The role of ABC transporters in drug resistance, metabolism and toxicity
Mini-Review Hristos Glavinas1, Péter Krajcsi1,2, Judit Cserepes3, and Balázs Sarkadi4 1 SOLVO Biotechnology, Szeged, Hungary 2 Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary 3 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary 4 National Medical Center, Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences, Budapest, Hungary
CONTENTS
Abstract 1. General overview of ABC transporters 1.1 The ABC Transporter Family 1.2 Molecular Mechanism, Cellular Distribution and Substrate Specificity of ABC Transporters 2. ABC Transporters as Therapeutic Targets 2.1 Circumvention of Multidrug Resistance in Cancer - MDR Transporters 2.2 Lipid Disorders 2.3 Other Therapeutic Targets 2.4 Gene Therapy Applications 3. Role of ABC Transporters in ADME/Toxicity properties of drugs 3.1 Drug Absorption 3.2 Drug Distribution 3.3 Drug Metabolism 3.4 Drug Excretion 3.5 Drug-drug interactions (DDI) 3.6 Toxicity 4. ABC transporter assay systems in drug research and development 4.1 Screening methods 4.2 Whole cell based assays 4.3 Membrane based assays 4.4 Screening strategy 5. Abreviations 6. Bibliography
Abstract
ATP Binding Cassette (ABC) transporters form a special family of membrane proteins, characterized by homologous ATP-binding, and large, multispanning transmembrane domains. Several members of this family are primary active transporters, which significantly modulate the absorption, metabolism, cellular effectivity and toxicity of pharmacological agents. This review provides a general overview of the human ABC transporters, their expression, localization and basic mechanism of action. Then we shortly deal with the human ABC transporters as targets of therapeutic interventions in medicine, including cancer drug resistance, lipid and other metabolic disrorders, and even gene therapy applications. We place a special emphasis on the three major groups of ABC transporters involved in cancer multidrug resistance (MDR). These are the classical P-glycoprotein (MDR1, ABCB1), the multidrug resistance associated proteins (MRPs, in the ABCC subfamily), and the ABCG2 protein, an ABC half-transporter. All these proteins catalyze an ATP-dependent active transport of chemically unrelated compounds, including anticancer drugs. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. Based on the physiological expression and role of these transporters, we provide examples for their role in Absorption-Distribution-Metabolism-Excretion (ADME) and toxicology, and describe several basic assays which can be applied for screening drug interactions with ABC transporters in the course of drug research and development.
1. General overview of ABC transporters
1.1 The ABC Transporter Family
ABC (ATP-binding cassette) proteins form one of the largest protein families, and members of this family are found in all living organisms from microbes to humans. The wild-spread presence of these proteins with a relatively conserved structure and function suggests a fundamental role. In the present review we focus on the expression, structure and function of ABC transporters present in humans and in related model organisms. The basic structure that defines the members of this protein family is the combination of conserved ATP-binding and transmembrane domains. In mammals the functionally active ABC proteins consist of four characteristic domains, two transmembrane domains (TMDs), and two nucleotide-binding domains (NBDs). These four domains may be present within one polypeptide chain ("full transporters"), or within two separate proteins ("half transporters").In this latter case the ABC transporters become functionally competent after specific dimerization (for reviews see 1-11). The membrane-spanning domains form the key structural background of the function of ABC transporters. The two TMDs contain polypeptide chains that span the membrane multiple times, typically forming six transmembrane ?-helices per domain, a total of twelve helices in a full transporter (7, 10). The twelve transmembrane ?