Introduction

During the past decade the extensive involvement of ATP-binding cassette (ABC) transporters in the pharmacokinetics and pharmacodynamics of drugs has been revealed. These findings established transport proteins as key determinants for drug transport and disposition. Their ubiquitous expression in various organs, particularly those involved in drug excretion (e.g., the liver and kidney) and tissue protection (e.g., the blood-brain barrier, BBB) suggest important physiological implications. Moreover, common pathways involving the same transporters have been identified for many drugs and essential nutrients. These findings point to the involvement of transport proteins in fundamental cellular processes, thus making them extremely significant subjects of investigation. ABC transporters are, furthermore, implicated in the development of de novo or acquired multidrug resistance (MDR) in many human malignancies. Through high overexpression in tumor cells, ABC transporters actively extrude chemotherapeutic drug substrates and reduce their concentration to subeffective intracellular levels, which in many cases results in failure of the cancer therapy. Investigational efforts have two significant aims: (1) the better understanding of the basic biological mechanisms involved and (2) the promotion of therapeutic interventions in medical treatments through identification of novel targets.

Independently of the progress achieved in recent years, the structure-function relationships of most transport proteins are unsolved, and their interactions with various ligands are poorly characterized at the molecular level. This makes it necessary to apply a more operative strategy of investigation that effectively combines experimental and in silico approaches.

This chapter presents an overview of the current approaches for molecular modeling of different transport proteins. As P-glycoprotein (P-gp) is the best characterized and the most significant MDR transport system, it will be in the main focus of discussion.

5.32.2 ATP-Binding Cassette Transporter Family

5.32.2.1 Physiological Significance and Role in Drug Distribution

The importance of drug transporters for intracellular drug concentration was discovered in tumor cells showing an MDR phenotype.1 The first MDR transporter, P-gp, initially discovered in resistant CHO cells in 1976,2 was found to be expressed in various resistant tumor cell lines of animal and human origin.3 To date, P-gp overexpression is thought to be mainly responsible for MDR in cancer drug therapy. The protein is, furthermore, naturally expressed in many tissues, especially those with barrier functions such as liver, BBB, kidney, intestine, and placenta (see 5.04 The Biology and Function of Transporters).

P-gp (the product of the human gene ABCB1) belongs to the highly conserved superfamily of ATP-binding cassette (ABC) transporters. Several hundred compounds have been reported to be its substrates and/or inhibitors. They belong to various chemical classes, and generally do not share structural homology. The characteristic features that relate a P-gp compound to the group of the substrates or inhibitors are currently not well defined.

Table 1 lists well-known P-gp substrates and inhibitors. Among the compounds listed, verapamil, trifluoperazine, and propafenone represent ones of the best studied first-generation MDR modulators. Verapamil is used as a reference compound in most MDR studies, a fact that can be explained by historical rather than scientific reasons. The intensive search for more specific and less toxic MDR modulators led to the development of second- and third-generation MDR modulators, some of which are shown in Figure 1.

Similarly to P-gp, several proteins encoded by genes of the subfamily C of the ABC transporters also transport a large variety of substrates. These transporters are named MDR-associated proteins (MRPs). The first subfamily member MRP1 (ABCC1) was discovered in 1987. In small quantities it is present in almost all mammalian cells, and it is expressed in the sinusoidal membrane of liver hepatocytes. MRP1 functions as a multispecific organic anion transporter. Both P-gp and MRP1 confer resistance to a similar but not identical spectrum of cytotoxic drugs.5'6 MRP1 transports cytotoxic drugs and other hydrophobic compounds only in the presence of glutathione. MRP2 (ABCC2) is expressed on the canalicular (apical) side of the hepatocyte, and promotes biliary transport of anionic conjugates with glutathione, sulfate, or glucuronic acid on one hand, and anticancer drugs such as vinblastine on the other hand. Overexpression of MRP2 is associated with cisplatin resistance in tumor cells.7 MRP3 is expressed in the intestine and liver, but little is known about its importance for drug distribution. MRP3 transports glucuronated metabolites of drugs.8 Its substrate spectrum appears to be much narrower than those of MRP1 and MRP2. The human MRP4 and MRP5 proteins are organic anion transporters that transport cyclic nucleotides and some nucleoside monophosphate analogs, including nucleoside-based antiviral drugs. MRP4 also transports prostaglandins.9

Table 2 summarizes compounds shown to interact with MRP family members that are expressed at the BBB and therefore could have implications for brain penetration of central nervous system (CNS)-active drugs.

A novel member of the ABC transporter superfamily is breast cancer resistance protein (BCRP) (ABCG2).54 It is expressed in the intestine, the bile canicular membrane, and the placenta.55 BCRP has been shown to play a role in the absorption and secretion of clinically important drugs. Table 3 lists substrates and inhibitors of BCRP. This transporter shows wide substrate recognition properties, including neutral, positively, and negatively charged compounds.

In summary, several ABC transporters, alone or in combination, are responsible for MDR and for the absorption, distribution, and elimination of drugs in humans. The next section presents a summary of experimental results on the structure and functioning of different transporters. The results are discussed in relation to their usefulness for in silico studies of the transport proteins, particularly P-gp.

5.32.2.2 Experimental Studies of P-Glycoprotein and Related Transporters

5.32.2.2.1 Structural data

High-resolution structures of human MDR proteins from the ABC family have not yet been resolved. However, x-ray structures of several other transporters have recently become available. These structures include MsbA from Escherichia colt90 and Vibrio cholerae,91 obtained in the absence of nucleotide at 4.5 A and 3.8 A resolution, respectively, and E. coli BtuCD resolved at 3.2 A.92

The lipid transporter MsbA is phylogenetically close to P-gp and the bacterial multidrug transporter LmrA from Lactococcus lactis; all believed to act as flipases. MsbA is formed of two monomers, each containing six a helical transmembrane domains (TMDs) and a nucleotide-binding domain (NBD) linked by an intracellular domain. The homodimer has 12 transmembrane a helices (six per monomer). MsbA closely resembles the topological organization of P-gp and the degree of sequence homology between both proteins approaches 30%.

Table 1 Main groups of P-gp substrates and inhibitors

Class

Anticancer drugs

Cytotoxic agents

Calcium channel blockers

HIV protease inhibitors

Steroids

Hydrophobic peptides

Other compounds

P-gp substrate/inhibitor

Anthracyclines (doxorubicin, daunorubicine, epirubicin)

Epipodophyllotoxins (etoposide, teniposide)

Taxanes (paclitaxel, docetaxel)

Vinca alkaloids (vincristine, vinblastine)

Actinomycin D

Topotecan

Colchicine

Puromycin

Dilthiazem

Nicardipin

Verapamil

Indinavir

Ritonavir

Saquinavir

Dexamethasone

Methylprednisolon

Progesterone

Testosterone

Cyclosporin A

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