1. Introduction
1.1. Multidrug Resistance
Multidrug resistance is a condition enabling a disease-causing organism to resist distinct drugs or chemicals of a wide variety of structure and function targeted at eradicating the organism. Organisms that display multidrug resistance can be pathologic cells, including bacterial, fungal and neoplastic (tumor) cells.
-Bacterial resistance to antibiotics
Various microorganisms have survived for thousands of years by their being able to adapt to antimicrobial agents. They do so via spontaneous mutation or by DNA transfer. It is this very process that enables some bacteria to oppose the assault of certain antibiotics, rendering the antibiotics ineffective. These microorganisms employ several mechanisms in attaining multidrug resistance:
• No longer relying on a glycoprotein cell wall
• Enzymatic deactivation of antibiotics
• Decreased cell wall permeability to antibiotics
• Altered target sites of antibiotic
• Efflux mechanisms to remove antibiotics
• Increased mutation rate as a stress response
Many different bacteria now exhibit multidrug resistance, including staphylococci, enterococci, gonococci, streptococci, salmonella, Mycobacterium tuberculosis and others. In addition, some resistant bacteria are able to transfer copies of DNA that codes for a mechanism of resistance to other bacteria, thereby conferring resistance to their neighbors, which then are also able to pass on the resistant gene.
-Antifungal resistance
Clinical resistance to antifungal agents was rare until the late 1980s, with only isolated cases in patients with chronic mucocutaneous candidiasis.1,2 The incidence of fungal infections, including resistant infections, has increased during the last 10 years, reflecting increased incidence of immunodeficiency associated with cancer chemotherapy, organ and bone marrow transplantation, and the HIV epidemic.1,3 Although the prevalence of drug resistance in fungi is below that observed in bacteria, many mycologists consider that selective pressure will, over time, lead to more widespread resistance.
There is considerable knowledge concerning the clinical, biochemicaland genetic aspects of resistance to antifungal agents. However, sample selection and inadequate information regarding denominatorslimit current epidemiological data. At present, there is noestablished national surveillance scheme to identify changes in antifungal susceptibility. In addition, there are no large-scale epidemiologicalsurveys of the extent of antifungal drug resistance in the publishedworld literature.
-Neoplastic resistance
Cancer cells also have the ability to become resistant to multiple different drugs, and share many of the same mechanisms:
• increased efflux of drug (as by P-glycoprotein, multidrug resistance-associated; protein, lung resistance-related protein, and breast cancer resistance protein);
• enzymatic deactivation (i.e., glutathione conjugation);
• decreased permeability (drugs cannot enter the cell);
• altered binding-sites;
• alternate metabolic pathways (the cancer compensates for the effect of the drug);
Because efflux is a significant contributor for multidrug resistance in cancer cells, current research is aimed at blocking specific efflux mechanisms.[1] Treatment of cancer is complicated by the fact that there is such a variety of different DNA mutations that cause or contribute to tumor formation, as well as myriad mechanisms by which cells resist drugs.
There are also certain notable differences between antibiotic drugs and antineoplastic (anticancer) drugs that complicate designing antineoplastic agents. Antibiotics are designed to target sites that are specific and unique to bacteria, thereby harming bacteria without harming host cells. Cancer cells, on the other hand, are altered human cells; therefore they are much more difficult to damage without also damaging healthy cells.
1.2. Multidrug Resistance to Anticancer Drugs (neoplastic resistance)
Next to cardiovascular disease, cancer is the leading cause of death in the United States. Whatever the cause, cancer is basically a disease of cells. In cancer cells, cell growth is uncontrolled as a result of imbalance between the rate of cell proliferation and apoptosis (programmed cell death). The intracellular biochemical pathways of tumor growth are intrinsically very complex and are regulated by several oncogenes and tumor suppressor genes as well as by apoptosis-related genes. Despite intensive research in the last three decades, effective chemotherapy remains elusive.
For many types of cancer, chemotherapy still provides palliative rather than curative therapy. A major problem in cancer chemotherapy is multidrug resistance. Multidrug resistance is the phenomenon in which cancer cells exposed to a single drug become resistant to an array of anticancer drugs with different chemical structures and molecular mechanisms of action. The ability of cancer cells to become simultaneously resistant to different anticancer drugs is, in fact, the primary reason for the high failure rate of cancer chemotherapy.
Many mechanisms are known to cause multidrug resistance. These include disruption of apoptotic signalling pathways, gene amplification, activation of DNA repair, and overexpression of efflux transporters.
Many anticancer drugs exert their antitumor effect against cancer cells by inducing apoptosis. Attenuation of proapoptotic genes and increase in antiapoptotic genes can cause resistance to apoptosis. Recent studies revealed that inactivation of p53 or reduction of ceramide (a second messenger in apoptotic signaling) could lead to decreased apoptosis. Ceramide glycosylation through glucosylceramide synthase allows cellular escape from
ceramide-induced programmed cell death, and providing another avenue for cancer cell resistance to cytotoxic anticancer drugs.
Another mechanism for drug resistance results from changes in DNA topoisomerase II and gene amplification. Reduction in nuclear DNA topoisomerase II (a nuclear enzyme) has been postulated as the main mechanism in doxorubicin induced drug resistance. An important feature of topoisomerase II is the formation of the enzyme-DNA complex referred to as the ‘‘cleavable complex’’ that facilitates DNA breaking. Drugs such as doxorubicin stabilize the enzyme-DNA complex by binding to topoisomerase II, leading to the inhibition of the cleavage of DNA strands.
Gene amplification is also a contributing factor to multidrug resistance. In some cases, tumor cells may survive via gene amplification. Methotrexate blocks the synthesis of DNA, RNA, and protein by inhibiting dihydrofolate reductase. Lymphoblasts obtained from myeloid leukemia patients undergoing methotrexate therapy showed both increased dihydrofolate reductase activity and three to six fold increase in dihydrofolate reductase gene copies.
In addition, resistance to certain classes of anticancer drugs is frequently linked to the increased level of glutathione (an intracellular scavenging thiol) and the activity of glutathione S-transferase in cancer cells. These results suggest that an increase in the level of glutathione may be another mechanism contributing to multifactorial drug resistance in tumor cells. Depletion of cellular glutathione and/or decreasing the activity for glutathione S-transferase has been postulated to be one avenue to overcome cellular resistance to several classes of anticancer drugs. Glutathione has been suggested to be involved in an MRP1-related cotransport system, and depletion of glutathione could impair the MRP1-mediated efflux transport.
Alterations in the binding of drugs to cellular proteins have also been shown in resistant cell lines. It is widely accepted that antitubulin agents, such as vinca alkaloids and taxanes, block cell division by inhibiting the formation of the mitotic spindle via binding to β-tubulins. Therefore, drug resistance to these agents may be attributed to alterations of microtubule structure and changes in microtubule dynamics. Acquired resistance to
paclitaxel has been shown to be associated with increased expression and mutation of β-tubulins.
