Role o f ABC multidrug tra nsporters innormal physiologyThe ABC drug efflux pumps Pgp, MRP1 andABCG2 play a central role in protecting organ-isms from the toxicity of a variety of endogenousand exogenous molecules [29]. The tissues ororgans that are protected depend on the specificpattern of expression and the activity of each pro-tein in normal tissues. These transporters are, thusmajor contributors to the absorption, distribution

    and excretion of clinically administered drugs.Studies on knockout mice lacking each of theseefflux pumps have confirmed these ideas [3033].

    Pgp is expressed at high levels in the apicalmembranes of epithelial cells lining the colon,small intestine, pancreatic and bile ductules,and the kidney proximal tubule. It is also foundin the endothelial cells lining capillaries in thebrain, testis and inner ear. Pgp-knockout ani-mals display a disrupted bloodbrain barrier,and can be up to 100-fold more sensitive tomany drugs, which often show neurotoxicitynot seen in wild-type animals [34]. The pregnantendometrium, the placenta and the adrenalgland also express high levels of Pgp. The loca-tion of Pgp suggests that its primary physiologi-cal role is to protect sensitive organs and thefetus from toxic xenobiotics [35]. In the intes-tine, Pgp extrudes many drugs into the lumen,thus reducing their absorption and oral bio-availability. It may export endogenous steroidhormones from the adrenal gland. The presenceof Pgp in hematopoietic progenitor cells pro-tects the bone marrow from the toxicity of chemotherapeutic drugs [36].

    Box 2. Clinically relevant drugs andother compounds that interact withMRP1 (ABCC1).Ant icancer drug s

    Vinca alkaloids (vinblastine and vincristine) A nthracyclines(doxorubicin and daunorubicin) Epipodophyllotoxins(etoposide and teniposide) Camptothecins (topotecan and irinotecan) M ethotrexateM etalloid s

    Sodium arsenate Sodium arsenite Potassium antimonite

    Peptides

    G lutathione (G SH, GSSG )

    Gluta th ione conjugates

    Leukotrienes C 4 , D4 and E 4 Prostaglandin A 2-SG

    Hydroxynonenal-SG Aflatoxin B 1-epoxide-SG M elphalan-SG Cyclophosphamide-SG Doxorubicin-SG

    Sulf ate conjugates

    Estrone-3-sulfate Dehydroepiandrosterone-3-sulfate Sulfatolithocholyl taurine

    Pesticides

    Fenitrothion M ethoxychlor

    Toxins

    Aflatoxin B 1Glucuron ide conjug ates

    G lucuronosylbilirubin Estradiol-17- -D-glucuronide Etoposide-glucuronide NS-38-glucuronide

    HIV prot ease inhibito rs

    Ritonavir Saquinavir

    Tyro sine kinase inhibit ors

    Imatinib mesylate Gef it inib

    Fluorescent compoun ds

    Calcein Fluo-3 BC EC F

    Ant ib io t ics

    Difloxacin G repafloxicin

    Folates

    Folic acid L-leucovorin

    Natura l products

    Curcuminoids

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    ABCG2 is expressed in a variety of normal tis-sues, including intestine, kidney and placenta, aswell as brain endothelial cells and hematopoieticstem cells. Its expression is strongly induced inthe mammary gland during pregnancy and lacta-tion. Similarly to Pgp, it is assumed to functionin protecting tissues from toxicants, and it likelyplays a role in intestinal absorption, brain pene-tration and transplacental passage of drugs. Inthe mouse, ABCG2 actively concentrates chem-otherapeutic drugs and a major dietary carcino-gen into milk [37]. A recent report indicates thatABCG2 is responsible for secretion of riboflavin(vitamin B2) into milk[38], indicating that it alsoplays an important physiological role. ABCG2 -knockout mice display the disease protoporphy-ria, which results from uptake from the gut of the chlorophyll breakdown product, pheophor-bide a, a normal constituent of food [39]. Theabsorption of this compound from the diet isnormally limited by ABCG2. The transporter isalso expressed in certain stem cells, where it actsas a marker of pluripotent stem cells (the sidepopulation). In these cells, ABCG2 appears tointeract with heme and prevent accumulation of porphyrins, enhancing cell survival underhypoxic conditions[40].

    In contrast to Pgp and ABCG2, MRP1 isexpressed at the basolateral membrane of polarized epithelial cells. It protects tissues such

    as the bone marrow, kidney collecting tubules,and oropharyngeal and intestinal mucosa, fromtoxicants, and is also involved in drug clearancefrom the cerebrospinal fluid, testicular tubulesand peritoneum [29]. MRP1 plays a central rolein glutathione homeostasis in vivo , and exportsLTC4 from mast cells. It may also be involved inprotecting cells from the toxicity of bilirubin.

    ATP bind ing & h ydrolysis The NBDs of all ABC proteins contain threehighly conserved sequence motifs that play a crit-ical role in ATP binding and hydrolysis; theWalker A and Walker B motifs, found in manyproteins that bind ATP or GTP, and a signature Cmotif unique to the ABC superfamily. Site-directed mutagenesis approaches have revealedthe importance of these three regions to catalyticfunction [41]. Structural studies of isolated NBDsubunits from several ABC proteins have yieldeduseful information on the catalytic cycle. In thestructures of BtuCD [42] and Rad50cd [43] (cata-lytic domains of a DNA-repair enzyme) the twoNBDs are in close contact to form a dimericstructure. Each ATP-binding site is formed from

    Box 3. Clinically relevant drugsand other compounds that interactwith ABCG2.Ant icancer drug s

    M itoxantrone

    Bisantrene (R482T mutant form) Epipodophyllotoxins (etoposide and teniposide) Camptothecins (topotecan and irinotecan) Flavopiridol A nthracyclines (doxorubicin and daunorubicin;

    R482T mutant form)

    A n t i f o l a t e s

    M ethotrexate

    Porphyrins

    Pheophorbide a Protoporphyrin IX Hematoporphyrin

    Tyrosine kinase inhibit ors

    Imatinib mesylate Gef itinib

    Flavonoids

    G enestein Q uercetin

    Carcinogens

    Aflatoxin B PhiP

    Fungal tox ins

    Fumitremorgin C K o143

    Drug & m etabol i te conjugates A cetominaphen sulfate Estrone-3-sulfate Dehydroepiandrosterone sulfate Estradiol-17- -D-glucuronide Di nitrophenyl-S-glutathione

    HM G CoA redu ctase inhibit ors

    Rosuvastatin Pravastatin Cerivastatin

    Ant ihyper tens ives

    Reserpine

    Ant ib io t ics Ciprofloxacin Norfloxacin

    Fluorescent compou nds

    Hoechst 33342 BOD IPY-prazosin Rhodamine 123 (R482T/G mutants)Ant iv i ra l drugs

    Zidovudine Lamivudine

    Natura l products

    Curcuminoids

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    the Walker A and B motifs of one NBD subunit,and the LSGGQ signature C motif of the part-ner NBD subunit. Two molecules of ATP arebound in these sites at the dimer interface. Thissandwich dimer structure has also beenobserved for the NBD subunit of the bacterialABC proteins MJ0796 and HlyB (Figure 2) .These NBDs form a stable dimer in the presenceof ATP when the catalytic activity is inactivatedby mutation [44,45]. It seems likely that this NBDdimerization process plays a critical role in thecatalytic cycle of all ABC proteins.

    All the ABC drug-efflux pumps display con-stitutive ATPase activity, which appears to beuncoupled from transport, and takes place inthe absence of substrates. In the case of Pgp,basal ATPase activity is as high as35 mol/min per mg for the purified protein,depending on the presence of detergent, lipidsand drugs [46,47]. The Km for ATP is high(0.20.5 mM) [48,49], indicating that Pgp has arelatively low affinity for nucleotides. ABCG2appears to have a similarly high K m value forATP, while MRP1 has a considerably lower Kmof approximately 100 M [50].

    The basal ATPase activity of Pgp is modulatedby drug substrates and modulators in a complexmanner. Many substrates show a biphasic pat-tern, stimulating activity at low concentrationsand inhibiting at higher concentrations, whereasothers show only stimulation or inhibition(e.g., [5153]). The presence of different deter-gents and lipids also affects the drug interactionpatterns [54,55] There is currently no satisfactoryexplanation for these observations, although ithas been suggested that the biphasic patternmight arise from the presence of two drug-bind-ing sites, a high-affinity stimulatory site and alow-affinity inhibitory site[56]. The increase inATPase activity (and presumably transport) onaddition of drug was correlated with the pre-dicted degree of hydrogen bonding of substrate

    in the drug-binding pocket [57]. Substrates withextensive H-bonding showed low ATPase stimu-lation and low transport rates, whereas thosewith low levels of H-bonding expressed highATPase stimulation and correspondingly hightransport rates. The stoichiometry of ATPhydrolysis relative to substrate transport is a con-troversial issue but, overall, the data suggest that12 ATP molecules are hydrolyzed for each drugmolecule transported by Pgp.