-helices probably form a pore-like structure across the membrane, and through this path a range of different substrates can be transported by these proteins. The conformational changes within the TMD domains are believed to be responsible for the opened or closed states of these transmembrane structures. ABC proteins can utilize the energy derived from ATP hydrolysis to perform a directed transmembrane movement of their substrates (primary active transporters), open or close a specific membrane channel (e.g. ion-channels), or regulate the permeability of multi-protein channel complexes (receptors). In the ABC proteins which act as primary active transporters, the transport function depends on the hydrolysis of ATP within the NBDs. These cytoplasmic domains are attached to the intracellular regions of the TMDs, and a close interaction provides the functional connection between these two different domains. The nucleotide-binding domains bind cytoplasmic ATP and, in the active transporters, ATP hydrolysis ensures the energy for the uphill transport of a substrate. The specific, close interaction of NBDs with the TMDs provide the transmission gear of the conformationalchanges caused by substrate binding and the ydrolysis of ATP (8-10). During the sequencing of the human genome the full range of the human ABC genes has been described and characterized. Table I. presents the full list of the 48 human ABC genes (9), including some of their basic features. After some confusion in naming and numbering of the newly identified ABC transporters, a consistent nomenclature has been introduced, based on the sequence homology between these proteins. In this system the ABC genes are grouped into seven subfamilies, based on the similarity in the gene structure, order of the domains, and sequence homology in the NBDs and TMDs. In the following sections we provide a general introduction for these families. We also describe examples for the expression and function of some key family members, especially for those with an already recognized medical or pharmacological importance. Table I. Human ABC transporters and their basic features
| Family | Member | Alias | Expression | Function |
|---|---|---|---|---|
| ABCA | ABCA1 ABCA2 ABCA3 ABCA4 ABCA5 ABCA6 ABCA7 ABCA8 ABCA9 ABCA10 ABCA11 ABCA12 |
ABC1 ABC2 ABC3,ABCC ABCR |
Ubiquitous Brain Lung Rod photoreceptors Muscle, heart, testes Liver Spleen, thymus Ovary Heart Muscle, heart Stomach Low in all tissues |
Removal of cholesterol and PLs onto HDL particles Drug resistance Surfactant protection N-retinydilester-PE efflux |
| ABCB | ABCB1 ABCB2 ABCB3 ABCB4 ABCB5 ABCB6 ABCB7 ABCB8 ABCB9 ABCB10 ABCB11 |
MDR1,PGP TAP1 TAP2 PGP3,MDR3 MTABC3 ABC7 MABC1 MTABC2 SPGP,BSEP |
Adrenal,kidney,brain Ubiquitous,ER Ubiquitous,ER Liver Ubiquitous Mitochondria Mitochondria Mitochondria Heart,brain Mitochondria Liver |
Multidrug resistance Peptide transport into the ER Peptide transport into the ER Phosphatidylcholine transport Iron transport Heme transport Heme transport Bile salt transport |
| ABCC | ABCC1 ABCC2 ABCC3 ABCC4 ABCC5 ABCC6 ABCC7 ABCC8 ABCC9 ABCC10 ABCC11 ABCC12 |
MRP1 MRP2, cMOAT MRP3, cMOAT-2 MRP4, MOAT-B MRP5, MOAT-C MRP6 CFTR SUR SUR2 MRP7 MRP8 MRP9 |
Ubiquitous Liver Lung, intestine, liver Prostate Ubiquitous Kidney, liver Exocrine tissues Pancreas Heart, muscle Low in all tissues Low in all tissues Low in all tissues |
Drug resistance Organic anion transport Drug resistance Nucleoside transport Nucleoside transport Chloride ion transport Sulfonylurea receptor |
| ABCD | ABCD1 ABCD2 ABCD3 ABCD4 |
ALD ALD1,ALDR PMP70, PXMP1 PMP69, P70R |
Peroxisomes Peroxisomes Peroxisomes Peroxisomes |
VLCFA transport regulation |
| ABCE | ABCE1 | OABP | Ovary, testes, spleen | Oligoadenylate-binding protein |
| ABCF | ABCF1 ABCF2 ABCF3 |
ABC50 | Ubiquitous Ubiquitous Ubiquitous |
|
| ABCG | ABCG1 ABCG2 ABCG4 ABCG5 ABCG8 |
ABC8, Human white ABCP, MXR, BCRP White2 Steroline 1 Steroline 2 |
Ubiquitous Placenta, intestine Liver Liver, intestine Liver, intestine |
Cholesterol transport Drug resistance Sterol transport Sterol transport |
HDL: high density lipoprotein
VLCFA: very long chain fatty acid
1.1.1 The ABCA subfamily This subfamily comprises of 12 full transporters; some of the largest ABC proteins belong to this family. This subfamily can be divided into two subgroups, based on a phylogenetic analysis (12). One subgroup comprises five proteins (ABCA5-6 and ABCA8-10), the genes encoding these transporters are clustered on chromosome 17q24. The other subgroup consists of seven proteins (ABCA1-4, A7, A12-13), encoded by genes in different chromosomes.The ABCA1 protein is relatively well characterized, and suggested to be involved in the removal of cholesterol and phospholipids from cells onto high-density lipoproteins (HDL) particles (13). Different mutations in the ABCA1 gene cause a well defined clinical syndrome, Tangier disease. Patients with this syndrome are characterized by deficient efflux of lipids from macrophages, resulting early atherosclerosis and low level of HDL in the blood (14, 15). The ABCA1 protein is heavily glycosylated and probably resides in the plasma membrane. Recent reports described that the regulation of the expression of ABCA7 is similar to that of ABCA1, suggesting a role for ABCA7 in the lipid homeostasis as well (16). ABCA4 is a retina-specific ABC transporter, expressed