Finally, reduction of intracellular drug concentration by increased efflux of drugs has also been recognized as a mechanism contributing to multidrug resistance in cancer cells. The P-gp was the first identified efflux transporter to confer multidrug resistance. In a study that used several tumor cell lines, Ling et al. demonstrated a good correlation between the expression of P-gp and degree of resistance and suggested that P-gp confers drug resistance by reducing intracellular drug concentrations via an efflux transport. However, P-gp is not the only efflux transporter that confers multidrug resistance. Another efflux transporter involved in drug resistance has been identified, referred to as the multidrug resistance-associated protein (MRP1). Recent studies revealed that at least two other members of the MRP subfamily (MRP2 and MRP3) may also act as efflux transporters in cancer cells. Another ABC transporter referred to as breast cancer resistance protein (BCRP) has also been identified in cancer cells. The BCRP confers resistance to many anticancer drugs, including mitoxantrone, topotecan, the active metabolite of irinotecan (SN-38), and anthracyclines.
Collectively, there are at least three efflux transporters that are known to contribute to multifactorial drug resistance by reducing intracellular drug concentrations.
As noted above, there are a wide variety of mechanisms by which human tumor cells can become resistant to anticancer drugs. Because of the heterogenous cell population in a tumor tissue, different cells may acquire different mechanisms of resistance. The mechanisms become even more complex for different types of tumors. Because of the multifactorial nature, it is very difficult to accurately assess the specific mechanisms and their relative contribution to drug resistance for all types of tumors.
1.3. Efflux transporters
To understand how drug accumulation can be reduced in cancer cells and what an efflux transporter is, it is first necessary to examine how drugs get into cells.
There are basically two mechanisms of drug uptake. For water-soluble, hydrophilic drugs such as cisplatin, nucleoside analogue and antifolates, drugs cannot cross the plasma membrane unless they put to use the on existing transporters or carriers, or enter through hydrophilic channels in the membrane.
Resistance to such drugs resulting from decreased accumulation occurs because of individual mutations in the carriers, which produce single-agent resistance. MDR can occur because of a generalized defect in the localization of transporters and carriers on the cell surface, as is seen when selecting for cisplatin resistance which results in MDR to many different hydrophilic anticancer drugs and other small molecules (Shen et al.,1998).
For hydrophobic drugs, such as the natural products vinblastine, vincristine, doxorubicin, daunorubicin, actinomycin D, etoposide, teniposide, and paclitaxel, entry occurs by diffusion across the plasma membrane, without any specific drug carriers.
The only way to keep such drugs out of the cells is by activation of energy-dependent transport system (reviewed in Ambudkar et al., 1999). The first of these transporters, to be identified and characterized know as the ATbinding cassette (ABC) transporters, was P-glycoprotein (P-gp), the product of the human MDR1 gene.
Multidrug transporters are proteins able to extrude a variety of chemicals of different structures and are expressed in all living cells. They fall into a limited number of families, some of them are present in microorganisms 1, such as the Major Facilitating Superfamily proteins (MFS), the Resistance Nodulation cell Division protein (RND), the Smallest Multidrug Resistance proteins (SMR) and Multidrug And Toxic compound Extrusion proteins (MATE). Other of them are present in both prokaryotic and eukaryotic organisms, such as the ATP-Binding Cassette type proteins (ABC).
Different families use different energy sources to function: for instance, MFS and SMR use an electrochemical proton gradient while the ABC superfamily uses ATP 2, 3. The ABC superfamily can be divided into two major clusters 4, the Pgp and the MRP clusters, to which a third one has recently been added: the MXR gene product, also known as breast cancer resistant protein (BCRP). This last protein contains about half of the aminoacids of Pgp (655 aa) and it is believed to act as a dimer (half transporter) 5.
In this thesis we will deal mainly with the most important mammalian ABC protein (P-gp). As all multidrug transporters do, this protein transport an amazing variety of substrates. However the characteristics that determine whether a compound is transported or not, are not completely understood, even if substrates share some common features such as high lipophilicity, relatively high molecular weight and an amphipathic nature 6,7.
1.4. ATP-binding cassette (ABC)
ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport chemically diverse substrates across the lipid bilayers of cellular membranes. Clinically relevant examples, including the human proteins MDR1 (also known as ABCB1 or P-glycoprotein) and MRP1 (also known as ABCC1), contribute to multidrug resistance of cancer cells by catalysing the extrusion of cytotoxic compounds used in cancer therapy.
FIG. 1.1. Function of the multidrug/xenobiotic ABC transporters. Multidrug/xenobiotic ABC transporters reside in the plasma membrane and extrude various hydrophobic and/or amphipatic xenobiotics and metabolic products. MDR1/Pgp transports hydrophobic compounds (X), while MRP1 and ABCG2 can extrude both hydrophobic drugs and intracellularly formed metabolites, e.g., glutathione or glucuronide conjugates (C-X).
The expression of several ABC transporters is under tight transcriptional regulation especially by the group of orphan nuclear receptors. Nuclear receptors constitute a family of transcription factors that act as heterodimers. These heterodimers bind to the promotor elements and induce gene expression. In these heterodimers, the obligatory partner is the retinoid receptor. It has long been known that the expression of the drug transporter MDR1 can be induced by various drugs8.
Fig. 1.2. Two-dimensional structural and topological models of Pgp, MRPs and BCRP. The models indicate the transmembrane segments, the glycosylation sites (GS) and the adenosine triphosphate (ATP) binding sites. It is emphasized that the figure presents schematic models, and that in reality the transmembrane segments come together and form more compact molecules. Two different general structures have been described for members of the MRP family. MRP 1, 2, 3 and 6 share the same structural assembly. The additional N-terminal domain, with five transmembrane segments is a characteristic feature of these multidrug transporters. MRP4 and MRP5 lack this N-terminal extension. Except for the location of the glycosylation sites their structure is comparable with that of Pgp. BCRP consists of only six transmembrane segments, and has only one nucleotide binding domain. It has been suggested that BCRP assembles in the membrane to functional homodimers.
A potential mechanism for this induction was provided recently, through the identification of another member of the family of nuclear receptors, the transcription factor SXR, as a regulator of MDR1 expression 9. SXR can bind many structurally different ligands such as rifampicin 10, phenobarbital, paclitaxel, clotrimazole, hyperforin, lithocholate, ritonavir. The role of SXR in MDR1 expression now explains why MDR1 is induced by these compounds.
The ABC transporters have been found to have a definite physiological role.
In man and mammals, Pgp is found in the mucosal epithelium of the intestine, where it makes an important contribution to the direct excretion of transported compounds into the intestinal lumen 11 and is also a major determinant for the reduced uptake of orally administered compounds 12.
Several ABC-transporter proteins, all homologues of either the Pgp or of the MRP subfamily, have been characterised in human hepatocytes where, by analogy with their intestinal functions, they may protect the hepatocyte by returning hydrophobic toxic bile components to the bile 13.
Pgp and MRP1 are also found in the epithelial cells of the proximal tubules of the kidneys, where they have both a direct excretory role for drugs, and a role in limiting the back diffusion of amphipathic compounds which have entered the pre-urine by ultrafiltration
14, 15
. Both proteins have been found in epithelial cells of pancreas, along endothelial cells in small blood capillaries of the brain and of the testis, and in several other cells and tissues 16,
17
.