    The use of the ATPase inhibitor, ortho-vanad-ate (Vi, an analog of P i), has led to some impor-tant mechanistic insights. The ATPase activity of Pgp is rapidly inhibited by the addition of Vi inthe presence of ATP. V i is trapped after a singlecatalytic turnover in only one NBD as the com-plex ADPViMg2+, which suggests that both cat-alytic sites must be functional for ATP hydrolysisto take place. Pgp was therefore proposed to oper-ate by a mechanism in which only one catalyticsite is in the transition state conformation at anytime, and the two sites alternate in catalysis [58].The Vi-trapped complex is very stable, and isbelieved to resemble the catalytic transition statestructurally. MRP1 and ABCG2 are also

    Figure 1. Predicted membrane topology of the drug-effluxtransporters of the ABC superfamily.

    Pgp is a full length transporter with 12 transmembrane (TM ) segments and twocytoplasmic NBDs, which arose from a gene duplication. Both the N- andC -termini are cytoplasmic, and the first extracellular loop contains three

    glycosylation sites [185] . T he topology of M RP1 is simi lar to that of Pgp, but itpossesses an addit ional N -terminal domain with five TM segments, which is ofunknown function. The extracellular N-terminus carries two oligosaccharidechains, whi le the C-terminus is cytoplasmic, with an additional glycosylation siteon an extracellular loop [186] . A BCG 2 is a half-transporter comprising six TMsegments and a cytoplasmic NBD that is located on the N-terminal side of theTM domain, in contrast wi th Pgp and M RP1. A single glycosylation site islocated on the third extracellular loop [187] . A BCG 2 is presumed tohomodimerize to form a functional transporter.M RP: M ultidrug-resistance-associated protein; NBD : Nucleotide-bindingdomain; Pgp: P-glycoprotein.This work i s protected by copyright and it is being used with the permi ssion ofA ccess C opyright. A ny alteration of its content or further copying in any formwhatsoever is strictly prohibited.

    N C

    Out

    In

    Pgp

    NBD NBD

    C

    N Out

    In

    MRP1

    NBD NBD

    C

    N

    Out

    In

    ABCG2

    NBD

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    inhibited by Vi, suggesting that the proposedmechanism may be common to these ABC pro-teins as well. However, the two NBDs of M RP1are clearly not functionally or structurally equiv-alent, and V i trapping occurs predominantly atthe C-terminal NBD [59].

    Nucleotide binding to ABC drug-effluxpumps has been investigated using photoaffinitylabeling with azido-nucleotide analogs and, inthe case of Pgp, fluorescence and electron para-magnetic resonance (EPR) spectroscopy. The lat-ter two techniques allowed estimation of theaffinity and stoichiometry of binding[60,61]. TheKd values for ATP binding to Pgp are in therange of 0.2 to 0.5 mM, similar to the Km forATP hydrolysis. Both NBDs are occupied withATP in the native protein, and one ATP mole-cule can bind to the vacant NBD in theVi-trapped state [60].

    Drug b ind ing Biochemical studies have revealed the mem-brane topology of the mammalian ABC drugpumps. Pgp is a single polypeptide that arosefrom an internal duplication, and comprises 12TM segments and two NBDs (Figure1) . ABCG2is a half-transporter with six transmembrane(TM) segments and a single NBD, which isassumed to homodimerize to form the trans-port-competent complex [9]. In Pgp, the NBDs

    are C-terminal to the TM regions in the pri-mary sequence, whereas in ABCG2 their orderis reversed. MRP1 resembles Pgp, but has anextra N-terminal domain, TMD0, consisting of five TM segments. The functional role of thisthird membrane-spanning domain is currentlyunclear. It is not required for transport orproper trafficking to the plasma membrane inpolarized cells [62], however, if the C-terminusof MRP1 is mutated, the TMD0 is essential fornormal targeting [63]. MRP1 appears to exist asa dimer in the membrane, and it has been sug-gested that TMD

    0 and the associated linker

    region may mediate dimerization [64].There are currently no high resolution struc-

    tures available for any eukaryotic ABC protein.The best existing information is a medium res-olution structure of Pgp determined by cryo-electron microscopy (Figure 2D) [65]. This struc-ture shows that Pgp has 12 TM helices and twoclosely apposed NBDs, supporting the pro-posed topology. Fluorescence spectroscopic andbiochemical cross linking studies are also com-patible with this structure [6668]. Electronmicroscopic structures have been reported for

    MRP1 [69] and ABCG2 [70], but these are of lowresolution and have added little to ourunderstanding of how these proteins function.

    The nature and location of the drug-bindingsite(s) have been extensively investigated for Pgpand M RP1, whereas less is known for ABCG2.The Pgp drug-binding pocket is made up by theTM helices of the protein [71], and is locatedwithin the cytoplasmic membrane leaflet. Severaldrug binding subsites or minipockets appear toexist that interact with each other sterically orallosterically in a complex fashion, so that trans-port of one drug is either stimulated or inhibitedby binding of a second drug [72,73]. The drug-binding pocket within the protein is shaped likea funnel that is narrower at the cytoplasmic side,and it appears large and flexible enough toaccommodate two drug molecules simultane-ously [74,75]. Substrates may enter this regionthrough gates formed by the cytoplasmic endsof TM helices 2/11 and 5/8, which are closetogether [76]. The amino acids lining the bindingpocket hold the drug in place via Van der Waalsforces, hydrophobic interactions and hydrogenbonding. The flexible binding site allows struc-turally unrelated drugs to establish interactionswith different subset of residues within the bind-ing pocket, so that binding takes place by aninduced-fit type of mechanism [77]. In this way,drug binding to Pgp resembles the process in the

    bacterial multidrug-binding transcriptional reg-ulators, such as QacR and BmrR. For these solu-ble proteins, the principles of multidrug bindinghave been well-established, due to the existenceof crystal structures showing how different drugsbind [78]. The drug-binding pocket of Pgp hasbeen suggested to be accessible to the externalaqueous solution [79], although fluorescencemeasurements indicate that the bound drugmolecule is in a relatively hydrophobicenvironment [80].

    Substrate binding to the ABC drug effluxpumps has been characterized using photoactivat-able drug analogs, radiolabeled drug-bindingstudies, and fluorescence-quenching approaches.The measured range of K d values for drug bindingto Pgp covers over four orders of magnitude [81],demonstrating that the transporter can effectivelydiscriminate between compounds, and is polyspe-cific rather than nonspecific. Attempts to generatea quantitative structure-activity relationship(QSAR) for Pgp have been challenging. The bestdescription of a Pgp substrate involves a set of structural elements, including twothree hydro-gen bond acceptors and hydrophobic groups,

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    Figure 2. High-resolution x-ray crystal structures.

    (A) Nucleotide sandwich dimer of the ATP-binding subunit, HlyB (catalytically inactive H662A mutant; takenfrom [45] , reprinted with permission from Elsevier Limited).(B) The bacterial multidrug efflux pump Sav1866 (taken from [92] , reprinted with permission).(C) The bacterial vitamin B 12 importer BtuCD (taken from [42] , reprinted with permission from A A A S).(D) The medium resolution cryoelectron microscopic structure of Pgp (taken from [65] , reprinted withpermission).ECL: Extracellular loop; IC D: Intracellular domain; IC L: Intracellular loop; NBD: Nucleotide-binding domain;Pgp: P-glycoprotein; TM D: Transmembrane domain.

    Walker B Walker A

    HLY (H662A mutant)

    Signature C

    Sav1866

    N-tor

    ECL2

    ECL1ECL3

    C-tor

    BtuCD

    Cytoplasm

    N-tor N-tor

    Pgp

    TMDs

    ICDs

    NBDs

    ICL1ICL2

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    arranged in a fixed spatial orientation [8284].Substrates with diverse structures are envisaged aspositioning themselves differently among theseelements so as to maximize their interactions.Binding strength would depend on the number of interaction points and the strength of the hydro-gen bonds. The TM helices of Pgp contain a largenumber of amino acid side chains that could serveas hydrogen bond donors to aid in binding drugmolecules. Aromatic amino acids, particularly Trpresidues, may play an important role in stackingwith substrates that contain aromatic rings [85],since their fluorescence properties are sensitive todrug binding [86]. The drug-binding pocket of ABCG2 may function in a similar way to that of Pgp. Radioligand binding studies showed thatthere appeared to be at least two symmetricsubstrate binding sites within the protein, withoverlapping specificity[87].

    The drug-binding pocket of MRP1 may bebipartite in nature, since it is able to accommodateboth a hydrophobic moiety and a negativelycharged glutathione group. The core region of theprotein, which contains the substrate-bindingregion, has been studied extensively using site-directed mutagenesis and crosslinking withphotoactivatable substrate analogs [88]. In addi-tion, molecular models of MRP1 have been devel-oped to integrate this data into a 3D structure [89].The TM helices 10, 11, 16 and 17 appear to be

    important in binding transport substrates, withlesser contributions from other TM regions andcytoplasmic loops. A basket of aromatic residueslocated close to the cytoplasm-membrane inter-face of MRP1 has been proposed to play a centralrole in the initial interaction of drugs with theprotein [89]. TM helix 6 may be involved inrecognition of glutathione and its conjugates.