exclusively in photoreceptors, where it probably transports retinal or its conjugates from the photoreceptor outer segment disks into the cytoplasm (17). Mutations in the ABCA4 gene have been associated with distinct chorioretinal disorders (18). Complete loss of ABCA4 function leads to retinitis pigmentosa, whereas patients with moderately severe mutations have the so called Stargardt disease (STGD). Heterozygous ABCA4 mutations may play a role in increased frequency of age-related macular degeneration (AMD), or recessive retinitis pigmentosa (RP19). The pathophysiology of these disorders is poorly understood, but the abnormal accumulation of retinoids caused by ABCA4 deficiency has been postulated to be the initial mechanism in these diseases. 1.1.2 The ABCB subfamily This is the most variable subfamily: it comprises of both full transporters and half transporters, includes transmembrane drug transporters, iron transporters and also specific peptide transporters, involved in antigen presentation. These proteins are localized in different membrane compartments, including the plasma membrane, endoplasmic reticulum, lysosomal and mitochondrial membranes. ABCB1 (MDR1/P-glycoprotein) was the first described membrane transporter characterized by its ability to confer multidrug resistance to cancer cells (19). MDR1 is glycosylated and localized in the plasma membrane, in the apical/luminal membrane in polarized cells. It is also present in the brush border membrane of intestinal cells, the biliary canalicular membrane of hepatocytes, the luminal membrane of proximal tubule epithelial cells of the kidney, and in the endothelial cells at the blood-brain barrier (1, 3, 4, 20 - see below). ABCB1 has a crucial physiological function in these barriers, namely the protection of he cells and organs against toxic compounds and metabolites. Due to its high transport capacity and broad substrate recognition, this transporter can confer a multidrug resistance phenotype to cancer cells (see below). ABCB2/TAP1 and ABCB3/TAP2 are half-transporters, and form heterodimers in order to function as peptide transporters involved in the MHC-I-dependent antigen presentation. They are localized in the membrane of endoplasmic reticulum, and pump degraded cytoplasmic peptides into the ER lumen where the MHC class I molecular complexes assemble (21).ABCB4/MDR3 and ABCB11/BSEP are involved in the transport of phosphatidylcholine (ABCB4) and bile salts (ABCB11), respectively, across the hepatocyte canalicular membrane into bile.Their mutations are responsible for various forms of progressive familial intrahepatic cholestasis (PFIC); mutations in ABCB4 were found in patients with PFIC3, resulting from a defective transport of phosphatidylcholine across the canalicular membrane (22-24). Mutations in ABCB11 cause PFIC2, in this case the biliary bile salt secretion is very low (25).ABCB6-8 are not well characterized as yet. These are ABC half-transporters, most probably localized in the mitochondrial inner membrane. They likely play a role in the mitochondrial metal homeostasis; ABCB6 probably mediates the transport of an iron complex, ABCB7 and ABCB8 are thought to be involved in the heme transport from mitochondria to the cytosol. They are also candidate genes for inherited metabolic disorders, ABCB6 for the lethal metabolic syndrome, ABCB7 for X-linked sideroblastic anemia with ataxia. ABCB9 is expressed in the lysosomes with unknown function as yet (26). 1.1.3. The ABCC subfamily The 12 members of this family are similar in their structure, all of them being full transporters. However, their functions are fundamentally different, they act with basically diverse molecular mechanisms. This subfamily includes active, ATP-dependent transporters (ABCC1-6, see 27-30), regulated ion channel (ABCC7/CFTR), and modulators of the permeability of specific K+ channels (SUR1/ABCC8 and SUR2/ABCC9) (4-6).The human multidrug resistance-associated protein ABCC1/MRP1 was discovered as the basis of a non-P-glycoprotein associated multidrug resistance (27). This protein is responsible for the transport of a remarkable range of drugs as it confers a cancer cell resistance to hydrophobic drugs, similarly to ABCB1/MDR1. However, further functional characterization revealed that the preferred substrates for MRP1 are organic anions, including drugs conjugated to glutathione (GSH), glucuronate, or sulfate (25-28, 30-32). MRP1 also mediates the cotransport of unconjugated amphiphilic anions, together with free GSH. The major physiological function of ABCC1 is presumably leukotriene C4 transport (28). ABCC2/MRP2 was discovered as an organic anion transporter in the canalicular membrane of hepatocytes, and its mutations were found in