One of the most important physiological actions of P-gp, and possibly MRP1, is at the level of brain penetration by drugs 18. The blood-brain barrier (BBB) maintains a nearly continuous physical barrier separating the brain compartment from the blood stream. Hydrophobic compounds can cross the BBB by passive diffusion and enter the brain compartment. However, there are drugs that exhibit poor brain penetration despite their high lipophilicity (e.g. doxorubicin and vincristine). The poor distribution of these compounds in the brain led to the seminal proposal that efflux transport systems such as P-gp and MRP1 can operate at these barriers and actively eliminate drugs from the brain 19, 20, 21. P-gp also
seems active in releasing functionally active neurotransmitters/ neuromodulators (e.g. opioids, b-endorphin, glutamate) directly from the brain into the blood 22, 23.
Fig.1.3. Putative localization of drug efflux proteins on brain capillary endothelial cells that form the blood–brain barrier
The placental barrier was also shown to express P-gp on the brush-border membrane (maternal side) of trophoblast cells, where it regulates the transfer of several substances from mother to foetus and protects the foetus from toxic compounds 24.
In short, while distribution of MRP1 and sister proteins is considered ubiquitous 25, Pgp has a more discrete expression, being preferentially localized at tissue–blood barriers, suggesting that Pgp is primarily involved in the extrusion of xenobiotics from cells into the luminal space and in the secretion of endogenous lipophilic molecules from secretory tissues
26, 27
.
The discovery of the physiological role of ABC transporter proteins has greatly complicated the development of safe and effective MDR modulators but, at the same time, has offered a sound basis to rationalize some previously unexplained pharmacokinetic events, and P-gp and MRP proteins are becoming increasingly important to explain the pharmacokinetics of drugs.
As a matter of fact, it has been shown that they have profound consequences on the absorption, distribution, and clearance of many families of drugs, including the chemotherapic itself.
Both Pgp and MRP1 are inducible 28 and it can be anticipated that any drug or foodstuff that induces or modulates the activity of transporter proteins is likely to alter the pharmacokinetic behavior of any other drug present in the organism that is a substrate thereof. As a matter of fact, the number of molecules (drugs, compounds of natural origin, food components) that have been shown to induce or modulate ABC transporter proteins is so huge that it is nearly impossible to keep track of all them.
1.4.1. ABC transporter architecture
The basic ABC transporter architecture consists of two transmembrane domains (TMDs) that provide a translocation pathway, and two cytoplasmic, water-exposed nucleotide-binding domains (NBDs) that hydrolyse ATP. These ABC transporter proteins are generally expressed as ‘half-transporters’ that contain one TMD fused to a NBD, which dimerize to form the full transporter.
Functionally important residues are highly conserved among the NBDs, suggesting that ABC transporters share a common mechanism of coupling ATP hydrolysis to substrate transport 29, 30. However, despite extensive biochemical, genetic and structural studies, a detailed understanding of the substrate acquisition and transport process has remained elusive.
The functional unit of these transporter is a dimer of two elongated subunits related by two-fold molecular and non-crystallographic symmetry (Fig. 1.4 a,b). The full transporter is 120 Å long, 65 Å wide and 55 Å deep. Each subunit consists of an amino-terminal TMD (amino-acid residues 1–320) and a carboxyterminal NBD (residues 337–578). The two subunits exhibit a considerable twist and embrace each other, with both the transmembrane and the nucleotide-binding domains tightly interacting.
The NBDs expose conserved ATP-binding and -hydrolysis motifs at the shared interface in a ‘head to- tail’ arrangement that has been widely accepted as physiologically relevant on the basis of structural 31, 32 and biochemical 33-35 data. The hallmark of this arrangement is that two ATP-hydrolysis sites are formed at the shared interface, between the P-loop of one NBD and the ABC signature motif of the other. This creates a direct link between the two sites and provides the molecular basis of the observed cooperativity in ATP binding and hydrolysis 36.
On the other hand each TMD crosses the lipid bilayer six times to yield 12 transmembrane helices for the homodimeric transporter, in agreement with the canonical ABC exporter topology. Around the middle of the membrane, bundles of transmembrane helices diverge into two discrete ‘wings’ that point away from one another towards the cell exterior, thus providing an outward-facing conformation.
Figure 1.4. Sav1866 structure. a, Backbone of the homodimeric protein in ribbon representation, with the subunits coloured yellow and turquoise. Bound ADP is in ball-and-stick representation. The view reveals the membrane-embedded ‘wings’ of the protein; the grey box depicts the probable location of the lipid bilayer on the basis of the hydrophobicity of the protein surface. b, Stereo view rotated with respect to a by 908 around the vertical two-fold molecular and non-crystallographic axis. The transmembrane helices of one subunit (turquoise) are numbered. TMDs, transmembrane domains; NBDs nucleotide-binding domains; ICL, intracellular loops (between transmembrane helices); ECL, extracellular loops (between transmembrane helices); N-ter, amino terminus; C-ter, carboxy terminus.
The TMDs of ABC transporters have been the more difficult regions to study because they show little amino acid homology between the different transporters. The large differences in the TMDs of the various ABC transporters is not surprising because these domains act as the substrate recognition sites and translocation pathways through the membrane for different substrates. Another factor that will make the TMDs different in other ABC proteins is that some prokaryotic transporters act as importers while other prokaryotic transporters and all human ABC transporters act as exporters. Although the database is small, crystal structures of complete prokaryotic ABC transporters suggest that the structures of importers may differ from exporters 37.
In addition a large cavity is present at the interface of the two transmembrane domains (Fig. 1.5.). Although shielded from the cytoplasm and the lipid bilayer at the level of the inner leaflet, the cavity is accessible from the outer leaflet and exposed to the extracellular space. The bottom of the cavity reaches beyond the intracellular membrane boundary, but no connection to the cytoplasm exists. At the level of the inner leaflet, the cavity features a hydrophilic surface that is primarily lined with polar and charged amino-acid side chains, with a slight surplus of negative charges and no significant hydrophobic patches. The observed cavity is consistent with an outward-facing conformation of the ATP-bound state of human MDR1, as revealed by electron microscopy 38, 39. The predominance of polar and charged amino acids suggests that rather than a high-affinity binding site, the observed cavity may reflect an extrusion pathway with little or no affinity for hydrophobic drugs The architecture of these transporter indicates that residues from all transmembrane helices contribute to the surface of the translocation pathway. It was noted recently that, despite extensive efforts, no well defined substrate binding sites have been identified in ABC exporters 40.
Figure 1.5. Substrate translocation pathway. a, Backbone traces of the Sav1866 subunits (green and yellow) in two orientations, rotated by 908 around a vertical axis. The molecular surface of the central cavity is coloured according to its calculated electrostatic potential (blue, positively charged; red, negatively charged), whereas that of the rest of the transporter is in transparent light grey. Lines indicate the approximate position of the two leaflets of the lipid bilayer. Access from the cavity to the outer leaflet of the lipid bilayer is visible in the right panel from the front and back, between the ‘wings’ formed by the TMDs. b, Cavity at the level of the inner leaflet viewed from the extracellular side. The transmembrane helices are numbered and the cavity is shown as a grey surface. c, Same as b but at the level of the outer leaflet. Note that owing to helix bending, different subsets of helices line the cavity when compared with b.
