Membrane transporter proteins may play a role both in the pharmacokinetics of several drugs (when expressed in normal tissues) and/or resistance to several anticancer agents (when expressed in cancers). Several of these proteins belong to the ABC (ATP Binding Cassette) protein superfamily, including, P-glycoprotein (Pgp, ABCB1), the Multidrug Resistance Protein (MRP1, MRP2, BCRP, ABCC1), the Mitoxantrone Resistance Protein (MXR, ABCG2), the canalicular Multispecific Organic Anion Transporter (cMOAT, ABCC2), the Bile Salt Export Pump (BSEP, ABCB11). Other transporter proteins, such the Organic Cation transporters (OCT1 and OCT2) and the Organic Anionic Transporter (OAT) do not belong to the ABC superfamily. These proteins seem to share several substrate drugs including, possibly, some anticancer agents. The partial sharing of anticancer and non-anticancer substrates between different transporters may explain some of the side effects of drugs used to inhibit the function of specific transporters. For example, Cyclosporin A has been tested clinically as an inhibitor of Pgp-caused multidrug resistance. One of its side effects is jaundice, which might be caused by its interference of the transport of conjugated bilirubin by cMOAT. So, the clinical efficacy of transporter inhibitors (whether used to reverse anticancer drug resistance, or to modulate pharmacokinetics) may be improved by optimizing their transporter selectivity. Pgp is the transporter that has been most thoroughly studied. Pgp effluxes its substrates from cells by transporting them from the inner cell membrane to the outer leaflet or to the extracellular space. Central to the biology of Pgp is its ability to bind a wide array of diverse substrates and inhibitors, suggesting the possible existence of multiple binding sites. The lack of definition of these sites and the unavailability of a crystal structure for Pgp have so far hindered a rational design of drugs targeting Pgp function. We propose that cellular membrane affinity chromatography (CMAC)can be used as a means to characterize the binding sites and transport cycle of the different transporters and, ultimately, to define effective and protein-selective pharmacophores for the pharmacological inhibition of transporter function. We are presently characterizing and optimizing CMAC models of Pgp. This process will also provide a prototype for the modeling of other ABC and non-ABC transporters. Membranes containing Pgp were obtained from a stabley trnasfected cell line (MDA435/LCC6MDR1 human breast cancer cells)and membranes which do not contain Pgp were obtained from the MDA435/LCC6 cell line). The membranes were immobilized on the surface of a 100 micron i.d. glass tube to form open tubnular columns, Pgp(+)-OT and Pgp(-)-OT. The columns were placed in a chromatographic system containing a mass spectrometer and rapid frontal chromatographic studies were performed with known Pgp substrates and compounds known not to bind to Pgp. The results indicate that the difference in the chromatographic retention on the two columns (the delta-time)can be used to classify compounds as Pgp substrates. The results are correlated with data obtained using cellular methods such as Caco-2 cell permeability and the method is a rapid, reproducible and accurate appraoch to screen for Pgp substrates. Cellular membranes from Sf9 insect cells transfected with cDNA coding the human multidrug resistance protein 1 (MRP1), multidrug resistance protein 2 (MRP2) and the breast cancer resistance protein (BCRP) have been utilized to create CMAC(MRP1), CMAC(MRP2) and CMAC(BCPR)columns and membranes from the non-transfected Sf9 cell line have been used to create CMAC(Sf9) columns. The data from the initial studies indicate that the CMAC columns contain functioning transporters and that they columns can be used to characterize the interactions of substrates and inhibitors with the immobilized transporters. Cellular membranes from a 1321N1 astrocytoma cell line have been immobilized to create a CMAC(1321N1) column. The initial studies demonstrate that the chromatographic approach developed in this project can be used to identify and distinguish between the multiple transporters expressed in this cell line. Full characterization of the properties and interactions of these transporters in underway. The data from this study indicate that human-derived astrocytoma and glioblastoma cell lines express Pgp, MRP1 and BCRP. The key observation is that Pgp and MRP1 are expressed on both the cellular membrane as well as the nuclear membrane. The latter observation is the first time that functional ABC transporters have been identified on the nuclear membrane and provide a new and key target for over coming multiple drug resistance in these tumors. The presence of BCRP in these tumors was confirmed using immunohistochemistry on tissues from glioblastoma tumors. Cell lines which express and do not express the human organic transporter (hOCT1)have been obtained and used to prepare CMAC(hOCT1(+))and CAMC(hOCT1(-))columns. The columns were prepared by immobilizing membranes obtained from the respective cell lines on the surface of immobilized artificial membrane (IAM) liquid chromatography stationary phases. Frontal affinity chromatography has demonstrated that the CMAC(hOCT1(+))column can be used to identify substrates and inhibitors of the hOCT1. The studies have also demonstrated that interactions with the hOCT1 are enantioselective and has been used to create a pharmacophore that describes inhibitor binding to the hOCT1 and can be used to identify these inhibitors in silico. CMAC columns containing mutated hOCT1 transporters created by single point mutations have been created and used to characterize the effects of these mutations on binding affintiy, selectivity and transport. An initial column containing an immobilized organic anion transporter has been developed and characterized.
|Habicht, K-L; Frazier, C; Singh, N et al. (2013) The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells. J Pharm Biomed Anal 72:159-62|
|Bhatia, Prateek; Bernier, Michel; Sanghvi, Mitesh et al. (2012) Breast cancer resistance protein (BCRP/ABCG2) localises to the nucleus in glioblastoma multiforme cells. Xenobiotica 42:748-55|
|Bhatia, Prateek A; Moaddel, Ruin; Wainer, Irving W (2010) The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) Talanta 81:1477-81|
|Moaddel, Ruin; Wainer, Irving W (2009) The preparation and development of cellular membrane affinity chromatography columns. Nat Protoc 4:197-205|
|Jozwiak, Krzysztof; Moaddel, Ruin; Ravichandran, Sarangan et al. (2008) Exploring enantiospecific ligand-protein interactions using cellular membrane affinity chromatography: chiral recognition as a dynamic process. J Chromatogr B Analyt Technol Biomed Life Sci 875:200-7|
|Wainer, Irving W (2008) Investigation of molecular recognition in biological systems using cellular membrane affinity chromatography. Chim Oggi 26:19-22|
|Bhatia, P; Kolinski, M; Moaddel, R et al. (2008) Determination and modelling of stereoselective interactions of ligands with drug transporters: a key dimension in the understanding of drug disposition. Xenobiotica 38:656-75|