    Transport m echan ism There has been increasing success in crystallizingintegral membrane proteins over the past fewyears, and the high-resolution structures of severalintact bacterial ABC proteins have been deter-mined, including BtuCD [42], HI1470/1471 [90],ModB2C2 [91] and Sav1866 (Figure 2) [92]. BtuCD,HI1470/1 and ModB2C2 are metal chelateimporters, and like all bacterial ABC importers,they are associated with a periplasmic bindingprotein that delivers the substrate to the mem-brane-bound complex. There is now a structuralbasis for understanding how these complexeswork to transport substrate from the periplasmicspace to the cytosol [91,93]. However, this class of proteins sheds little light on the structure or

    possible mechanism of action of mammalianABC drug-efflux pumps. Three high-resolutionstructures of the bacterial lipid A transporter,MsbA [94], were recently withdrawn [95]. Thehomology of M sbA to Pgp had made thesestructures attractive to other researchers in thefield, and they were widely used as the basis forhomology models and molecular dynamics sim-ulations [96,97]. At this time, it is not clear howvalid these models and simulations are.Sav1866 is a putative bacterial multidrugexporter with 12 TM helices, and thus moreclosely resembles mammalian drug effluxpumps. It remains to be seen how useful thisstructure will be in its application to themammalian multidrug transporters.

    Drug transport involves two interconnectedcycles [98]. First there is the catalytic cycle of ATPhydrolysis, which drives transport. Second, thereis the substrate transport cycle, whereby a drugmolecule is moved from the cytoplasmic side tothe extracellular side of the membrane. Details of the catalytic and transport cycles, and how theyare coupled, remain enigmatic. In many studies,Pgp has appeared to behave as a symmetricalmolecule, with two equivalent NBDs (althoughthis is controversial, see [99]), and it is likely thatthe ABCG2 homodimer also functions symmet-rically. On the other hand, there is considerableevidence that the NBDs of the ABCC subfamily

    are not structurally or functionally equivalent,and the two NBDs of M RP1 may each performa different role in the catalysis and transportcycle [59].

    The catalytic cycle involves low affinity bind-ing of ATP to both NBDs, which induces forma-tion of a putative nucleotide sandwich dimer. Inthe case of Pgp, this may involve bringing theNBDs into very close apposition to form twocomposite catalytic sites at the interface of thedomains. One molecule of ATP appears tobecome bound very tightly (occluded) at thisstage [100], and is probably committed to enter thecatalytic transition state, thus being hydrolyzed toADP and Pi. After ATP hydrolysis, which appearsto be the rate-limiting step for ABC proteins, thealternating sites mechanism [58] proposes thatADP and Pi are released from one NBD, whichthen reloads with ATP. The second round of ATPhydrolysis then takes place at the catalytic site of the partner NBD. It is not known how this coop-eration between the two active sites is achieved.An alternative, processive clamp mechanism hasbeen proposed [101] in which both ATP moleculesare hydrolyzed in succession before release of

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    ADP and Pi and reloading with two more ATPmolecules. Mechanistic schemes have beenproposed in which either one or two molecules of ATP are hydrolyzed for each drug moleculetransported [58,102].

    Drug transport by the MDR efflux pumpsstarts with entry of the substrate into the bindingpocket on the cytoplasmic side, followed by pro-tein conformational changes (driven by ATPbinding or hydrolysis), and release of the druginto either the extracellular aqueous space or theopposing membrane leaflet. The drug is thoughtto initially interact with a high-affinity bindingsite, and then be moved to a low-affinity site forrelease. The coupling between drug transportand ATP hydrolysis involves communicationbetween the two domains, linked by conforma-tional changes, which have been demonstratedfor Pgp by fluorescence spectroscopy [103]. Thereis an ongoing debate about whether the energyfor drug transport by Pgp is provided by ATPhydrolysis [102] or ATP binding (the ATP switchmodel [104]).

    Role of th e lipid bilayer in drug binding&eff luxThe lipid bilayer plays an important part in theefflux function of Pgp (and probably ABCG2),whose substrates are typically hydrophobic andlipid-soluble. The idea that the transporter acts

    as a vacuum cleaner for hydrophobic moleculespresent within the membrane was suggested [105]and is now widely accepted. In intact cells, drugsentering the cell from the extracellular side areintercepted at the plasma membrane and trans-ported to the exterior without entering thecytosol (Figure 3A) . The binding process com-prises two steps; partitioning of drug from waterinto the membrane, and subsequent transfer of drug from the lipid to the binding pocket of theprotein. Because the membrane concentratesdrugs up to 1000-fold for presentation to thetransporter, the intrinsic affinity of the trans-porter for its substrates may be quite low. Thiswas confirmed by a thermodynamic analysis of the drug-binding process within a lipid bilayer[106]. The free energy of binding of a drug to Pgpwithin the lipid milieu correlates well with thecross-sectional area of the molecule, which inturn likely reflects the number of favorable inter-actions that the drug can make with the proteinTM helices [106]. The physical properties of themembrane modulate drug binding and transportby Pgp reconstituted into lipid bilayer vesicles.Drugs with high lipidwater partition

    coefficients demonstrated higher apparent bind-ing affinities, in keeping with the vacuumcleaner model (Figure3B) [107]. The initial rate of substrate transport was also dependent on thelipid fluidity, and reached a maximum at thelipid melting temperature [108].

    Drugs approaching the extracellular side of the plasma membrane will rapidly partition intothe outer leaflet, then flip-flop into the innerleaflet, a process that is known to be slow formany Pgp substrates [109]. Pgp may expel drugsfrom the membrane into the aqueous phasedirectly, or it may flip them from the inner tothe outer leaflet of the bilayer, from which theycan rapidly partition into the aqueous phase(Figure 3A) . Reconstituted Pgp is able to flip fluo-rescent analogs of both phospholipids and sim-ple glycosphingolipids [110], and interacts withmany lipid-based drugs [14], indicating that itmay also be a lipid transporter. The closelyrelated ABCB4 protein exports phosphatidyl-choline into the bile, likely by a flippase mecha-nism. Given the resemblance in function andsubstrate profiles between Pgp and ABCG2, it ispossible that the latter operates by a similarmode of action.

    Modulators that inhibit Pgp transport mayfunction within the milieu of the bilayer. It hasbeen proposed that the feature which distin-guishes this group of compounds is their high

    rate of spontaneous movement across the mem-brane [109]. Substrates are suggested to crossmembranes at a low rate. After they have beeneffluxed, they will rapidly repartition into theouter leaflet, cross slowly to the inner leaflet andinteract with Pgp once again, but the trans-porter will be able to keep pace and maintain asubstrate concentration gradient. Modulators,on the other hand, appear to cross membranesvery rapidly, faster than the rate of Pgp-medi-ated transport. Thus, the protein engages in afutile cycle of modulator transport, but cannoteither generate a modulator gradient or trans-port other substrates. Therfore, although mod-ulators inhibit transport of other drugs, it isdifficult to measure the rate at which they arethemselves transported.

    ABC drug-efflux pumps in tumorsOur understanding of the involvement of ABCtransport proteins in drug-resistant cancers isstill evolving. High expression of Pgp has beenobserved prior to chemotherapy treatment inmany different tumor types, including kidney,colon, liver, breast and ovarian cancers. In

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    Mod ulation of MDR efflux pumps incancer t reatme ntLarge numbers of modulators have been identi-fied for Pgp over the past 25 years [25]. In con-trast, only a few modulators have been describedfor MRP1 [116], including VX-710 (biricodar)MK571 (a leukotrieneD4-receptor antagonist),flavonoids, raloxifene analogs, isoxazole-basedcompounds and glutathione derivatives. ABCG2inhibitors have only recently been investigated,and include VX-710, GF120918 (elacridar),XR9576 (tariquidar), fumitremorgin C (a myco-toxin) and its derivative Ko143, pantoprazole,flavonoids, estrogens and antiestrogens [117]. Themost sought-after characteristic of modulatorshas been their ability to reverse resistance tocommonly used anticancer drugs, and manyclinical trials targeting Pgp have been carriedout. However, translation of basic biochemicalknowledge of the ABC efflux pumps to clinicalapplications at the bedside has proved difficult.Very few of the hundreds of modulators identi-fied in vitro are suitable for clinical application incancer treatment (Box 4). The development of modulators that are effective against MDRtumors, yet nontoxic, has been a major challenge

    over the past two decades. For example, fumitre-morgin C, which is a highly specific ABCG2inhibitor, is too neurotoxic for clinical use.

    The extent of involvement of ABC effluxpumps in anticancer drug resistance, andwhether modulation can result in increasedpatient survival, remains controversial. The firstgeneration of Pgp modulators used clinically(verapamil and cyclosporine A) were generallydrugs already in use for treatment of other medi-cal conditions, and they suffered from the dualproblems of high toxicity and low efficacy at tol-erable doses. Second-generation modulators(PSC833 and VX-710) showed improved effi-cacy at low doses, but serious adverse pharmaco-kinetic interactions were often noted in caseswhere both the treatment drug and the modula-tor were substrates for cytochrome P450 3A.Reduced clearance of the anticancer drug led toincreased toxicity to the patient. Third-genera-tion modulators, including LY335979 (zosuqui-dar), XR9576 and OC14409 (ontogen), havelow toxicity and show both increased selectivityand high potency against Pgp. There is hope thatthese new agents will prove more successful inclinical trials of Pgp modulation in cancer, whichhave so far been disappointing [118].