patients with Dubin-Johnson syndrome (33-34). This protein is present in the liver, kidney and gut epithelium apical membranes, where it can mediate the efflux of bilirubin glucuronides and other organic anions, playing a role in the detoxification for many endogenous and xenobiotic compounds. In Dubin-Johnson syndrome the inherited mutations of ABCC2 cause a defect in the hepatobiliary secretion of amphiphilic anionic conjugates, resulting in severe conjugated hyperbilirubinemia. By now it has been documented that ABCC2 may also cause multidrug resistance in tumor cells.ABCC3/MRP3 is also an organic anion transporter (35), and this protein prefers glucuronate conjugates as transported substrates. This protein exhibits a similar tissue distribution as MRP2, but unlike MRP2, that is present in the apical surface of cells, MRP3 is located in the basolateral membranes of polarized cells (35-36). The human MRP4, MRP5 and MRP6 have been recently characterized as primary active transporters. According to these data MRP4 can mediate the transport of several nucleoside analogs, including anti-human immunodeficiency virus drugs, such as 9-(2-phosphonylmethoxyethyl) adenine (PMEA) and azidothymidine monophosphate (AZTMP). This protein can also confer resistance to 9-(2-phosphonylmethoxyethyl) guanine (PMEG), a compound with neoplastic activity (37, 38). Similarly to MRP4, MRP5 can also catalyze the efflux of nucleoside monophosphate analogues, including nucleoside-based antiviral drugs, and drugs used in the treatment of acute lymphoblastic or myeloid leukemia (ALL/AML) (39). However, the physiological functions of MRP4 and MRP5 are unknown as yet. Mutations affecting the human MRP6 gene were described in patients with pseudoxanthoma elasticum (PXE), a rare heritable disorder, resulting in the calcification of elastic fibers. Functional studies demonstrated that MRP6 can mediate the transport of glutathione conjugates, e.g. LTC4 and N-ethylmaleimide S-glutathione (40-41). However, the physiological function and the role of ABCC6 in the pathogenesis of this disease are still unknown. Several recently recognized ABCC proteins (ABCC10, 11 and 12) may also be involved in as yet ill-defined conjugate transport processes (42). The gene coding for ABCC7/CFTR was identified in the late 1980-es as the gene in which mutations are responsible for the development of cystic fibrosis (CF). This is the most frequent autosomal recessive disease within Caucasian populations, with a prevalence of 1 in 2,500 births. The CF gene has been identified by positional cloning (43) and the 'cystic fibrosis transmembrane conductance regulator' protein (ABCC7/CFTR) was documented to function as a chloride ion channel, probably playing a role in exocrine secretions. The CF symptoms mostly relate to the disruption of exocrine functions: in the sweat glands (high sweat electrolyte), pancreas, bronchial glands (chronic bronchopulmonary infection), intestinal glands (meconium ileus), etc. However, additional functional role(s) of the CFTR may be involved in the development of the CF disease (44). ABCC8/SUR1 has been documented to regulate the cell surface expression and function of ATP-sensitive potassium channels in the pancreatic islets and thus modulate insulin secretion. This protein provides a high-affinity binding site for sulfonylurea, which is widely used to treat patients with non-insulin-dependent diabetes mellitus (45). 1.1.4. The ABCD subfamily This subfamily consists of four human proteins, all ABC half-transporters, that require a partner half-transporter to form functional homo- or heterodimeric transporters. They are localized in the peroxisomal membrane, and mutations in these genes cause different peroxisomal disorders. Adrenoleukodystrophy (ALD) is a severe X-linked disorder, the most frequent peroxisomal disorder, with a prevalence of 1 in 20,000 males, caused by mutations in the ABCD1 gene (46). This neurodegenerative disease is characterized by the accumulation of the unbranched saturated fatty acids with a chain length of 24 to 30 carbons, as well as of cholesterol esters in the brain and in adrenal cortex. The ABCD1/ALD protein is located in the peroxisomal membrane and most likely plays a role in the transport of very long chain fatty acids. 1.1.5-6. The ABCE and ABCF subfamily The members of these subfamilies have NBDs, characteristic for ABC proteins, but have no TMDs identified, and thus not known to be involved in any membrane transport functions. 