We recently reported the synthesis and quantitative structure–activity relationships of a new breast cancer resistance protein (BCRP) inhibitor class. In the study presented herein, we investigated the possibility to better define the scaffold of this compound class by removing or modifying the aromatic ring A with various substituents selected on the basis of their electronic and lipophilic properties. The results show that this aromatic ring is important, but not essential, for activity. Many of the selected substituents led to compounds with low activity, but in some cases activity was retained. Among these, a phenolic hydroxy group proved to impart as much potency to the molecule as a hydroxyethyl side chain, initially considered necessary for activity. This derivative is one of the most active compounds in this class, maintaining an inhibitory activity similar to that of the reference compound; it is also selective for BCRP.

Tariquidar and elacridar are among the most potent inhibitors of the multidrug resistance transporter P-glycoprotein (P-gp), but how they interact with the protein is yet unknown. In this work, we describe a possible way in which these inhibitors interact with P-gp. We rely on structure–activity relationship analysis of a small group of tariquidar and elacridar analogues that was purposefully selected, designed, and tested. Structural modifications of the compounds relate to the presence or absence of functional groups in the tariquidar and elacridar scaffolds. The activity of the compounds was evaluated by their effects on the accumulation of P-gp substrates rhodamine 123 and Hoechst 33342 in resistant tumor cells. The data allow estimation of the ability of the compounds to interact with the experimentally proposed R- and H-sites to which rhodamine 123 and Hoechst 33342 bind, respectively. Using an inward-facing homology model of human P-gp based on the crystallographic structure of mouse P-gp, we demonstrate that these binding sites may overlap with the binding sites of the QZ59 ligands co-crystallized with mouse P-gp. Based on this SAR analysis, and using flexible alignment and docking, we propose possible binding modes for tariquidar and elacridar. Our results suggest the possibility for the studied compounds to bind to sites that coincide or overlap with the binding sites of rhodamine 123 and Hoechst 33342. These results contribute to further understanding of structure–function relationships of P-gp and can help in the design of selective and potent P-gp inhibitors with potential clinical use.

The breast cancer resistance protein (BCRP/ABCG2) is a member of the ABC transporter superfamily. This protein has a number of physiological functions, including protection of the human body from xenobiotics. The overexpression of BCRP in certain tumor cell lines causes cross-resistance against various drugs used in chemotherapeutic treatment. In a previous work we showed that a new class of compounds derived from XR9576 (tariquidar) selectively inhibits BCRP. In this work we synthesized more members of this class, with modification on the second and third aromatic rings. The inhibitory activities against BCRP and P-gp were assayed using a Hoechst 33342 assay for BCRP and a calcein AM assay for P-gp. Finally, quantitative structure–activity relationships for both aromatic rings were established. The results obtained show the importance of the electron density on the third aromatic ring, influenced by substituents, pointing to interactions with aromatic residues of the protein binding site. In the second aromatic ring the activity of compounds is influenced by the steric volume of the substituents.

The primary aim of this work was to analyze the contacts between residues in the nucleotide binding domains (NBDs) and at the interface between the transmembrane domains (TMDs) and the NBDs in the inward-open homology model of human P-glycoprotein (P-gp). The analysis revealed communication nets through hydrogen bonding in the NBD and at the NBD–TMD interface of each half involving residues from the adenosine triphosphate (ATP) motifs and the coupling helices of the intracellular loops. Similar networks have been identified in P-gp conformations generated by molecular dynamics simulation. Differences have been recorded in the networking between both halves of P-gp. Many of the residue contacts have also been observed in the X-ray crystal structures of other ATP binding cassette (ABC) transporters, which confirms their validity. Next, possible binding pockets involving residues of importance for the TMD–NBD communication were identified. By studying these pockets, binding sites were suggested for rhodamine 123 (R-site) and prazosin (regulatory site) at the NBD–TMD interface that agreed with the experimental data on their location. Additionally, one more R-site in the protein cavity was proposed, in accordance with the available biochemical data. Together with the previously suggested Hoechst 33342 site (H-site), all sites were interpreted with respect to their effects on the protein ATPase activity, in correspondence with the experimental observations. Several residues involved in key contacts in the P-gp NBDs were proposed for further targeted mutagenesis experiments.

Multidrug resistance observed in cancer chemotherapy is commonly attributed to overexpression of efflux transporter proteins. These proteins act as ATP-dependent drug efflux pumps, actively extruding chemotherapeutic agents from cells and causing a decrease in intracellular drug accumulation. Besides the well-recognized role of P-glycoprotein (P-gp, ABCB1), the breast cancer resistance protein (BCRP, ABCG2) is becoming increasingly accepted as playing an important role in multidrug resistance. In contrast to P-glycoprotein, only a few inhibitors of ABCG2 are known. According to the literature, tyrosine kinase inhibitors (TKIs) can be considered to be broad-spectrum inhibitors, interacting with ABCB1, ABCC1 and ABCG2. Here, we investigated seven different TKIs, gefitinib, erlotinib, AG1478, PD158780, PD153035, nilotinib and imatinib, for their potential to restore ABCG2 sensitivity to cells. Furthermore, we analyzed the alteration of ABCG2 expression caused by TKIs and demonstrated that EGFR inhibitors such as gefitinib and PD158780 reduced both total and surface expression of ABCG2 in EGRF-positive MDCK BCRP cells by interaction with the PI3K/Akt signaling pathway. The reduced ABCG2 content led to an increased effect of XR9577, a well-known ABCG2 modulator, lowering the concentration required for half maximal inhibition. On the other hand, BCR-ABL inhibitors had no influence on ABCG2 expression and modulator activity. Interestingly, a combination of an EGFR inhibitor with the PI3K/Akt inhibitor LY294002 led to a significant reduction of ABCG2 expression at low concentrations of the drugs. Based on our results, we assume that EGFR exerts a post-transcriptional enhancing effect on ABCG2 expression via the PI3K/Akt signaling pathway, which can be attenuated by EGFR inhibitors. Blocking the key signaling pathway regulating ABCG2 expression with EGFR inhibitors, combined with the inhibition of ABCG2 with potent modulators might be a promising approach to circumvent MDR in cancer cells.