    Several ABCG2 modulators that could beused in patients have been identified in the pastfew years (Box 4) [114], however, none has yet been

    used in a clinical trial. Given the fact that manyof the compounds identified as ABCG2 inhibi-tors also act on Pgp (e.g., GF120918), the possi-bility of using dual PgpABCG2 modulatorsclinically appears to be a realistic goal. Somemultifunctional modulators have been identifiedthat inhibit the activity of all three efflux pumps,for example, VX-710 appears to block Pgp,MRP1 and ABCG2 [119].

    Role o f ABC multidrug tra nsporters indrug the rapyMany drugs commonly used in clinical therapyare transport substrates for Pgp, MRP1 andABCG2 (Boxes 1, 2 & 3) . The ABC proteins thusplay an important role in absorption and dispo-sition of these drugs in vivo . Both ABCG2 andPgp can limit the uptake of many drugs in theintestine. The presence of these efflux pumps isa serious problem in drug discovery, since manynew drug candidates may not be able to crossthe intestinal barrier in vivo , making them clin-ically useless. Drug discovery screening at manypharmaceutical companies now includes testingfor interactions with ABC transporters. The

    Box 4. Clinically relevant modulatorsthat interact with Pgp, MRP1and ABCG2.

    P-glycopro tein: f irst generation

    Verapamil Cyclosporin A Tamoxifen

    Second generation

    PSC833 (valspodar) VX -710 (biricodar)

    Third generatio n

    LY335979 (zosuquidar) XR9576 (tariquidar) G F120918 (elacridar)

    O C144093 (ontogen)M RP1

    VX -710 (biricodar)

    ABCG2

    G F120918 (elacridar) K o143 Pantoprazole XR9576 (tariquidar) VX -710 (biricodar) Gefitinib? Imatinib? Q uercetin?

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    presence of efflux pumps in the endothelialcells of the brain capillaries also has a far-reach-ing impact on pharmacotherapy of braindiseases, including cancer, AIDS, Parkinsonsdisease, epilepsy and schizophrenia [120,121].Several ABC transporters (the most importantare Pgp and ABCG2) are present in the luminalmembrane of endothelial cells, where theyimmediately pump drugs back into the blood,thus greatly reducing their accumulation inbrain tissue. By contrast, M RP1 is present inthe choroid plexus epithelium, and contributesto the bloodcerebrospinal fluid (CSF) drug-permeability barrier by preventing transfer of drugs into the CSF.

    Studies using knockout mice have demon-strated that access of many drugs to the braincan be increased five- to 100-fold in the absenceof Pgp and ABCG2 [122,123]. In M RP1 -knock-out mice, levels of etoposide in the CSF areincreased tenfold compared with wild-typemice [124]. These observations have generatedmuch interest in using Pgp and ABCG2 modu-lators in conjunction with therapeutic drugs toenhance their oral bioavailability and deliveryto the brain [125]. This approach is known to besuccessful in a mouse model, and future appli-cation to humans may improve the therapeuticeffectiveness of drugs targeted to the centralnervous system.

    Polymo rphisms in A BC drug -eff lux pum ps In recent years, there has been considerableinterest in how polymorphisms in ABC effluxpumps may affect drug therapy in these individ-uals [126]. Many SNPs in Pgp, MRP1 andABCG2 have been identified in different humanpopulations (see below for details), and arethought to play a major role in the variations indrug responses observed in different individualsand ethnic groups. A point mutation that occursin at least 1% of the population is considered tobe an SNP, which may be nonsynonymous (giv-ing a change in the coding sequence) or synony-mous (silent). SNPs may result in differences inboth protein expression level and transport func-tion, which are in turn expected to affect drugabsorption, plasma concentration, distribution,and elimination. More extensive changes in thegenome sequence encoding these proteins arealso possible. For example, some dog breeds(e.g., Collie) lack a Pgp arising from a frame-shift mutation and are hypersensitive to certaindrugs. Despite the widespread clinical use of drugs that are substrates for Pgp, ABCG2 and

    MRP1, there have been no reports of humannull alleles. Details of naturally occurring humanABC transporter polymorphisms are available inseveral databases [201203].

    Effe ct of po lymo rphisms &mut at ions onPgp expression &f unctionPgp variants carrying spontaneous mutationshave been found in cultured cell lines; the firstone to be identified was G195V, which causedincreased resistance to colchicine, while resist-ance to several other drugs was unchanged [127].Another cell line with a spontaneous deletion of F335 showed altered resistance to severalsubstrates [128].

    Genetic polymorphisms in ABCB1 have beenreported to change the mRNA expression, pro-tein expression and function of Pgp [129]. Thefirst SNP reported for human Pgp was theG2677T variant, which is a nonsynonymousSNP resulting in a change in the codingsequence, A893S. Over 50 SNPs and inser-tion/deletion polymorphisms in the ABCB1 gene have been reported to date [126,129133] . Dis-tinct haplotypes exist [134], with considerableheterogeneity within various populations,although all ethnic groups appear to have thethree most common haplotypes. One commonhaplotype includes the SNPs C1236T (exon 12,synonymous), G2677T (exon 21, nonsynony-

    mous, A893S) and C3435T (exon 26, synony-mous), and is found frequently in EuropeanAmericans, whereas C1236C-G2677T-C3435Chaplotype is common in Africans [135].

    The synonymous C3435T polymorphismwas reported to be associated with reduced PgpmRNA expression in a few studies, but this wascontradicted by others, and the overall consensusis that such an association is not significant [129].Several other single polymorphisms in Pgp havealso failed to show an association with levels of protein expression [129], leading to inconclusiveresults overall. More recent work examiningprotein expression levels has focussed on asso-ciations based on haplotypes, however, theresults have again been inconclusive, so that anassociation between haplotype and Pgp mRNAor protein expression has not been demon-strated. Confounding factors in these studiesinclude medications, diet, interindividual dif-ferences in drug metabolism and the presenceof underlying disease.

    Recent work has demonstrated that expressionof C3435T results in Pgp that has a slightly dif-ferent tertiary structure and altered interactions

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    with drugs and modulators, despite having thesame amino acid sequence [136]. These functionalchanges were suggested to arise from alteredfolding kinetics during protein biosynthesis as aresult of rare codon usage.

    Expression in cultured mammalian cell linesof Pgps carrying SNPs identified in humanpopulations has shown that many of these vari-ants have little or no effect on either Pgp sur-face expression or transport function [137139].In recent reports, several non-synonymous pol-ymorphisms expressed in mammalian andinsect cells displayed modest changes in sub-strate specificity and drug-stimulated ATPaseactivity [139,140]. However, the nonsynonymousmutations of G2677T/A/C, which result in theamino acid changes A893S, A893T and A893P,gave changes in both substrate specificity andATPase kinetic properties as measured with 41different test compounds [139]. The extent of the observed functional change varied with theparticular drug tested. The polymorphisms atamino acid 893 also show wide differences intheir allele frequency in different ethnic groups.This location (which is within the second intra-cellular loop in the C-terminal half of Pgp) thusappears to be a hotspot for mutations, and thesepolymorphisms could indeed influence the dis-position and therapeutic efficacy of variousdrugs administered clinically.

    Influence of Pgp polymorphisms ondrug the rapyPolymorphisms that affect Pgp expression orfunction would be predicted to be associatedwith changes in both the pharmacokinetics of administered drugs and the clinical outcome of drug therapy. Since Pgp is known to affect drugoral absorption, renal clearance and penetrationinto organs such as the brain, polymorphismsmight alter all of these parameters. Hoffmeyerreported a twofold reduction in the levels of duodenal Pgp in C3435T subjects, which wasassociated with increased oral absorption andhigher plasma levels of digoxin [141]. However,the majority of subsequent studies with otherPgp drug substrates (tacrolimus, fexofenadineand cyclosporine A) failed to confirm this[129].Later work showed that the linked nonsynony-mous SNP G2677T might be responsible forthe observed association with reducedfexofenadine uptake, suggesting that this vari-ant has increased activity in vivo [135]. Themajority of attempts to demonstrate an asso-ciation between ABCB1 genotype/haplotype

    and pharmacokinetics for other drugs believed tobe Pgp substrates have also been inconclusive[129]. Even the few studies that showed positiveassociations also reported contradictory data,and replication has been difficult.