1.4.2. Transport mechanism
The simplest scheme of the transport mechanism invokes two states: an inward-facing conformation with the substrate binding site accessible from the cell interior, and an outward-facing conformation with an extrusion pocket exposed to the external medium. Tight interaction of the NBDs in the ATP-bound state is coupled to the outward-facing conformation of the TMDs. In this conformation, bound substrates may escape into the outer leaflet of the lipid bilayer or into the aqueous medium surrounding the cell, depending on their hydrophobicity. Hydrolysis of ATP is expected to return the transporter to an inward-facing conformation, again granting access to the binding site from the cell interior.
ABC transporters may thus use an “alternating access and release” mechanism first postulated for major facilitator transport proteins 41, with the distinction that ATP binding and hydrolysis, rather than substrate acquisition, may control the conversion of one state into the other. Most ABC transporters bind and hydrolyse two ATP molecules in each reaction cycle 42, 43. However, the number of bound substrates can vary, and depending on the molecular mass up to two substrates have been found to enter the binding pockets of human MDR1 and MRP2 44, 45. If two small substrates are bound, the apparent stoichiometry of hydrolysed ATP per transported substrate will be one, whereas for a single, larger substrate, it will be two.
Figure 1.6. | ABC exporter schematics. a, Earlier cartoons depict two compact transporter halves (subunits) arranged side-by-side, suggesting separation during the transport cycle. b, Schematic of Sav1866 in the observed, outward-facing conformation.
1.5. Characterstics of the MRP1 and MRP2 Transporters
Studies using cell lines selected for MDR demonstrate an association between expression of MRP1 or MRP2 and MDR. Like P-gp, MRP (multidrug resistance protein) has the capacity to mediate transmembrane transport of many (conjugated) drugs and other compounds.
Nine members have been identified in the MRP family. However, the study of MRP has mainly focused on MRP1 and MRP2. Several cell lines that display a multidrug resistance phenotype have been isolated without detectable P-gp expression, despite having undergone drug selection similar to that elevating expression of P-gp. MRP1 was discovered in these cells by a differential hybridization screen aimed at identifying mRNA species whose expression is associated with the gain or loss of the multidrug resistance phenotype 46.
An isoform of MRP1 has been cloned and localized predominantly to the hepatocyte canalicular membrane. This apical conjugate-transporting ATPase has been termed as multidrug resistance protein 2 (MRP2) because there is some similarity in substrate specificity and sequence with the multidrug resistance protein (MRP1). MRP2 is also known as canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT). It is important to understand the mechanism of MRP1 and MRP2 in the disposition of endogenous compounds, drugs and other xenobiotics in many organs in order to selectively target these transporters.
Human MRP1 and MRP2 are composed of 1531 and 1545 amino acids, respectively. The
MRP1 gene is localized to chromosome 16p13.1 and the MRP2 gene is found at
chromosome 10q24. Both of them are amplified in various anti-tumor drug-resistant cell lines 47, 48. Analysis of their amino acid sequences identified the MRP1 and MRP2 proteins as members of the ATP-binding cassette (ABC) superfamily of transporter proteins. However, the well known P-gp is only distantly related to MRP1 and MRP2. The amino acid sequence identity between MRP1 and P-gp is only 15%, while comparison of the MRP2 sequence with that of P-gp indicates that there is 25% identity between them. MRP2 has an overall amino acid sequence identity of 49% to MRP1, and the region with highest identity of amino acid sequence is located in the carboxylterminal domain 49. Human MRP1 and
MRP2 have a 172 kDa 50 and 174 kDa molecular mass of unglycosylated form, respectively
51
. Both MRP1 and MRP2 are N-glycosylated in their mature form and have an apparent molecular mass of about 190 kDa. MRP1 and MRP2 have a similar core structure to that of MDR, i.e. an internally duplicated structure of two cytosolic nucleotide ATP-binding sites and two putative six transmembrane segments.
Fig. 1.7. A topology model of MRP1. The trans membrane helices labeled with a star are involved in substrate recognition.
In addition to an MDR1-like core, MRP1 and MRP2 contain an additional N-terminal segment of about 280 amino acids embedded with five transmembrane helices, while a small cytoplasmic loop (~ 80 amino acids) connects this area to the core. Studies revealed a special role of the small cytoplasmic loop region for the transport activity and the proper intracellular routing of the MRP1 and MRP2 proteins.
MRP1 and MRP2 are predominantly localized to the plasma membrane, particularly in cancer cells, with detectable levels present in intracellular membrane compartments of some cell types.
MRP1 is expressed at a low level in the liver, and the expression is restricted to the basolateral membrane in polarized cells. The expression of MRP2 is predominant in the
canalicular membrane of hepatocytes, and highly expressed MRP2 is found in the apical membranes of kidney proximal tubules 52.
MRP2 is predominantly expressed in the liver and to a lesser extent in the kidney, whereas none is detectable in the brain, heart, lung, testis or skeletal muscle 53, 54. In humans, MRP1 appears to be more ubiquitously distributed than MRP2; it has been detected in several types of epithelia, lung bronchioles, smooth and skeletal muscle cells, and in the heart, adrenal cortex, epidermis, salivary gland ducts and alveolar macrophages. This generalized tissue distribution makes it difficult to evaluate normal functions of MRP1. Because of the presence of MRP1 in many epithelia, MRP1 may have an excretory function in protecting the organism against xenobiotics. Recent evidence indicates that subcellular MRP1 is active but its physiological function in this subcellular location, if any, is not well understood 55. In addition, MRP1 expression is detected in almost every tumor including lung, gastrointestinal and urothelial carcinomas, neuroblastomas, gliomas, retinoblastomas, melanomas, tumors of the breast, endometrium, ovary, prostate and thyroid, as well as in hematological malignancies. MRP1 is particularly highly expressed in the major histologic forms of non-small cell lung cancer. MRP2, described as the canalicular multispecific organic anion transporter, is also expressed at the renal proximal tubules and small intestinal villi 56. MRP1 and MRP2 proteins play an important physiological role in the protection of the body against xenobiotics occurring in the environment, which is accomplished by active efflux of these toxic agents.
Therefore, inhibition of MRP1 would not probably lead to severe abnormalities. However, it cannot be excluded that humans lacking MRP1 might show a phenotype due to a broad exposure to toxins in food and the use of various drugs. High level of MRP1 in the oropharyngeal mucosa protects against drug-induced oral mucosit is caused by direct damage of the epithelium of the tongue and cheek 57, 58. MRP1 deficient also exhibit markedly increased damage of bone marrow, oropharyngeal mucosal surfaces, and the testes by cytotoxic drugs and impaired inflammatory response 59, 60, 57. A high level of MRP1 in the epithelium of the urinary collecting ducts protects against diabetes insipidus, and in the basal plasma membrane of the epithelium of the testicular tubules and sertoli cells, it protects against the abrogation of spermatogenesis.
