Substrate-Dependent Breast Cancer Resistance Protein (Bcrp1/Abcg2)-Mediated Interactions: Consideration of Multiple Binding Sites in in Vitro Assay Design

Abstract

ABSTRACT:In vitro assays are frequently used for the screening of substrates and inhibitors of transporter-mediated efflux. Examining directional flux across Madin-Darby canine kidney (MDCK) II cell monolayers that overexpress a transporter protein is particularly useful in identifying whether or not a candidate compound is an inhibitor or substrate for that transport system. Studies that use a single substrate or inhibitor in competition assays can be challenging to interpret because of the possible multiple mechanisms involved in substrate/inhibitor-protein interactions. During our previous studies of substrate-inhibitor-transporter interactions, we observed differences in breast cancer resistance protein (BCRP) inhibition, depending on the substrate and the inhibitor. Therefore, we investigated BCRP-mediated interactions with a 4 ؋ 4 matrix of substrates and inhibitors using monolayers formed from MDCKII cells transfected with murine BCRP (Bcrp1/Abcg2). The selective BCRP inhibitor 3-(6-isobutyl- Multidrug resistance observed in cancer therapy is commonly attributed to ATP-binding cassette (ABC) efflux transporter proteins, such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP). These ABC transport proteins are localized not only in drug-resistant cancer cells, but also in the plasma membranes of epithelial cells in various normal tissues such as intestine, liver, kidney, placenta, and the blood-brain barrier where they can influence absorption, distribution, metabolism, and excretion of their substrates (Leslie et al., 2005). The efflux transport protein BCRP is expressed in various organs, and because of its transmembrane orientation, this transporter is able to extrude substrates out of the cell (Maliepaard et al., 2001). BCRP is considered a half-transporter because of the presence of only six transmembrane domains, and it has been shown to exist in a multisubunit oligomeric structure that may be critical for its function (Bhatia et al., 2005).Numerous in vitro studies have characterized substrate-transporter interactions and these effects have also been observed in vivo (Merino et al., 2006;Shaik et al., 2007). Interaction of BCRP with various substrate molecules in vitro and in vivo has provided evidence that BCRP-mediated efflux could play a major role in the absorption, distribution, metabolism, and excretion of its substrate molecules (Staud and Pavek, 2005). Identifying inhibitors that modulate BCRPmediated efflux of substrates could be valuable in enhancing target exposure to the drug. Various molecules have been shown to have inhibitory effects on BCRP-mediated transport of its substrates. GF120918 is a potent P-gp inhibitor that was shown to also have an inhibitory effect on BCRP-mediated transport. For instance, GF120918 inhibits BCRP-mediated transport of abacavir, zidovudine, STI-571 (imatinib), and prazosin (Breedveld et al., 2005;Ejendal and Hrycyna, 2005;Pan et al., 2007). Fumitremorgin C was identified as the first select...

Overlapping Substrate and Inhibitor Specificity of Human and Murine ABCG2

“…This was attributed to species differences of ABCG2. We observed a significant fold-increase in Pp-18 accumulation (comparable to inhibition by FTC or Ko143) in mouse and human ABCG2-expressing cell lines when adding the same concentrations of chrysin and benzoflavone used by Zhang et al Due to the existence of multiple binding sites on ABCG2 (Giri et al, 2009), there may be different transporter-mediated interactions, and competitive substrates may not inhibit the efflux of all transport substrates. As such, caution should be taken that an inhibitor of ABCG2 is dependent on the substrates tested.…”

The Need for Human Breast Cancer Resistance Protein Substrate and Inhibition Evaluation in Drug Discovery and Development: Why, When, and How?

“…However, their ERs in M-BCRP cells were insensitive to Ko143. This can be due to differences in binding sites and substrate-dependent inhibition (Muenster et al, 2008;Giri et al, 2009). Those examples well illustrate that not only the ER value in M-BCRP cells should be taken into consideration, but the impact of Ko143 and the ER in the control cells to avoid falsenegative or false-positive results (Fig.…”

“…Likewise, polarized transcellular assays are not suitable for low permeable compounds, since they do not sufficiently penetrate the cells and therefore do not reach the export protein, potentially resulting in false-negative results. The existence of multiple binding sites on transport proteins could also add to the complexity of identifying substrate and inhibitor molecules in in vitro assay systems, leading to incorrect interpretation (Muenster et al, 2008;Giri et al, 2009). Careful selection of substrates and inhibitors is therefore critical for designing definitive studies, and may circumvent the current issues with very high experimental interlaboratory variability in in vitro assays .…”

How to overcome ATP-binding cassette drug efflux transporter-mediated drug resistance?

“…In vitro assays showed that ABCG2 might have more than two binding sites, since ABCG2 inhibitors displayed different inhibition profiles, depending on the tested substrate [71][72][73] . For instance, the inhibitor Ko143 inhibited the transport of all the substrates tested by Giri et al [72] , which suggested its binding to a region that allosterically inhibits the transport of these substrates. However, the ABCG2 inhibitors elacridar, nelfinavir and Pluronic P85 seemed to bind to a different region, which inhibited the transport of the nucleoside analogs abacavir and zidovudine, but showed no or a partial effect on the transport of prazosin and imatinib.…”

“…Examples are a wide range of anticancer drugs, sulfate and glucuronide conjugates of xenobiotics, tyrosine kinase inhibitors (TKIs), statins, fluorescent dyes and flavonoids [10,63] . In vitro assays showed that ABCG2 might have more than two binding sites, since ABCG2 inhibitors displayed different inhibition profiles, depending on the tested substrate [71][72][73] . For instance, the inhibitor Ko143 inhibited the transport of all the substrates tested by Giri et al [72] , which suggested its binding to a region that allosterically inhibits the transport of these substrates.…”