    If an association between ABCB1 genotypeand Pgp expression/activity is real, the clinicaloutcome of drug treatment with a Pgp substratewould also display this dependence. HIV-pro-tease inhibitors are known to be Pgp substrates,and transport activity would be expected toreduce both oral uptake of these drugs, andtheir penetration into the brain. The G1199Apolymorphism (S400N) affected the trans-epi-thelial transport of five HIV protease inhibitors[140]. An association appears to exist betweencertain Pgp variants and the outcome of treat-ment with antiretroviral therapy for AIDS.Patients with 3435TT phenotypes who initi-ated antiretroviral therapy showed better recov-ery of immune function after 6 months [142],while those with the 3435CC genotype tendedto have earlier treatment failure due to highviral load [143]. A more rapid response to nelfi-navir therapy was also noted in HIV-1-infectedchildren with the 3435CT genotype comparedto those with the CC genotype [144]. However,several studies reported no significant asso-ciation of genotype with clinical outcome forthis group of drugs [129].

    A more well-documented link has beenreported between epilepsy that is refractory totreatment with multiple drugs and the C3435Tpolymorphism in the MDR1 gene [145]. Patientswith drug-resistant epileptic seizures were morelikely to show homozygosity for the CC geno-type, a polymorphism that is associated withincreased Pgp-transport function. These resultssuggest that drugs are less able to cross thebloodbrain barrier in this patient group. How-ever, these findings have not been confirmed bymore recent studies [129,146148] .

    Two common Pgp polymorphisms,G2677T/A and C3435T, may be involved inthe differential response of patients to the chol-esterol-lowering statins. The C3435T variantwas associated with a lower response to ator-astatin in female patients, and haplotype analy-sis identified a subgroup of individuals with aremarkable response to treatment that was notlinked to a single polymorphism [149]. Responseto treatment with fluvastatin was associatedwith a haplotype containing the G2677T/Aallele [150]. Another group also reported a linkbetween responses to several statins and the

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    polymorphism at position 2677, however, theassociation seen for the C3435T allelecontradicted that of the first study [151].

    The effects of Pgp polymorphisms on theoutcome of anticancer drug treatment havebeen described for several different tumor typesand treatment regimens [129]. Again, althoughsome positive associations have been reported,no clear pattern has emerged to date.

    Influence of Pgp polymorphisms ondisease susceptibilitySince Pgps play a central role in tissue defenceagainst toxic substrates, it would be predictedthat polymorphisms might alter susceptibilityof individuals to disease states. In particular,Pgp may protect the GI tract from bacterialflora and their toxins, which is supported by thefact that mdr1a -knockout mice spontaneouslydevelop colitis that progresses to dysplasia [152].It is, therefore, not surprising that Pgp poly-morphisms have been linked to inflammatorybowel disease, both Crohns disease and ulcera-tive colitis [153155]. Susceptibility to develop-ment of colon cancer is also increased by certainPgp polymorphisms [156158], and carriers of the3435TT and 3435T genotype were at substan-tially increased risk in an under-50 patient pop-ulation [157]. The same polymorphisms alsoappear to increase the risk of renal epithelial

    tumors [159].A link to Parkinsons disease may also exist.Transport of the anti-Parkinsons drug,budipine, out of the brain is mediated by Pgpin mice [160], and susceptibility to the diseasewas reported to be associated with Pgp poly-morphisms. The haplotype G2677T/C3435Tappears to confer protection to Parkinsons dis-ease in Chinese populations, although thesefindings have been disputed [146,161,162] , andthe association remains controversial.

    Polymo rphisms in M RP1 A number of M RP1 gene polymorphisms havebeen identified and its haplotypes investigated[134,163165]. The M RP1 gene displays high hap-lotype diversity, and distinct differencesbetween ethnic groups were noted [164]. Thesignificance of these variants, and their poten-tial role in drug delivery and disease suscepti-bility, has been explored by transfection of these proteins into mammalian cell lines, fol-lowed by characterization of their expressionlevels and transport function [163,166]. Mostmutant MRP1 proteins were expressed at levels

    comparable to the wild-type, and only a fewshowed altered function. These results suggestthat the majority of M RP1 polymorphisms areunlikely to have effects on drug disposition.The G1299T polymorphism (exon 10) resultsin the change R433S in a cytoplasmic loop of MRP1, and was observed to increase doxoru-bicin resistance, but decrease transport of sev-eral organic anions [167]. The nonsynonymouschange G128C (C43S) impaired the plasmamembrane localization of the protein, and alsodecreased resistance to doxorubicin andsodium arsenite [168]. Very few M RP1 polymor-phisms have been associated with clinicaldisease or altered drug responses.

    Muta tions &po lymorphisms in ABCG2 The oral bioavailability and clearance of drugsthat are ABCG2 substrates is highly variable [169],suggesting that interindividual variations arisingfrom polymorphisms might be important. Over80 SNPs, missense, nonsense and frameshiftmutations in the ABCG2 gene have been identi-fied in different ethnic groups [23,170], includingV12M (N-terminal cytosolic region), Q141K(NBD) and Q126stop (in which no active proteinis produced). Functional characterization of sev-eral of these polymorphisms has been carried out;some show increased transport activity, while oth-ers display reduced expression and/or function. In

    a study of six different SNP variants, the C421Apolymorphism (nonsynonymous, Q141K) wasexpressed at lower levels, and the S441N varianthad both lower expression and altered localiza-tion [171]. The Q141K mutation is locatedbetween the Walker A and signature motifs, soaltered ATPase activity is possible. Comparedwith wild-typeABCG2 , the Q141K variant dis-played lower ATPase activity and lower mitox-antrone efflux when expressed in HEK-293 cells,whereas the V12M and D620N proteins showedlittle change[172]. Somewhat different results werereported by another group for the V12M andQ141K variants [173]. A recent study examinedseven ABCG2 variants in detail, and found thatcells expressing both V12M and Q141K hadreduced resistance towards the drug SN-38 [174].Several different studies that examined the expres-sion level of the Q141K variant, both in humantissues and in transfected cell lines, have yieldedcontradictory results [175].

    The frequency of the Q141K polymorphismvaries considerably among different ethnicpopulations, being commonly found in Chinaand Japan, and was found to be part of a

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    common haplotype [176,177]. Individuals carry-ing this polymorphism are predicted to showaltered responses to anticancer agents andother drugs due to reduced efflux activity of ABCG2, and indeed, changes in thepharmacokinetics of several drugs were noted,with increased levels in the plasma [178180].The accumulation of the tyrosine kinase inhib-itor gefitinib was higher in patients hetero-zygous at the C421A (Q141K) locus comparedwith those with a wild-type genotype, indicat-ing that this polymorphism may indeed affectthe outcome of anticancer drug treatment [181].However, no significant effect of this polymor-phism on irinotecan pharmacokinetics wasobserved [177]. The frequency of the CC geno-type for the C421A polymorphism was signifi-cantly higher in renal cell carcinoma patients,suggesting that ABCG2 may be a susceptibilitygene for this cancer [182]. A recent study charac-terized the activity of 18 ABCG2 variants, andconcluded that Q126stop, F208S, S248P,E334stop, S441N and F489L are defective inhematoporphyrin transport [170], which mayincrease the risk of disease in individualscarrying these polymorphisms.

    A spontaneous mutation in ABCG2 wasidentified in drug-selected cultured cell lines.Changes to R482 in the third TM segmentresulted in a gain-of-function mutant that had

    altered substrate specificity [183]. Several otheramino acid substitutions at R482 also led tolarge changes in drug transport and substratespecificity [184]. The R482T and R482G vari-ants were able to efflux rhodamine 123 anddoxorubicin, whereas the wild-type was not,however, all three forms of ABCG2 transportedmitoxantrone. These results indicate that asingle amino acid change at position 482 canalter the substrate specificity and drug-resist-ance phenotype, and suggest that this residuemay play a critical role in ABCG2 function.However, to date, no SNP at this position hasbeen found in human populations.

    Fut ure perspectiveThe central role played by ABC multidrugefflux pumps in protecting tissues from exoge-nous and endogenous toxins is now widelyrecognized. These related transporters play acentral role in the uptake of drugs and deliveryto their tissue targets, however, we have muchto learn about the complex network of

    interactions between them. Substantial progresshas recently been made in determining the highresolution structures of bacterial ABC proteins,however, structural information for themammalian family members is very sparse.Much more structural and biochemical infor-mation will be necessary for a detailed under-standing of the catalytic cycle and drug-transport mechanism of the ABC multidrugefflux pumps. Such understanding may promptthe use of completely new approaches tomanipulating the function of these transportersin a way that is helpful for drug therapy. Theuse of modulators to block MDR during cancerchemotherapy treatment has been an attractivegoal, however, the clinical trials carried out todate have proved disappointing. It is still notclear whether this approach has enough prom-ise to be pursued in the future with the highlyspecific and efficacious third-generation modu-lators that have been developed recently. Natu-rally-occurring polymorphisms of the ABCmultidrug efflux pumps have only been identi-fied relatively recently, and much more workremains to be done to resolve some of the cur-rent controversies and determine their trueimpact on transporter function. The majorityof studies in this area have suffered from seriousexperimental limitations, such as sample selec-tion, sample size, confounding factors and

    genotype/phenotype errors, and a comprehen-sive set of recommendations to avoid theseproblems in the future has been presented [129].The role of polymorphisms in responses to drugtherapy and disease susceptibility is a develop-ing field that will clearly be important in thefuture as the ultimate goal of personalizedmedicine is pursued.