1.1.7. The ABCG subfamily These five half-transporters, composing the ABCG subfamily, have a unique domain arrangement, with NBDs located at the N-termini and TMDs, located at the C-termini. The first described member of this subfamily was the human homolog of the Drosophila white gene, ABCG1 (47). The expression of this gene is regulated by cholesterol and lipoproteins in macrophages, suggesting that this protein is involved to the lipid metabolism in humans (48).ABCG5 and ABCG8 are also playing a role in the lipid metabolism, and mutations of either of these genes cause a rare heritable disorder, called sitosterolemia (49-50). In these patients the increased absorption of plant and fish sterols and their decreased biliary excretion cause a high level of toxic sitosterols in the blood, resulting in premature atherosclerosis and cardiac infarct.ABCG2, a half-transporter causing mitoxantrone resistance in carcinoma cells and multidrug resistance in breast cancer, was first found in multidrug-resistant tumor cells not expressing either MDR1 or MRP1 (51-53). ABCG2 functions as a high capacity drug transporter with a wide substrate specificity, and confers resistance to mitoxantrone, topotecan, and probably even methotrexate.
1.2 Molecular mechanism, cellular distribution, and substrate specificity of ABC transporters
Members of the ABC superfamily are associated with a broad spectrum of physiological functions, including detoxification (ABCB1/MDR1, ABCC1/MRP1), defense against xenobiotics and oxidative stress (ABCCs/MRPs), absorption and secretion processes (MDRs, MRPs), lipid metabolism (ABCA1, MDR3, ABCGs), antigen presentation (ABCB2/TAP1 and ABCB3/TAP2), etc. Many of the ABC transporters function as active transporters, they mediate the transport of substrates against a concentration gradient. This transport activity requires the energy of ATP hydrolysis,controlled by drug interaction, and coupled to the actual substrate translocation. Most probably the substrate recognition and binding occurs in some parts of the transmembrane domains (3-6, 54). According to several reports, the hydrophobic substrates of MDR1 are recognized within the membrane bilayer or in its vicinity (55). Similarly, in the case of MRP1 it has also been suggested that the transmembrane domains are involved in the drug interactions (56). But the specific sequences responsible for the substrate recognition have not been identified as yet. Interestingly, in case of MDR1 and MRP1, where the substrate-specificities are comparable, the two proteins have only 15% amino-acid identity (57). Recent reports have revealed a few key amino acids playing a role in the recognition of certain substrates in MDR1 (58-59), but the corresponding amino acids have not been found in MRP1. ATP binding and hydrolysis occurs in the nucleotide-binding domains, and in ABC transporter the close interaction of two NBDs results in the formation of a fully competent ATP hydrolytic site. In active transporters the specific substrate binding enhances the ATPase activity of the transporter, so the transported substrate can promote the liberation of the energy required for its translocation. Due to the molecular connection of NBDs to TMDs, the conformational changes resulted by the ATP hydrolysis can be directly transferred within the protein. Experiments analyzing the ATPase cycle of MDR1 or MRP1 revealed that the interaction of the two NBDs is an essential requirement for the catalytic reaction (60-61).These results suggest that both NBDs can bind ATP and both catalytic sites are active, and at least in the case of MDR1, the two ABC domains enter into the catalytic cycle alternatively (62-63). The ATPase activity of all ABC transporters studied so far is inhibited by vanadate. Vanadate probably replaces phosphate during ATP hydrolysis, and stabilizes a specific, transition state conformation (see ref. 64). Thus the ATPase activity of ABC transporters reflects their catalytic, transport activity. This substrate-modulated, vanadate-sensitive ATPase activity, representing the full transport cycle, can be directly measured and thus used in various ABC transporter assay systems. In ABC transporters the catalytic reaction involves the formation of a transition state complex that contains an occluded nucleotide. This complex, when containing ADP, can be stabilized by vanadate (64), and this vanadate-dependent nucleotide trapping is significantly modulated by compounds interacting with the transporter (6, 60, 65). Again, this reaction can be used to study drug-protein interactions, as the concentration dependent modulation of nucleotide trapping correlates with the interaction of a given compound with the ABC proteins.Still, we know relatively little about the detailed catalytic mechanism of ABC transporters, although several models have already been suggested to describe this mechanism (64).As shown in Table I, ABC transporters with multidrug transporter function (ABCB1/MDR1, ABCC1/MRP1, ABCG2) show a widespread expression profile, providing a cellular defense mechanism throughout the organism. The tissue distribution of these three major multidrug resistance proteins is overlapping but different. ABCC1 expression is high in all tissues, it is elevated in lung and the testis, and reduced in the liver. MDR1 is more restricted to tissues involved in the absorption and secretion (1-4). MDR1 is also highly expressed in tissues with pharmacological barrier function, such as the blood-brain barrier, and the choroid plexus (66-67). Recent reports have demonstrated that ABCG2 is highly expressed in the placenta, liver, intestine and in various stem cells (68-69).In tissues where different multidrug transporters are present, the subcellular localization of these proteins can be a discriminating feature. All multidrug transporters are localized predominantly in the plasma membrane. ABCB1/MDR1 exhibits apical (luminal) expression in polarized cells, e.g. epithelial cells of the intestine and the proximal tubules of kidney, or the canalicular membranes of hepatocytes (21-22, 70). In contrast, MRP1 in polarized cells is localized solely in the basolateral membrane. The expression of ABCG2 was reported to be mostly apical (71). The substrate specificity of MDR-ABC proteins exhibits as wide a variety as their tissue distribution and they share the ability to transport a wide range of compounds out of cells. The substrate specificities of these three proteins are again overlapping, but each transporter can handle unique compounds as well.ABCB1/MDR1 favors uncharged or positively charged hydrophobic compounds, whereas the preferred substrates for ABCC1/MRP1 are organic anions, including drugs conjugated to glutathione (GSH), glucuronate, or sulfate. Typical high affinity substrates of MRP1 are leukotriene C4 (LTC4),17 ß-glucuronosyl estradiol and bisglucuronosyl bilirubin. ABCG2 has also a remarkable range of transported substrates (mitoxantrone, anthracyclins, topotecan, flavopiridol, and methotrexate - see 11, 68, 72). In some cases, when different transporters with overlapping substrate profiles are located in the same cells, their affinity, capacity, and the cellular drug-concentrations determine the actual fate of the substrates. In hepatocytes, where MRP2 and MRP3 are both expressed, different conjugates are transported according to the transporters affinity and capacity. MRP2 transports glucuronate conjugates with a relatively low affinity, but with high capacity, in contrast to MRP3, which is a high affinity, but low capacity transporter for these conjugates. Moreover, MRP-mediated transport is greatly increased by the presence of intracellular bile salts. Thus, under normal conditions glucuronate conjugates are extruded into the bile by MRP2, located in the apical membrane. However, at increasing cellular concentrations of glucuronate conjugates MRP3 can also remove a certain amount of these compounds into the blood stream, and defend the cells from the toxic materials (73-74).
2. ABC Transporters as Therapeutic Targets
2.1 Circumvention of Multidrug Resistance in Cancer - MDR Transporters
The multidrug resistance phenotype in tumors is associated with the overexpression of certain ABC transporters, termed MDR proteins. The P-glycoprotein (Pgp, MDR1, ABCB1) mediated multidrug resistance was the first discovered and probably still is the most widely observed mechanism in clinical multidrug resistance (1, 3, 5, 75). There are two other ABC transporters, which have been definitely demonstrated to participate in the multidrug resistance of tumors: the multidrug resistance protein 1 (MRP1/ABCC1, 27, 29, 76), and the mitoxantrone resistance protein (MXR/BCRP, ABCG2, 68). Furthermore, other human ABC proteins capable of actively transporting various compounds out of the cells, may also be players in selected cases of multidrug resistance. These include the homologues of MRP1, MRP2-MRP5. MRP2 and MRP3 seem to be key players in organic conjugate transport in various tissues, while MRP4 and MRP5, may have special functions as nucleoside transporters (36, 39, 77-78, 80). The three major proteins involved in cancer multidrug resistance are MDR1 (P-glycoprotein, ABCB1), MRP1 (multidrug resistance protein 1, ABCC1) and the ABCG2 multidrug transporter (BCRP/ MXR). MDR1 and MRP1 can recognize and transport a large variety of hydrophobic drugs, and MRP1 can also extrude anionic drugs or drug conjugates (28-29, 81-82). The substrate specificity of ABCG2 partially overlaps with that of MDR1 and MRP1, that is the compounds transported by ABCG2 are also large, hydrophobic molecules, including mitoxantrone, topotecan, flavopiridol, methotrexate and Hoechst 33342 (81).