MRP1 contributes to another barrier, the blood-CSF (cerebrospinal fluid) barrier together with P-gp, which separates the brain from the spinal cord and prevents many compounds from moving from the blood stream into the CSF. In fact, MRP1 is an important contributor to the blood-tissue barrier since high levels of MRP1 have been detected in the choroid plexus. The absence of MRP1 results in a 10-fold higher transport of compounds into the CSF, whereas the plasma and brain concentration was not significantly different 58, 61.
Transport across the hepatocyte canalicular membrane into bile is a decisive step in the elimination of endogenous and xenobiotic substances from the mammalian organism. Transferases in the hepatocyte catalyze the conversion of many of these substances into anionic conjugates with glutathione, glucuronate, or sulfate. Excretion of these conjugates into bile is mediated by MRP2. Functional studies in normal and transport-deficient mutant rats indicated a broad spectrum of endogenous and xenobiotic anions as substrates for MRP2, including many glutathione S-conjugates, glucuronides, and sulfate conjugates. In the liver, MRP2 plays an important role in the biliary excretion of multiple conjugated and unconjugated organic anions across the canalicular apical membrane. Another physiological role of MRP2 is to maintain the homeostasis of reduced folates. MRP2 provides a major route for the secretion of organic anions from the liver, and rats and humans lacking this transporter develop a mild liver disease, mainly due to the inability of the liver to excrete bilirubin-glucuronides.
1.6. Breast cancer resistance protein (BCRP)
BCRP was first discovered in a chemotherapy resistant breast cancer cell line, but there is no indication that its expression is specific for breast cancer cells or that BCRP should play a significant role in chemotherapy-resistance in breast cancer. The tissue distribution of BCRP shows extensive overlap with that of P-gp, suggesting that both transporters similarly confer protection from potentially harmful xenobiotics in various tissues.
In the brain, BCRP has been detected in capillary endothelial cells of pigs, mice, and humans, mainly at the luminal surface. Based on mRNA analysis, BCRP was more strongly expressed at the BBB than Pgp or MRP1.
It might also play a role in regulating stem cell differentiation. BCRP is involved in multidrug resistance in cancer, especially with regard to acute myeloid leukemia 62. When overexpressed in cell lines, BCRP has the ability to confer high levels of resistance to anthracyclines, mitoxantrone, bisantrene, and the camptothecins topotecan.
The mechanism by which substrates are recognized by BCRP and how the energy of ATP hydrolysis is transduced into transport is unclear 63. BCRP is expressed abundantly in the placenta, breast, as well as in liver, intestine, and stem cells. This transporter has several substrates in common with P-gp and may pose an additional barrier to drug access to the brain 64, 65.
1.6.1. Structure and cellular localization
BCRP is a plasma membrane glycoprotein, in polarized cell types localizing to the apical regions 66, 67. BCRP is extensively glycosylated (from among the three predicted N-glycosylation sites) on asparagine-596, which is located within the third extracellular loop of the polypeptide 68, 69, 70. The extent of glycosylation of BCRP is variable in different tissues, but it has been clearly established that BCRP glycosylation, similarly to that found for P-gp or MRPs, is not required either for the proper expression, function, or routing of this protein. Despite the lack of glycosylation, BCRP could be functionally expressed both in insect cells
71, 72 and in Lactococcus bacteria 73, and the removal of the predicted glycosylation sites did
not modify either the membrane localization or the transport function of the protein in mammalian cells 68, 70.
An interesting feature of the BCRP protein that its structure and/or dimer form is stabilized by S-S bridges within the third extracellular loop of its transmembrane domain.
Recent biochemical studies established that alterations or removal of the NH2-terminal 5–10 amino acid regions are not harmful for BCRP localization or function, while the COOH terminus (which, according to the membrane topology model has only a few amino acids
outside the transmembrane region) is highly sensitive. Decoration of this area by conventional tags (e.g., 10-His or GFP) drastically reduces transport function and in most cases alters the membrane localization of BCRP (370 and C. Ozvegy-Laczka and B. Sarkadi, unpublished experiments).
1.6.2. Transport properties
BCRP is an active transporter for many different drugs and metabolites, by extruding these compounds from the cells through a process energized by ATP hydrolysis. The transported substrates of ABCG2 include many cytotoxic drugs, their partially detoxified metabolites, toxins, and carcinogens found in food products, as well as endogenous compounds. Similarly to all ABC multidrug transporters, drug extrusion by BCRP is closely coupled to a drug-stimulated, vanadate-sensitive ATPase activity, which requires the presence of Mg2+ 71. The direct, ATP-dependent transport of several less-hydrophobic substrates of BCRP, e.g., methotrexate 74, 75, glucuronidated methotrexate, or sulfated estrogens and xenobiotics 76, 77, 78, 75 has been directly demonstrated in insideout membrane vesicles.
The substrate transport and ATP cleavage cycle of BCRP has not been investigated as yet in such detail as for P-gp or MRP1, but we suspect no major differences in the basic steps. In the case of BCRP we have the advantage of having a specific, high-affinity inhibitor molecule. Rabindran and co-workers 79,80 first observed that a micotoxin, Fumitremorgin C, was a strong inhibitor of drug resistance in BCRP-expressing tumor cells, and Koomen and co-workers 81 developed derivatives of this molecule with potent and selective inhibitory action on BCRP. One of these molecules, Ko143, inhibits BCRP in nanomolar concentrations, whereas it has practically no effect on other multidrug transporters or on cellular functions up to micromolar levels. ATP cleavage and the transport cycle, as in all ABC multidrug transporters, seem to be coupled to events in the BCRP protein.
1.6.3. Dimer formation
According to our current knowledge the minimum requirement for a functional ABC transporter is the cooperation of two ABC and two transmembrane domains; thus the functional form of BCRP must be at least a dimer. Most experimental data indicate that in the case of BCRP, the prevalent form is a homodimer; at least a homodimer can carry out all the relevant transport functions. In drug-selected cell lines, the upregulation of the BCRP gene expression alone was sufficient to cause drug resistance 82, 83, and mtransfection by the BCRP cDNA alone produced massive drug resistance in various mammalian cells 84.
Homodimerization of the BCRP protein was directly demonstrated by using nonreducing SDS gels, where a disulfide-bridged dimer of BCRP was found to occur 85, 86, 87. Due to the reducing intracellular environment, such a physiological disulfide link can only be formed between SH groups present on the extracellular loops of the protein. Indeed, three cysteines, conserved in most mammalian ortholog BCRP sequences in the third extracellular loop, were found to play a key role in the dimerization, expression, and localization of this protein
88, 87
. According to our unpublished studies (O¨ zvegy-Laczka and Sarkadi, unpublished data), chemical cross-linking of BCRP dimers may still preserve transport function, while various modifications of the cell surface S-S bridges may result in the loss of BCRP transport activity.
Several sequence motifs within BCRP have been suggested to influence BCRP function and dimerization 86, but a clear-cut picture has not yet been established in this regard. As suggested by one experimental study, higher order oligomerization of BCRP may be involved in the function of this protein 89.