    It is possible that pharmacogenomics may, inthe future, be able to predict individualresponses to drug treatment, leading toimproved treatment and clinical outcome.

    Financial &competing interests disclosureThe author has no relevant affi li ations or fi nancial i nvolve-

    ment wi th any organizati on or enti ty wit h a fi nancial int er-

    est i n or f inancial confl ict wi th t he subject matter or

    materials di scussed in the manuscri pt. Thi s includes employ-

    ment, consultancies, honorari a, stock ownership or options,

    expert testi mony, grants or patents received or pendi ng or

    royalti es.

    No wr iti ng assi stance was uti li zed in the producti on of

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    Executive summary

    Three ABC superfamily multidrug efflux pumps play a central role in protecting sensit ive tissues from exogenous and endogenoustoxic compounds: P-glycoprotein (Pgp, A BCB1), M RP1 (A BCC1) and A BCG 2 (BC RP). These transporters show overlappingsubstrate specif icity, and affect the uptake and distributi on of many clinically important drugs.

    Pgp and the A BCG 2 homodimer comprise two transmembrane (TM ) domains and two nucleotide binding domains (N BDs),whereas M RP1 has an extra N-terminal TM domain. The NBDs likely dimerize during the catalytic cycle to form a nucleotidesandwich with two bound ATP molecules, whose hydrolysis drives active drug transport.

    Substrates for Pgp and A BCG 2 are lipid-soluble, and they are likely expelled from the membrane into either the extracellularaqueous phase (hydrophobic vacuum cleaner) or the outer membrane leaflet (drug flippase).

    M any chemical modulators that inhibit the transport activity of Pgp are known, while fewer have been identified for M RP1 andA BC G 2; however, only a small number of these compounds are suitable for clinical use.

    Expression of A BC drug transporters in human tumors has been linked to resistance to anticancer agents and poor prognosis,however, attempts to inhibi t their activity in clinical trials using modulators have been disappointing.

    Polymorphisms in A BC efflux pumps have been implicated in di fferent responses to chemotherapy and drug therapy, as well asdisease susceptibility. However, overall results have been inconclusive, and the impact of genotype on the expression and functionof these transporters remains controversial.

    BibliographyPapers of special note have been highlighted aseither of interest (*) or of considerable interest(**) to readers.1. Dassa E, Bouige P: The ABC of

    ABCs: a phylogenetic and functionalclassification of ABC systems in livingorganisms. Res. Microbiol. 152, 211229(2001).

    2. Dean M, Rzhetsky A, Allikmets R:Thehuman ATP-binding cassette (ABC)transporter superfamily. Genome Res. 11,11561166 (2001).

    3. Sheps JA, Ralph S, Zhao Z, Baillie DL,Ling V: The ABC transporter gene family ofCaenorhabditis elegans has implications forthe evolutionary dynamics of multidrugresistance in eukaryotes. Genome Biol. 5,R15 (2004).

    4. Borst P, Oude Elferink RP: MammalianABC transporters in health anddisease. Annu. Rev. Bi ochem. 71, 537592(2002).

    5. Schinkel AH , Jonker JW: Mammalian drug

    efflux transporters of the ATP bindingcassette (ABC) family: an overview.Adv. Drug Deliv. Rev. 55, 329 (2003).Review of the role in physiology andpharmacology of the mammalianATP-binding cassette (ABC) transportersknown to mediate transport of clinicallyrelevant drugs.

    6. Gottesman MM, Ling V: The molecularbasis of multidrug resistance incancer: theearly years of P-glycoproteinresearch. FEBS Lett. 580, 9981009(2006).

    7. Huang Y, Sadee W: Membrane transportersand channels in chemoresistance and sensitivity of tumor cells. Cancer Lett. 239,168182 (2006).

    8. Litman T, Druley TE, Stein WD,Bates SE: From MDR to MXR: newunderstanding of multidrug resistancesystems, their properties and clinicalsignificance. Cell . M ol. Li fe Sci . 58, 931959(2001).

    9. zvegy C, Litman T, Szakcs G et al. :Functional characterization of the humanmultidrug transporter, ABCG2,expressed in insect cells. Biochem.Bi ophys. Res. Commun. 285, 111117(2001).

    10. Bosch I, Dunussi-Joannopoulos K, Wu RL,Furlong ST, Croop J: Phosphatidylcholineand phosphatidylethanolamine behave assubstrates of the human MDR1P-glycoprotein. Biochemistr y 36, 56855694(1997).

    11. Romsicki Y, Sharom FJ: Phospholipidflippase activity of the reconstituted

    P-glycoprotein multidrug transporter.Biochemistr y 40, 69376947 (2001).

    12. Eckford PD, Sharom FJ: Thereconstituted P-glycoprotein multidrugtransporter is a flippase forglucosylceramide and other simpleglycosphingolipids.Biochem. J. 389,517526 (2005).

    13. Ernest S, Bello-Reuss E: Secretion ofplatelet-activating factor is mediated byMDR1 P-glycoprotein in cultured humanmesangial cells. J. Am. Soc. Nephrol. 10,23062313 (1999).

    14. Eckford PD, Sharom FJ: P-glycoprotein(ABCB1) interacts directly with lipid-basedanti-cancer drugs and platelet-activatingfactors. Biochem. Cell Bi ol. 84, 10221033(2006).

    15. Bello-Reuss E, Ernest S, Holland OB,Hellmich MR: Role of multidrug resistanceP-glycoprotein in the secretion ofaldosterone by human adrenal NCI-H295cells. Am. J. Physiol. Cell Physiol. 278,C1256C1265 (2000).

    16. Liu Y, Huang L, Hoffman T, Gosland M,Vore M: MDR1 substrates/modulatorsprotect against -estradiol-17-D-glucuronide cholestasis in rat liver. CancerRes. 56, 49924997 (1996).

    17. Lam FC, Liu R, Lu Pet al. : -amyloid effluxmediated by p-glycoprotein. J. Neurochem. 76, 11211128 (2001).

    18. Pawlik A, Baskiewicz-Masiuk M,Machalinski B, Safranow K,Gawronska-Szklarz B: Involvement of P-glycoprotein in the release of cytokines fromperipheral blood mononuclear cells treated

    with methotrexate and dexamethasone.J. Pharm. Pharmacol. 57, 14211425(2005).

    19. Ruetz S, Gros P: Phosphatidylcholinetranslocase: a physiological role for the mdr2gene. Cell 77, 10711081 (1994).

    20. Smith AJ, van Helvoort A, van Meer Get al. : MDR3 P-glycoprotein, aphosphatidylcholine translocase, transportsseveral cytotoxic drugs and directly interactswith drugs as judged by interference withnucleotide trapping. J. Biol. Chem. 275,2353023539 (2000).

  • 8/13/2019 ABC Multidrug Transporters

    18/23

    REVIEW Sharom

    122 Pharmacogenomics (2008) 9(1) future science grouputure s ien e group

    21. Bakos E, Homolya L: Portrait ofmultifaceted transporter, the multidrugresistance-associated protein 1(MRP1/ABCC1).Pflugers Arch. Eur. J.Physiol. 453, 621641 (2007).

    22. Cole SP, Sparks KE, Fraser K et al. :

    Pharmacological characterization ofmultidrug resistant M RP-transfected humantumor cells. Cancer Res. 54, 59025910(1994).

    23. Ishikawa T, Tamura A, Saito H,Wakabayashi K, Nakagawa H:Pharmacogenomics of the human ABCtransporter ABCG2: from functionalevaluation to drug molecular design.Naturwi ssenschaften 92, 451463 (2005).

    ABCG2 function and polymorphisms, andtheir relationship to design of new drugs.

    24. Staud F, Pavek P: Breast cancer resistanceprotein (BCRP/ABCG2).Int. J. Biochem.Cell Biol. 37, 720725 (2005).

    25. Robert J, Jarry C: Multidrug resistancereversal agents. J. M ed. Chem. 46,48054817 (2003).

    26. Dayan G, Jault JM, Baubichon-Cortay Het al. : Binding of steroid modulators torecombinant cytosolic domain from mouseP-glycoprotein in close proximity to theATP site. Biochemistr y 36, 1520815215(1997).

    27. Martin C, Berridge G, Mistry P, Higgins C,Charlton P, Callaghan R: The molecularinteraction of the high affinity reversal agent

    XR9576 with P-glycoprotein.Br. J.Pharmacol. 128, 403411 (1999).

    28. Sauna ZE, Peng XH , Nandigama K,TekleS, Ambudkar SV: The molecular basisof the action of disulfiram as a modulator ofthe multidrug resistance-linked ATPbinding cassette transporters MDR1(ABCB1) and MRP1 (ABCC1).Mol.Pharmacol. 65, 675684 (2004).

    29. Leslie EM, Deeley RG, Cole SPC:Multidrug resistance proteins: role ofP-glycoprotein, MRP1, MRP2, and BCRP(ABCG2) in tissue defense. Toxicol. Appl.Pharmacol. 204, 216237 (2005).

    Discusses the role of four ABC-transporterproteins in protecting tissues from a varietyof toxic agents.

