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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.
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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
thi s manuscri pt.
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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.
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