1.7. P-glycoprotein (P-gp)
The first identified and so far best characterized multidrug resistance transporter is P-gp. P-gp is a typical ABC transporter protein composed of two homologous halves, each containing six transmembrane (TM) domains and an ATP-binding/utilization domain, separated by a flexible linker region. The interaction of the two halves of P-gp is critical for
the functioning of the molecule, and a flexible linker region is sufficient for the proper interaction of the two halves, most likely for the communication between the two ATP sites. The two halves of human P-gp interacts to form a single transporter, and the major drug-binding domains reside in transmembrane domains 4, 5, 6, 10, 11, and 12.
Both ATP sites are capable of hydrolysing ATP but not simultaneously; the stoichiometry of ATP hydrolysis and drug transport are obligatorily linked.
P-gp is glycosylated at three sites in the first extracellular loop. The glycosilation appears to be required for the proper trafficking of the transporter to the cell surface, but is not required for the transport function of P-gp.
Fig. 1.8. A topology model of Pgp. The trans membrane helices labeled with a star are involved in substrate recognition.
The resolved structures and the developed homology models, as well as the many other studies carried out on the structure of extruding pumps, do show that the recognition sites of P-gp are large, flexible and rich in amino acids able to give productive interactions with substrates. In particular, the recognition sites appear to contain an unusual number of
aromatic amino acids that, besides electrostatic bonds, are able to produce hydrophobic and π-stacking interactions 90-95.
These findings, suggest an amazingly simple answer to some crucial questions that have bothered scientists since the discovery of extrusion proteins: how do these proteins recognize so many different substrates? Do they contain several separated drug-recognition sites or a single, promiscuous one? If so, what is its nature? To answer to these questions, it was proposed that P-gp presents two or even three distinct substrate binding sites with distinct but overlapping substrate specificity 96-101. However, at present, it seems unrealistic to think that the binding sites of these transport proteins are multiple and scattered along the whole molecule 102.
As quite a number of tumors express P-gp, multidrug resistance may be very important with regard to the development of resistance to chemotherapy. P-gp is also expressed in a variety of normal tissues such as the epithelial cells of the liver, the kidney, the choroids plexus and the intestine, and the capillary endothelial cells of the brain 103-106. AlthoughPgp is predominantly in the cell membrane and acts as a drug efflux transporter in diverse cell types, morerecent studies have shown that it is also expressed in intracellular compartments.
P-gp is localized incytoplasmic vesicles, in which is oriented so that drugs are transported and concentrated in the interior of the vesicles, leading to sequestration of drugs away from their subcellular targets.
Numerous investigations with drugs such as cyclosporine, digoxin, domperidone, etoposide, loperamide, ondansteron, taxol, and vinblastine have demonstrated that P-gp has an important role in determining the concentration–time profiles of P-gp substrates in the different parts of the body 107, 108.
Most of these P-gp substrates are also substrates of the major drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). This explains that competitive inhibitors of P-gp, which are transported by P-gp, also inhibit CYP3A4.
Figure 1.9.. Homology model ofP-gp. A) Face views (front and side); the protein backbone is colored by the secondary structure; the ADP molecules and Na ions are presented in space filled form and colored according to the atom types (C: gray; O: red; N: blue, Na: big blue spheres); B) A membrane incorporated model with the electrostatic potential mapped on the Gaussian contact surface of the structure: red–negative values; blue-positive values; gray- neutral (close to zero); the POPC lipids are orange.
1.7.1. P-gp at the level of blood-brain barrier (BBB)
Drug delivery to the brain is often hindered by a barrier system, namely, the blood-brain barrier (BBB). The BBB consists of capillary endothelial cells with tight junctions that lack small aqueous pores. Astrocytes, pericytes, and the extracellular matrix components are believed to control the integrity of the BBB 109. In human adults, the vascular capillaries account for about 1% of the brain volume and the surface area of capillary endocellular cells is estimated to be approximately 10m2.
Fig. 1.10. Schematic comparison of a brain capillary with a capillary in the periphery (modified from Kandel et al., 2000).
The functional role of the BBB is to protect the brain against toxic xenobiotics by separating the brain from the cerebral blood circulation. As a result, only lipophilic xenobiotics and drugs can cross the barriers and enter the brain by way of passive diffusion. Studies by many investigators have shown that there is a strong positive correlation between lipophilicity and brain penetration of drugs 110, 111.
Although lipophilicity is an important factor in determining the brain penetration of drugs, many lipophilic drugs exhibit poor brain penetration. Moreover other factors than lipophilicity may also play an important role in the transport of drugs across the brain. It has been suggested that hydrogen bonding may also influence brain penetration. A negative correlation was found between the brain penetration of lipophilic compounds and the total number of hydrogen bonds; derivatives which could interact with many hydrogen bonds posse lower permeability 112. In addition to lipophilicity and number of hydrogen bonds, brain penetration studies in animals have revealed that the molecular size of drugs is also an important determinant for brain penetration 113.
However, the poor brain penetration of some lipophilic drugs still cannot be explained by factors such as hydrogen bonds and molecular size alone. Without knowing an exact cause, these compounds were regarded as ‘‘outlier compounds’’. No connection was made between the possible efflux function of P-gp in the BBB and the poor brain penetration of these lipophilic compounds observed in animals until the findings of P-gp in brain capillaries 114,
115
. Using monoclonal antibodies, they demonstrated that P-gp was highly expressed on the apical surface of the endothelial cells of the brain capillaries. Recognizing these findings, it is now clear that the observed poor brain penetration of some lipophilic drugs is due mainly to the efflux transport of P-gp.
Thus, P-gp substrate drugs entering the endothelial cells from the blood are immediately pumped back into the blood. As a consequence, the net penetration of substrate drugs and other substrate compounds from the blood into brain tissue can be strikingly decreased under physiological conditions.
The function of P-gp as a BBB efflux transporter is especially impressive for drugs that exhibit pronounced CNS side effects, when they enter the brain.
The brain is known to be a site of viral replication for the human immunodeficiency virus (HIV) and thus is an important target tissue for antiretroviral agents. However, HIV protease inhibitors, which brought considerable progress in the treatment of HIV infection, have only limited ability to reach the CNS, with the majority of this class of drugs not detected in human CSF after administration 116, 117.
First generation antihistamines are often associated with CNS side effects such as sedation, whereas second generation antihistamines are generally non-sedating. In an in vitro study using rat brain endothelial cells, all second generation antihistamines tested proved to be substrates of P-gp, whereas all first generation antihistamines tested showed no affinity for P-gp 118, 119. Based on these data, it was postulated that P-gp mediated efflux at the BBB explains the lack of CNS side effects of modern antihistamines.
P-gp seems to be involved in immuno-modulation by modulating the secretion of several cytokines from normal peripheral T lymphocytes and the migration of dendritic cells from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity 120.
Other data from different laboratories have indicated a drug efflux-independent role for P-gp in the inhibition of apoptosis 121-126. Modulation of cytokine efflux, signalling lipids and intracellular pH have all been suggested as ways by which P-gp may affect cellular resistance to apoptosis 123.