    30. Schinkel AH, Smit JJ, van Tellingen O et al. :Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency inthe blood-brain barrier and to increasedsensitivity to drugs. Cell 77, 491502(1994).

    31. Borst P, Schinkel AH : What have we learntthus far from mice with disruptedP-glycoprotein genes? Eur. J. Cancer 32A,985990 (1996).

    32. Wijnholds J, Scheffer GL, van der Valk Met al. : Multidrug resistance protein 1protects the oropharyngeal mucosal layerand the testicular tubules against drug-induced damage. J. Exp. Med. 188, 797808(1998).

    33. Zhou S, Morris JJ, Barnes YX, Lan L,Schuetz JD, Sorrentino BP: Bcrp1 geneexpression is required for normal numbersof side population stem cells in mice,and confers relative protection tomitoxantrone in hematopoietic cellsin vivo . Proc. Natl Acad. Sci . U SA 99,1233912344 (2002).

    34. Doran A, Obach RS, Smith BJ et al. : Theimpact of P-glycoprotein on the dispositionof drugs targeted for indications of thecentral nervous system: evaluation using theMDR1A/1B knockout mouse model. DrugMetab. D ispos. 33, 165174 (2005).

    35. Fromm MF: Importance of P-glycoproteinat blood-tissue barriers. Trends Pharmacol.Sci . 25, 423429 (2004).

    36. van Tellingen O, Buckle T, Jonker JW,van der Valk MA, Beijnen JH:P-glycoprotein and Mrp1 collectivelyprotect the bone marrow from vincristine-induced toxicity in vivo . Br. J. Cancer 89,17761782 (2003).

    37. Jonker JW, Merino G, Musters S et al. :Thebreast cancer resistance protein BCRP(ABCG2) concentrates drugs andcarcinogenic xenotoxins into milk.Nat. M ed. 11, 127129 (2005).

    38. van Herwaarden AE, Wagenaar E,Merino Get al. : Multidrug transporterABCG2/breast cancer resistanceprotein secretes riboflavin (vitamin B-2) intomilk.M ol. Cell . Biol. 27, 12471253(2007).

    39. Jonker JW, Buitelaar M, Wagenaar E et al. :The breast cancer resistance proteinprotects against a major chlorophyll-deriveddietary phototoxin and protoporphyria.Proc. Natl Acad. Sci . U SA 99, 1564915654(2002).

    40. Krishnamurthy P, Ross DD, Nakanishi Tet al. : The stem cell marker Bcrp/ABCG2enhances hypoxic cell survival throughinteractions with heme. J. Biol. Chem. 279,2421824225 (2004).

    41. Frelet A, Klein M: Insight in eukaryoticABC transporter function by mutationanalysis. FEBS Lett. 580, 10641084(2006).

    42. Locher KP, Lee AT, Rees DC: TheE. coli BtuCD structure: a framework forABC transporter architecture andmechanism. Science 296, 10911098(2002).

    43. Hopfner KP, Karcher A, Shin DSet al. :Structural biology of Rad50 ATPase:ATP-driven conformational control in DNAdouble-strand break repair and theABC-ATPase superfamily. Cell 101,789800 (2000).

    44. Smith PC, Karpowich N, Millen Let al. :ATP binding to the motor domain from anABC transporter drives formation of anucleotide sandwich dimer. Mol. Cell 10,139149 (2002).

    45. Hanekop N, Zaitseva J, Jenewein S,Holland IB, Schmitt L: Molecular insightsinto the mechanism of ATP-hydrolysis bythe NBD of the ABC-transporter HlyB.FEBS Lett . 580, 10361041 (2006).

    46. Liu R, Sharom FJ: Fluorescence studies onthe nucleotide binding domains of theP-glycoprotein multidrug transporter.Biochemi str y 36, 28362843 (1997).

    47. Lerner-Marmarosh N , Gimi K,Urbatsch IL, Gros P, Senior AE: Largescale purification of detergent-solubleP-glycoprotein from Pichia pastoriscells and characterization of nucleotidebinding properties of wild-type, Walker A,and Walker B mutant proteins. J. Biol.Chem. 274, 3471134718 (1999).

    48. Sharom FJ, Yu X, Chu JWK, Doige CA:Characterization of the ATPase activity ofP-glycoprotein from multidrug-resistantChinese hamster ovary cells. Biochem. J. 308, 381390 (1995).

    49. Urbatsch IL, al-Shawi MK, Senior AE:Characterization of the ATPaseactivity of purified Chinese hamsterP-glycoprotein.Biochemistr y 33, 70697076(1994).

    50. Mao QC, Leslie EM, Deeley RG, ColeSPC:ATPase activity of purified and reconsti tutedmultidrug resistance protein MRP1 fromdrug-selected H69AR cells. Biochim.Biophys. Acta 1461, 6982 (1999).

    51. Doige CA, Yu X, Sharom FJ: ATPaseactivity of partially purified P-glycoproteinfrom multidrug-resistant Chinese hamsterovary cells. Biochim. Bi ophys. Acta 1109,149160 (1992).

    52. Garrigos M, Mir LM, Orlowski S:Competitive and non-competitiveinhibition of the multidrug-resistance-associated P-glycoprotein ATPase-furtherexperimental evidence for a multisite model.Eur. J. Biochem. 244, 664673 (1997).

    53. Litman T, Zeuthen T, Skovsgaard T,Stein WD: Competitive, non-competitiveand cooperative interactions betweensubstrates of P-glycoprotein as measured byits ATPase activity. Biochim. Bi ophys. Acta 1361, 169176 (1997).

  • 8/13/2019 ABC Multidrug Transporters

    19/23

    ABC multidrug transporters: structure, function and role in chemoresistance REVI EW

    123future science grouputure s ien e group www.futuremedicine.com

    54. Doige CA, Yu X, Sharom FJ: The effectsoflipids and detergents on ATPase-activeP-glycoprotein.Biochim. Biophys. Acta 1146,6572 (1993).

    55. Urbatsch IL, Senior AE: Effectsoflipids on ATPase activity of purified Chinese

    hamster P-glycoprotein.Arch. Biochem. Biophys. 316, 135140(1995).

    56. Litman T, Zeuthen T, Skovsgaard T,Stein WD: Structure-activity relationships ofP-glycoprotein interacting drugs:kinetic characterization of their effectsonATPase activity. Biochim. Biophys. Acta 1361,159168 (1997).

    57. Omote H, al-Shawi MK: Interaction oftransported drugswith the lipid bilayer andP-glycoprotein through a solvation exchangemechanism. Biophys. J. 90, 40464059(2006).

    58. Senior AE, al-Shawi MK, Urbatsch IL:The catalytic cycle of P-glycoprotein.FEBS Lett . 377, 285289 (1995).

    59. Gao M, Cui HR, Loe DWet al. : Comparisonof the functional characteristicsof thenucleotide binding domainsof multidrugresistance protein 1. J. Biol. Chem. 275,1309813108 (2000).

    60. Qu Q, Russell PL, Sharom FJ: Stoichiometryand affinity of nucleotide binding toP-glycoprotein during the catalytic cycle.Biochemistr y 42, 11701177 (2003).

    61. Delannoy S, Urbatsch IL, Tombline G,

    Senior AE, Vogel PD: Nucleotidebinding to the multidrug resistance P-glycoprotein asstudied by ESR spectroscopy.Biochemistr y 44, 1401014019 (2005).

    62. Bakos E, Evers R, Szakcs G et al. :Functional multidrug resistance protein(MRP1) lacking the N-terminaltransmembrane domain. J. Biol. Chem. 273,3216732175 (1998).

    63. Westlake CJ, Cole SPC, Deeley RG:Role of the NH2-terminal membranespanning domain of multidrug resistanceprotein 1/ABCC1 in protein processingand trafficking.Mol. Biol. Cell 16, 24832492(2005).

    64. Yang YY, Liu Y, Dong ZZet al. : Regulationof function by dimerization through theamino-terminal membrane-spanningdomain of human ABCC1/MRP1. J. Biol.Chem. 282, 88218830 (2007).

    65. Rosenberg MF, Callaghan R, Modok S,Higgins CF, Ford RC: Three-dimensionalstructure of P-glycoprotein thetransmembrane regions adopt anasymmetric configuration in thenucleotide-bound state. J. Biol. Chem. 280,28572862 (2005).

    66. Qu Q, Sharom FJ: FRET analysis indicatesthat the two ATPase active sites of theP-glycoprotein multidrug transporter areclosely associated. Biochemi str y 40,14131422 (2001).

    67. Urbatsch IL, Gimi K, Wilke-Mounts S

    et al. : Cysteines 431 and 1074 areresponsible for inhibitory disulfidecross-linking between the twonucleotide-binding sites in humanP-glycoprotein. J. Biol. Chem. 276,2698026987 (2001).

    68. Loo TW, Bartlett MC, Clarke DM:The LSGGQ motif in each nucleotide-binding domain of human P-glycoproteinis adjacent to the opposing Walker Asequence. J. Biol. Chem. 277,4130341306 (2002).