A new class of specific breast cancer resistance protein (BCRP) inhibitors was identified, showing no inhibition of the ATP binding cassette (ABC) transporters P-gp and MRP1. Some of these modulators inhibit BCRP with high potency; they are only slightly less potent than Ko143 and could serve as promising lead structures for the design of novel effective BCRP inhibitors. These inhibitors are structurally related to tariquidar (XR9576) and belong to a library of multidrug-resistance modulators synthesized by our research group. The absence of the tetrahydroisoquinoline substructure appears to play a crucial role for specificity; we found that the presence of this substructure is not essential for interaction with BCRP. To determine the type of interaction between pheophorbide A and compounds with and without the tetrahydroisoquinoline substructure, various substrate pheophorbide A concentrations were used in enzyme kinetics assays. The resulting data show that these compounds share a noncompetitive-type interaction with pheophorbide A. Experiments with imatinib and pheophorbide A revealed a mixed-type interaction. The combination of imatinib and compounds with and without the tetrahydroisoquinoline substructure resulted in a positive cooperative effect, indicating that imatinib engages a binding site distinct from that of the new compounds on one side and distinct from that of pheophorbide A on the other side as well. The results of this study suggest that the category of BCRP-specific inhibitors, which includes only fumitremorgin C, Ko143 and analogues, and novobiocin needs to be extended by this new class of inhibitors, which possess three key characteristics: specificity, potency, and low toxicity.

Quinazolinones, indolo- and pyrrolopyrimidines with inhibitory effects toward ABCB1 (P-gp) and ABCC1 (MRP1) transporters were studied by pharmacophore modeling, docking, and 3D QSAR to describe the binding preferences of the proteins. The pharmacophore overlays between dual and/or highly selective inhibitors point to binding sites of different topology and physiochemical properties for MRP1 and P-gp. Docking of selective inhibitors into the P-gp binding cavity by the use of a structural model based on the recently resolved P-gp structure confirms the P-gp pharmacophore features identified, and reveals the interactions of some functional groups and atoms in the structures with particular protein residues. The 3D QSAR analysis of the dual-effect inhibitors allows satisfactory prediction of the selectivity index of the compounds and outlines electrostatics as most important for selectivity. The results from the combined modeling approach complement each other and could improve our understanding of the protein–ligand interactions involved, and could aid in the development of highly selective and potent inhibitors of P-gp and MRP1.

P-glycoprotein (P-gp), a 170 kDa plasma membrane protein, is one of the most relevant ABC transporters involved in the development of multidrug resistance (MDR). Understanding its mechanism of transport as well as its interactions with various substrates are basic requirements for the development of adequate therapeutic approaches to overcome this kind of resistance against a broad spectrum of structurally unrelated cytostatic drugs. P-gp modulators (activators) that exert various effects on the intracellular accumulation of distinct P-gp substrates are useful tools for investigating the interactions between multiple drug binding sites of this transport protein. In this study, a series of 27 different imidazobenzothiazoles and imidazobenzimidazoles structurally related to the known P-gp activators QB102 and QB11 was designed, and their modulating properties were investigated. Most of them were able to stimulate P-gp-mediated efflux of daunorubicin and rhodamine 123 in a concentration-dependent manner, but some compounds also displayed weak inhibitory effects. Additionally, P-gp-mediated efflux of vinblastine and colchicine was inhibited by several compounds. Therefore, we concluded that the novel compounds bind to the H site of P-gp and activate the efflux of specific substrates of the R site in a positive cooperative manner, whereas binding of H-type substrates is inhibited competitively. This hypothesis is confirmed by the observation that the modulators do not influence hydrolysis of ATP or its affinity toward P-gp.

A homology model of P-glycoprotein based on the crystal structure of the multidrug transporter Sav1866 is developed, incorporated into a membrane environment, and optimized. The resulting model is analyzed in relation to the functional state and potential binding sites. The comparison of modeled distances to distances reported in experimental studies between particular residues suggests that the model corresponds most closely to the first ATP hydrolysis step of the protein transport cycle. Comparison to the protein 3D structure confirms this suggestion. Using SiteID and Site Finder programs three membrane related binding regions are identified: a region at the interface between the membrane and cytosol and two regions located in the transmembrane domains. The regions contain binding pockets of different size, orientation, and amino acids. A binding pocket located inside the membrane cavity is also identified. The pockets are analyzed in relation to amino acids shown experimentally to influence the protein function. The results suggest that the protein has multiple binding sites and may bind and/or release substrates in multiple pathways.