P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization timeof-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino-and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.Multidrug resistance represents a serious obstacle to successful cancer chemotherapy. Although multifactorial in etiology, one type of multidrug resistance is associated with the overexpression of energy-dependent membrane-bound pumps, which intercept and efflux drugs before they reach their intracellular target structures. P-glycoprotein (ABCB1) represents a paradigm ATP-dependent efflux pump expressed in human cancer cells. In addition to its expression in cancer cells, P-gp is also physiologically expressed in a number of tissues such as intestinal epithelial cells, at the brush border of renal tubule epithelial cells, the canalicular side of hepatocytes, and in capillary endothelial cells forming the blood-brain barrier. It thus interferes with oral drug absorption and drug delivery to the brain, and it enhances renal and biliary excretion. P-gp has therefore attracted considerable attention as a nontarget in the field of drug development, because for a large number of active compounds, interaction with P-glycoprotein might compromise their future development into a drug. Considerable energy has therefore been devoted to the characterization of molecular features that make compounds P-gp substrates and to the definition of the molecular mechanism of drug transport by P-gp. A number of studies have dealt with the kinetics and thermodynamics of the transport process

The aim of the present review is to summarize recent progress in identifying substrate binding domains of P-glycoprotein by photoaffinity labeling. Preferred substrate binding regions have been identified using a number of photoaffinity ligands, including anthracyclines, the quinazoline iodoarylazidoprazosine (IAAP), dihydropyridines, taxanes and propafenones. These studies allowed identification of protein regions, which are involved in ligand interaction.

In line with our studies on propafenone-type inhibitors of P-glycoprotein (P-gp), we applied several methods to approach virtual screening tools for identification of new P-gp inhibitors on one hand and the molecular basis of ligand-protein interaction on the other hand. For virtual screening, a combination of autocorrelation vectors and selforganising artificial neural networks proved extremely valuable in identifying P-gp inhibitors with structurally new scaffolds. For a closer view on the binding region for propafenone-type ligands we applied a combination of pharmacophore-driven photoaffinity labeling and protein homology modeling. On LmrA, a bacterial homologue of P-gp, we were able to identify distinct regions on transmembrane helices 3, 5 and 6 which show significant changes in the labeling pattern during different steps of the catalytic cycle.

aLipophilicity is one of the major determining physicochemical descriptors for P-glycoprotein (P-gp) inhibitory activity. Recently, Pajeva and Wiese showed a Department of that in case of P-gp interaction, lipophilicity may be regarded as space-directed Pharmaceutical Chemistry, property. In the present study, a series of propafenone-type P-gp inhibitors with University of Vienna, systematically varying hydrophobicity distribution within the molecules were synWien, Austria thesised and pharmacologically tested. QSAR studies on the basis of multiple b Department of Medical linear regression analysis showed that with increasing lipophilicity of the Chemistry, University of substituents on the amine moiety, the statistical significance of the indicator variVienna, ables, denoting the substitution pattern on the central aromatic ring system, also Wien, Austria increases. This indicates that the distribution of hydrophobicity within the molecules influences the mode of interaction with P-gp.Keywords: Multidrug resistance; P-Glycoprotein, Propafenone; Hydrophobicity distribution IntroductionThe phenomenon of multidrug resistance (MDR) has become a major obstacle in the treatment of cancer with chemotherapeutic drugs. The development of broad specificity mechanisms of resistance to multiple classes of drugs is strongly associated with the overexpression of membrane-bound drug efflux pumps such as P-glycoprotein (P-gp) [1]. P-gp is a 170-kDa membrane protein that belongs to the family of ABC (ATP-Binding Cassette) transporters and functions as an ATP-dependent efflux protein for a large variety of structurally and functionally diverse drugs and natural products. These include anthracyclines, epipodophyllotoxines, actinomycin D, vinca alkaloids, colchicine and taxol [2]. All ABC transporters share a common architecture consisting of four domains. Two transmembrane domains form a pathway across the membrane through which solutes can move. . The low-resolution three-dimensional structure of P-gp in the presence and absence of ATP has recently been resolved at 10-Å resolution by electron cryomicroscopy of negatively stained crystals [12]. Very recently, two protein homology models of P-gp on the basis of the X-ray structure of MsbA were published [13,14]. However, both the detailed mechanism of transport and the ligand/protein interaction remain unresolved up to now.It has been shown that drugs with the ability to inhibit P-gp lead to resensitisation of resistant tumour cells. Among them are numerous structurally and functionally diverse drugs, such as verapamil, dihydropyridines, phenothiazines, thioxanthenes, amiodarone, and even flavonoids, steroids and detergents. In an attempt to systematically explore structure-activity relationships within the class of P-gp inhibitors, we used the class Ic anti-arrhythmic agent propafenone as template ( Figure 1A).Our results obtained so far show that pharmacophoric substructures such as H-bond acceptors and one or more aromatic rings seem to be important for the biological activity....