    69. Rosenberg MF, Mao QC, Holzenburg A,Ford RC, Deeley RG, Cole SPC: Thestructure of the multidrug resistanceprotein 1 (MRP1/ABCC1) crystallizationand single-particle analysis. J. Biol. Chem. 276, 1607616082 (2001).

    70. McDevitt CA, Collins RF, Conway Met al. : Purification and 3D structuralanalysis of oligomeric human multidrugtransporter ABCG2. Structure 14,16231632 (2006).

    71. Loo TW, Clarke DM: Recent progress inunderstanding the mechanism ofP-glycoprotein-mediated drug efflux.J. M embr. Bi ol. 206, 173185 (2005).

    72. Shapiro AB, Ling V: Positivelycooperative sites for drug transport byP-glycoprotein with distinct drugspecificities. Eur. J. Biochem. 250, 130137(1997).

    73. Martin C, Berridge G, Higgins CF,Mistry P, Charlton P, Callaghan R:Communication between multiple drugbinding sites on P-glycoprotein.M ol. Pharmacol. 58, 624632(2000).

    74. Loo TW, Bartlett MC, Clarke DM:Simultaneous binding of two differentdrugs in the binding pocket of the humanmultidrug resistance P-glycoprotein.J. Biol. Chem. 278, 3970639710(2003).

    75. Lugo MR, Sharom FJ: Interaction ofLDS-751 and rhodamine 123 withP-glycoprotein: evidence for simultaneousbinding of both drugs. Biochemistry 44,1402014029 (2005).

    76. Loo TW, Clarke DM : Do drugsubstrates enter the commondrug-binding pocket of P-glycoproteinthrough gates? Bi ochem. Biophys. Res.Commun. 329, 419422 (2005).

    77. Loo TW, Bartlett MC, Clarke DM:Substrate-induced conformational, changesin the transmembrane segments of humanP-glycoprotein direct evidence for thesubstrate-induced fit mechanism for drugbinding. J. Biol. Chem. 278, 1360313606

    (2003).78. Schumacher MA, Miller MC,Brennan RG: Structural mechanism of thesimultaneousbinding of two drugsto amultidrug-binding protein.EMBO J. 23,29232930 (2004).

    79. Loo TW, Bartlett MC, Clarke DM:The drug-binding pocket of thehuman multidrug resistanceP-glycoprotein is accessible to theaqueous medium. Biochemistr y 43,1208112089 (2004).

    Summarizes what is known of the structureand transport mechanism ofP-glycoprotein (Pgp), focusing on thestructure of the drug-binding site.

    80. Lugo MR, Sharom FJ: Interaction ofLDS-751 with P-glycoprotein and mappingof the location of the R drug binding site.Biochemistr y 44, 643655 (2005).

    81. Sharom FJ, Liu R, Qu Q, Romsicki Y:Exploring the structure and function of theP-glycoprotein multidrug transporter usingfluorescence spectroscopic tools. SeminarsCell Dev. Biol. 12, 257266 (2001).

    82. Seelig A: A general pattern for substraterecognition by P-glycoprotein. Eur. J.Biochem. 251, 252261 (1998).

    83. Cianchetta G, Singleton RW, Zhang Met al. : A pharmacophore hypothesis forP-glycoprotein substrate recognition usingGRIND-based 3D-QSAR. J. Med. Chem. 48, 29272935 (2005).

    84. Pajeva IK, Wiese M: Pharmacophoremodel of drugs involved in P-glycoproteinmult idrug resistance: Explanation ofstructural variety (hypothesis). J. M ed.Chem. 45, 56715686 (2002).

    85. Pawagi AB, Wang J, Silverman M,Reithmeier RA, Deber CM: Transmembranearomatic amino acid distribution inP-glycoprotein. A functional role in broadsubstrate specificity. J. Mol. Biol. 235,554564 (1994).

    86. Liu R, Siemiarczuk A, Sharom FJ:Intrinsic fluorescence of the P-glycoproteinmultidrug transporter: sensitivity oftryptophan residues to binding of drugsandnucleotides. Biochemistr y 39, 1492714938(2000).

    87. Clark R, Kerr ID, Callaghan R: Multipledrugbinding siteson the R482G isoform ofthe ABCG2 transporter. Br. J. Pharmacol. 149,506515 (2006).

  • 8/13/2019 ABC Multidrug Transporters

    20/23

    REVIEW Sharom

    124 Pharmacogenomics (2008) 9(1) future science grouputure s ien e group

    88. Deeley RG, Cole SPC: Substrate recognitionand transport by multidrug resistance protein1 (ABCC1).FEBS Lett . 580, 11031111(2006).

    89. Campbell JD, Koike K, Moreau C,Sansom MSP, Deeley RG, Cole SPC:

    Molecular modeling correctly predictsthefunctional importance of Phe594 intransmembrane Helix 11 of the multidrugresistance protein, MRP1 (ABCC1). J. Biol.Chem. 279, 463468 (2004).

    90. Pinkett HW, Lee AT, Lum P, Locher KP,Rees DC: An inward-facing conformation ofa putative metal-chelate-type ABCtransporter. Science 315, 373377 (2007).

    91. Hollenstein K, Frei DC, Locher KP:Structure of an ABC t ransporter in complexwith its binding protein.Nature 446,213216 (2007).

    92. Dawson RJ, Locher KP: Structure of abacterial multidrug ABC transporter. Nature 443, 180185 (2006).

    93. Locher KP, Borths E: ABC transporterarchitecture and mechanism: implicationsfrom the crystal structures of BtuCD andBtuF. FEBS Lett . 564, 264268 (2004).

    94. Reyes CL, Ward A, Yu J, Chang G: Thestructures of MsbA: Insight into ABCtransporter-mediated multidrug efflux.FEBS Lett . 580, 10421048 (2006).

    95. Chang G, Roth CB, Reyes CL, Pornillos O,Chen YJ, Chen AP: Structure of MsbA fromE. coli: A homolog of the multidrug

    resistance ATP binding cassette (ABC)transporters (Retraction of vol 293, pg1793, 2001). Science 314, 1875 (2006).

    96. Seigneuret M, Garnier-Suillerot A: Astructural model for the open conformationof the mdr1 P-glycoprotein based on theMsbA crystal structure. J. Biol. Chem. 278,3011530124 (2003).

    97. Stenham DR, Campbell JD, Sansom MS,Higgins CF, Kerr ID, Linton KJ: Anatomic detail model for the human ATPbinding cassette transporter P-glycoproteinderived from disulfide cross-linking andhomology modeling. FASEB J. 17,22872289 (2003).

    98. Callaghan R, Ford RC, Kerr ID: Thetranslocation mechanism of P-glycoprotein.FEBS Lett . 580, 10561063 (2006).

    Recent review that dissects the detailedsteps of Pgp-mediated drug transport andits coupling to ATP hydrolysis.

    99. Hrycyna CA, Ramachandra M,Germann UA, Cheng PW, Pastan I,Gottesman MM: Both ATP sites ofhuman P-glycoprotein are essential but notsymmetric. Biochemistr y 38, 1388713899(1999).

    100. Tombline G, Bartholomew LA,Urbatsch IL, Senior AE: Combinedmutation of catalytic glutamate residues inthe two nucleotide binding domains ofP-glycoprotein generates a conformationthat binds ATP and ADP tightly. J. Biol.

    Chem. 279, 3121231220 (2004).101. Janas E, Hofacker M , Chen M,Gompf S, Van der Does C, Tamp R:The ATP hydrolysis cycle of thenucleotide-binding domain of themitochondrial ATP-binding cassettetransporter Mdl1p. J. Biol. Chem. 278,2686226869 (2003).

    102. Ambudkar SV, Kim IW, Sauna ZE: Thepower of the pump: mechanisms of actionof P-glycoprotein (ABCB1).Eur. J. Pharm.Sci . 27, 392400 (2006).

    103. Liu R, Sharom FJ: Site-directedfluorescence labeling of P-glycoprotein oncysteine residues in the nucleotide bindingdomains. Biochemistr y 35, 1186511873(1996).

    104. Higgins CF, Linton KJ: The ATP switchmodel for ABC transporters. Nature Str uct.Biol. 11, 918926 (2004).

    105. Higgins CF, Gottesman MM: Is themultidrug transporter a flippase? TrendsBiochem. Sci . 17, 1821 (1992).

    106. Gatlik-Landwojtowicz E, Aanismaa P,SeeligA: Quantification andcharacterization of P-glycoprotein-substrateinteractions. Biochemistr y 45, 30203032

    (2006).107. Romsicki Y, Sharom FJ: The membrane

    lipid environment modulates druginteractions with the P-glycoproteinmultidrug transporter. Biochemistr y 38,68876896 (1999).

    108. Lu P, Liu R, Sharom FJ: Drug transport byreconstituted P-glycoprotein inproteoliposomes. Effect of substrates andmodulators, and dependence on bilayerphase state. Eur. J. Bi ochem. 268,16871697 (2001).

    109. Eytan GD, Regev R, Oren G, Assaraf YG:The role of passive transbilayer drugmovement in multidrug resistance and itsmodulation. J. Biol. Chem. 271,1289712902 (1996).

    110. Sharom FJ, Lugo MR, Eckford PD:New insights in