Upregulation of P-gp in astrocytes and neurons as observed in neurodegenerative brain diseases such as epilepsy might represent an important adaptive mechanism that protects surviving brain cells from cytotoxicity by endogenously released compounds, for instance the excitatory and excitotoxic neurotransmitter glutamate that is a substrate for P-gp 127. In contrast to normal astrocytes, Pgp-overexpressing astrocytes from brain tissue of epileptic patients did not express the pro-apoptotic proteins p53 and p21 and other apoptotic markers, substantiating a role of P-gp in the protection from apoptotic cell death 125.
Another recently reported function is that P-gp acts as a β-amyloid efflux pump with a potential role in the pathogenesis of Alzheimer’s disease. In glial cells, ABC proteins such as P-gp and several MRPs are involved in nucleotide efflux, which represent the main source of cerebral extracellular purines 128.
In addition, P-gp has been implicated in the export of endogenous brain peptides, including opioid ligands, from the brain to peripheral sites of action 129 and in the mechanisms involved in the development of morphine tolerance 130.
1.7.2. P-gp at the level of placenta
Placenta is the organ that brings into close apposition the blood circulations of two human beings, mother and fetus, while maintaining separation of the two blood systems. It performs many functions essential for the maintenance of pregnancy and normal development of the fetus. One of the placenta’s major roles is to regulate the exchange of nutrients and gases between mother and fetus and to remove fetal waste products; placenta is also considered to be the first fetal organ exposed to exogenous substances including drugs. It is generally accepted that any chemical substance administered to mother is able to permeate, to some degree, across the placenta 131, 132. Therapeutic compounds cross the placenta depending on their lipid solubility, molecular size, degree of ionization and plasma protein binding. Non-ionized and lipid molecules with molecular weight up to 600 Da cross the placenta via passive diffusion. Drugs and xenobiotics that are structurally related to endogenous compounds can be additionally recognized as substrates of nutrient transporters like monoamine, carnitine, nucleoside and organic ion transporters133,134.
Fig. 1.11. Schematic structure of the human placenta. (A) Cross section of the uterus at the term of pregnancy showing fetus connected with the placenta via the umbilical cord. In the detailed schema, structure of cotyledon, placental functional unit, is depicted. Chorion, the fetal part of the placenta, consists of chorionic plate and chorionic villi that are washed by maternal blood entering intervillous space through spiral arteries in decidua basalis. The oxygen and nutrients from maternal blood cross the surface trophoblast layer of chorionic villi, enter the fetal blood and are brought to the fetus via umbilical vein (red). The deoxygenated blood is conducted from the fetus through two umbilical arteries (blue). (B) Hematoxylin–eosin stained paraffin sections of terminal villi in human third trimester placenta (microphotograph courtesy of Dr. Nachtigal). (C) Schematic description of the terminal villi section showing localization of P-gp at the apical microvillous membrane of syncytiotrophoblast and presence of other placental drug efflux transporters.
Over the last decade, increasing effort has been devoted to investigation of ABC drug efflux proteins in the placenta, including P-glycoprotein (P-gp, MDR1, encoded by ABCB1), multidrug resistance-associated proteins (MRPs encoded by ABCC1-6 and
ABCC10-12) and breast cancer resistance protein (BCRP, encoded by ABCG2). Some of these transporters were originally found to be associated with multidrug resistance in cancer cells, but are currently recognized also for their substantial role in modulation of drug absorption, distribution and metabolism 135-137.
Immunohistochemical study with three different monoclonal antibodies detected P-gp in the syncytiotrophoblast microvillous border of the human first-trimester placentas 138, implying presence of P-gp over the whole period of pregnancy. However, later immunohistochemical studies confirmed intense staining for P-gp in the trophoblast layer of human term placenta
139,140
.
On the other hand the decrease of P-gp in human placenta with advancing gestation was demonstrated by recent work of Sun et al. 141.
From the above studies, it seems obvious that P-gp is present in the placenta even at the early stages of pregnancy and changes up to the term, which may influence disposition of xenobiotics to the fetus.
Based on the placental expression, P-gp was expected to play a role in protection of fetus against toxic xenobiotics. The first study confirming functional activity of P-gp in the placenta was carried out by Nakamura et al. 140, who demonstrated P-gp-mediated uptake of vincristine in the membrane vesicles prepared from trophoblast of human placenta.
P-gp is likely to provide protection to the fetus by limiting the penetration of xenobiotics from maternal to the fetal circulation and moreover, it helps remove its substrates back to the maternal compartment once they have reached the fetal circulation.
Hormonal regulation of pregnancy is associated with increased concentrations of estrogens and progesterone in the third trimester. Recently, expression of human P-gp and its rodent orthologues has been proposed to be controlled by estrogens and progesterone both in in vivo and in vitro models142, 143.
Human P-gp expression in the endometrium has been found to correspond with that of nuclear progesterone receptor and plasma and tissue levels of progesterone [97]. Similarly, Arceci et al. demonstrated that P-gp is induced at high levels in the uterine secretory epithelium by the combination of estrogen and progesterone 144.
1.7.3. Potential role of P-gp and MRP1 at the blood-cerebrospinal fluid barrier
There is a shift in the appreciation of the contribution of the BCSFB cerebrospinal fluid barrier) to drug transport between blood and brain. The BBB (blood-brain barriers) has been considered to be the most important barrier, because its surface is thought to be about 3 orders of magnitude larger than that of the BCSFB. However, the characteristics of the BBB and BCSFB are different, and therefore, relative contributions of the two barriers in the exchange of compounds between blood and brain cannot be judged solely on the ratio of the respective surfaces of these blood–brain barriers. The BCSFB is considered to have a significant role in the exchange of xenobiotics between blood and brain for the following reasons 145.
First, the organization of the tight junctions of the epithelial cells of the choroid plexus is parallel by sparsely interconnected strands, which makes these tight junctions slightly more permeable than those between the brain endothelial cells.
Second, the choroidal epithelial cells contain a high level of a number of detoxifying metabolizing enzymes.
In fact the BCSFB has a relatively high expression level of several cytochrome P450 isoenzymes.
Third, the BCSFB can also rapidly and specifically handle the efflux of a quite a number of xenobiotics.
Fig. 1.12. Schematic representation of the blood–cerebrospinal fluid (blood–CSF) barrier.
Actually, the presence of P-gp and MDR1 expression at the choroid plexus have been indicated. It was concluded that P-gp localizes subapically at the choroid plexus epithelium, with transport into the direction of the CSF (celebrospinal fluid). Conversely, MRP localizes basolaterally, conferring transport to the blood side of the epithelial cells.
Nishino et al. 146found a four- to five fold higher level of MRP1 expression at the choroid plexus compared with that in the lung, one of the tissues exhibiting high expression of MRP1. This indicated the presence of MRP in the choroids plexus, at a basolateral position based upon the direction of the transport.
In the choroidal epithelium, P-gp and MRP1 seem to have opposite transport directions. This indicates different situations for compounds that are (a) substrates for both P-gp and MRP1, (b) substrates for P-gp alone, (c) substrates for MRP1 alone without need for a conjugation, and (d) substrates for MRP1 following conjugation (Fig. 1.13.a–d).