Prevention of clinical MDR should significantly improve therapeutic response in a large number of tumor patients. One way to achieve this goal would be to develop anticancer agents which do not interact with any of the multidrug transporters. However, as cytotoxic drugs have to penetrate the cell membrane and the MDR proteins have an extremely wide recognition pattern, this seems to be a remote possibility. There are several suggested methods to prevent the expression or function of multidrug transporters, but pharmacological modulation seems to be the first choice at present.MDR modifying agents which inhibit the function of the MDR proteins, either competitively or non-competitively, are good candidates for such a pharmacological modulation. These compounds are expected to increase the cytotoxic action of MDR-related drugs by preventing the extrusion of anticancer drugs from the target cells.Studies involving various in vitro assay systems (see below) led to the identification of several Pgp modulator compounds, as diverse in structure as the transported drugs. The first generation consisted of drugs that were already in clinical use, while the second generation modulators were derivatives of the above compounds. These, e.g. R-verapamil and PSC-833, had less pronounced effects on their original targets, but retained their modulatory effects. The third generation of MDR modifiers are molecules specifically devised to interact with specific MDR transporters(for recent reviews see 4, 32, 58, 81, 84-85).As these agents are mostly in the early development phase in various research laboratories, their clinical efficiency has yet to be proven. Small hydrobhobic peptide derivatives (Reversins), interacting with P-gp/MDR1 with high affinity and selectivity, were also developed in our laboratory (86).
2.2 Lipid Disorders
ABC transporters have been documented to play a major role in lipid transport and lipid-related disorders. The association between cholesterol and atherosclerosis is thought to involve the cellular uptake and deposition of cholesterol. By removing cholesterol from the cells, the reverse cholesterol pathway provides protection for the artery wall against unwanted lipid deposition. It has been suggested that ABCA1 is the key protein in controlling the cellular apolipoprotein-mediated lipid removal pathway (87-88). Mutations in the ABCA1 gene result in Tangier disease, a genetic disorder characterized by an abnormal lipoprotein profile and the accumulation of cholesterol esters in various tissues (89-91). Cholesterol efflux from Tangier-fibroblasts to lipid-poor apolipoproteins is defective, suggesting that ABCA1 has a key role in the modulation of the reverse cholesterol transport (for reviews see 87, 89, 93). Several other ABC transporters have also been implicated in lipid and/or cholesterol transport. The expression level of ABCG1 in human macrophages is greatly increased by cholesterol loading and by lipoproteins, suggesting that this protein is involved in the metabolism of these lipids (48). There are no published functional and localization data available as yet regarding this ABC transporter in various cell types. However, it has been documented in detail, that close relatives of ABCG1, that is ABCG5 and ABCG8, play a key role in lipid metabolism, as mutations of either of these genes was found to cause a heritable disorder altering the absorption and excretion of plant- and fish-derived steroid molecules (sitosterolemia, 49-50). In these patients high levels of blood sitosterols, and an altered cholesterol metabolism leads to atherosclerosis and cardiac diseases. ABCG5 and ABCG8 were documented to work as obligate heretodimers, and both their function and correct cellular localization depends on this dimerization process (94). ABCB4 (MDR3) is a specific phosphatidyl choline transporter residing in the bile canalicular membrane in the liver, and has an essential function in the proper bile formation (22).Modulation of the expression and function of these lipid transporter proteins may soon become an important pharmacological target. We know very little as yet about the molecular mechanism and structure of these proteins, thus this seems to be a rapidly developing and promising area of ABC transporter research.
2.3. Other Therapeutic Targets
There are numerous ABC transporters in which specific mutations cause severe inherited diseases. These are all natural therapeutic targets in medicine and in the present review we just shortly point to some of these target proteins. In the case of the lethal inherited