The compounds that are substrates for both P-gp and MRP1 (a) actually are cleared from the choroidal epithelial cells and may therefore protect these cells, thereby contributing to the detoxification of the choroid plexus itself 147. Such compounds may be etoposide and
colchicin, but so far, no report has indicated an increased vulnerability of the choroidal epithelial to toxicity of these compounds.
For compounds that are substrates for MRP1 alone without need for a conjugation (b), failure of MRP1 efflux will lead to a higher net CSF influx of potentially toxic compounds. In fact, the opposite will occur upon failure of P-gp-mediated transport at the choroid plexus, namely, a decrease in CSF concentrations for those compounds that are in fact selective substrates to P-gp (c). Under normal conditions, P-gp at the choroid plexus would contribute to higher concentrations of its substrates in the CSF, while P-gp mediated efflux at the BBB counteracts the distribution into the rest of the brain.
For compounds that need to be conjugated before being transported by MRP1 (d), there is a need for a concerted action of glutathione metabolism and MRP1-mediated efflux. A failure in either MRP1 efflux or glutathione metabolism may lead to higher choroidal epithelial intracellular levels of potentially neurotoxic compounds. Under normal conditions, glutathione metabolism and MRP1 efflux act as a potential detoxification of the choroid plexus cells, but also contribute, via lowering of CSF concentrations, to the detoxification of mostly the periventricular organs of the brain.
Fig. 1.13. P-gp and MRP1 have opposite transport directions. This indicates different situations for compounds that are (a) substrates for both P-gp and MRP1, (b) substrates for P-gp alone, (c) substrates for MRP1 alone without need for a conjugation, and (d) substrates for MRP1 following conjugation.
1.8. Identification of P-gp binding regions and pockets
The low-affinity binding site is thought to be exposed to extracellular medium in the post-hydrolytic state (vanadate trapped) 148. In this state the protein is suggested to have already undergone the most essential conformational changes and might be in a transition from the “open to inside” to the “close to inside” conformation of the TMs, correspondingly the drug release must have already occurred.
Three main membrane-related binding regions in P-gp were outlined (Figure 1.15.A and B). Binding region 1 is located at the interface between the membrane and the cytosol and two other binding regions are located in the transmembrane parts of the protein.
The regions contain multiple binding pockets and involve amino acids from all TMs but also from other structural units of the protein. In the regions the pockets are close to each other (Figure 1.15.); thus it is possible that drugs, depending on their structural properties, may bind to either more hydrophobic or more hydrophilic pockets or even to more than one pocket simultaneously. Additionally, a big binding pocket has been found in the protein
cavity (Figure 1.15. C). Inspection shows that it involves residues from binding regions 1, 2, and 3 and may represent an “escaping” site, where the compounds that bind to any of these regions are released from the protein.
The analysis of the cavity illustrates that it maintains hydrophobic to neutral surface properties and could possess potential affinity for hydrophobic drugs (Figure 1.14.).
Figure 1.14. Lipophilic potentials of the P-gp model mapped on Connolly surfaces (A, B, and C) and Connolly channel (D, E, and F). Left: side view; Middle: front view; Right: top view (from outside, corresponds to side view); brown: highest hydrophobic area; blue: highest hydrophilic area; magenta: the Ca-chain of the protein.
Figure 1.15. Binding regions ofP-gp. A) Binding pockets identified by SiteID;[30] the pockets are filled with spheres set around the oxygen atoms of the water molecule; the same color is used for filling the spheres belonging to the same pocket. B and C) Binding pockets identified by Site Finder;[31] the pockets are filled with alpha spheres; gray spheres indicate hydrophobic atoms and red spheres, hydrophilic atoms; the protein backbone is shown as a blue tube; the membrane-related binding regions and the cavity pocket are outlined with dotted lines; the horizontal dotted lines show the approximate borders of the membrane.
Binding regions 2 and 3 contain pockets exposed simultaneously to the pore and to the membrane (Figure 1.17., 1.19.). Thus, it can be presumed that more hydrophobic drugs may also be released by the protein to the outer leaflet of the membrane. Analysis of the binding pockets revealed that they contain a large number of amino acids found to have an important impact on functioning of the protein in various mutational, cross-linking, and labeling experiments. The residues are shown to influence the transport activity for a number of substrates and inhibitors of P-gp, thus implying that the pockets identified might be reasonable. Unfortunately, from the experimental data no consensus conclusion about possible involvement of particular amino acids in the binding of particular drugs can be outlined.
Figure 1.17. Binding region 2 filled with alpha spheres: A) Side view from the membrane; B) Side view from the pore; C) Top view (from outside); gray spheres indicate hydrophobic atoms and red spheres hydrophilic atoms; the amino acids are shown in sticks and colored according to the atom types (C: gray; O: red; N: blue; S: yellow; the nonpolar hydrogens are hidden); the TM backbones are shown as a blue tube.
Figure 1.18. Binding region 3 with labeled amino acids shown in experimental studies to affect the transport function of P-gp; the orientation ofthe helices corresponds to Figure 5B; the residues are presented as balls and sticks (the nonpolar hydrogens are hidden).
In the absence of 3D structural data of a ligand bound to P-gp, it is difficult to precisely define the location of the binding sites. The presence of residues in the pockets which have not directly been proven to influence the transport function of the protein in experimental studies, does not necessarily mean that they are not involved in binding.
Residues that affect the transport of P-gp substrates and inhibitors (anthracyclines, Vinca alkaloids, colchicine, propafenones, rhodamine 123, verapamil, and their derivatives) have
been found in different binding regions. It is likely that big compounds, such as Vinca alkaloids, cyclosporine A, and their analogues, may interact with P-gp within the cytoplasmic compartment.
In this region, as suggested by Gruol et al. 149 initial interactions of smaller drugs with the cytoplasmic loops of the protein, may also take place and these interactions can further influence the interaction of the protein with the ATP molecules. According to the location of the labeled amino acids verapamil may have two binding sites in binding regions 2 and 3. However, it cannot be excluded that these substrates, similar to the other ones, may interact with the protein in binding region 1 and use the pore for release from the cell. It is feasible that small drugs may have binding sites in more than one binding region of the protein. These results are in agreement with the assumption that P-gp has multiple drug-binding sites which may behave differently 148.
In conclusion, P-glycoprotein has multiple binding sites and may bind and/or release substrates in multiple pathways. The binding regions and pockets identified may help further experimental studies, molecular modeling, and dynamics simulations that aim at a more precise location and identification of the protein binding sites.
1.9. The transport cycle of P-glycoprotein
Essentially there are two different models hypothesied to explain the mechanisms of P-gp transporter. The most significant difference between the two models is in the nature of the power stroke that drives the drug from a high affinity site to a low affinity site.
In one model (Fig. 1.20. A, Step II), the formation of the nucleotide sandwich dimer results in conformational changes that are communicated to the drug binding site 150.
Two sequential ATP hydrolysis events then reset the P-gp molecule (Steps IV–VI; Fig. 1.20. B). The alternate model 151-153 also requires two ATP hydrolysis events, but one powers the efflux of the drug (Fig. 1.20. B, Step II) and the other resets the protein to its ground
