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Abstract: Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1G465R) and CMAC(hOCT1R488M). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [3H] MMP+ (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1. [Copyright &y& Elsevier]

Background and Purpose: The human organic cation transporter-1 (hOCT1) is a polyspecific transporter that plays a role in drug distribution, metabolism and excretion. Previous studies have demonstrated that hOCT1 binding can be stereoselective, but the mechanism for stereochemical recognition has not been described. The purpose of this study was to develop a pharmacophore model to describe stereoselective binding to hOCT1.Experimental Approach: A set of 22 compounds including 8 pairs of enantiomers and five pairs of diastereomers was used to develop a pharmacophore model. The pharmacophore modeling was carried out using Catalyst version 4.11 and HypoGen and was based upon the correlation of the structures and activities (K(i) values) of the compounds used in the study.Key Results: The resulting model contained a positive ion, hydrophobic and two hydrogen-bond acceptor interaction sites. The relative enantioselectivity of 8/8 enantiomeric pairs and diastereoselectivity of 5/5 diastereomers was described by mapping to a combination of at least 3 of the 4 functional feature sites of the model.Conclusions and Implications: The pharmacophore model describes stereoselective interactions with hOCT1 at one of the binding sites on the molecule. [ABSTRACT FROM AUTHOR]

Abstract: Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [3H]-methyl phenyl pyridinium ([3H]-MPP+) as the marker ligand and MPP+, verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The Kd values calculated from the chromatographic studies correlated with previously reported Ki values (r2=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP+ and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil. [Copyright &y& Elsevier]

The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. ( Lycopodiaceae ) and Trigonella foenum graecum L. ( Fabaceae ) against both the α3β4 and α3β4α5 nAChRs. [ABSTRACT FROM AUTHOR]

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities ( K d ) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities ( K i ) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. [ABSTRACT FROM AUTHOR]

Cellular membrane affinity chromatography stationary phases have been extensively used to characterize immobilized proteins and provide a direct measurement of multiple binding sites, including orthosteric and allosteric sites. This review will address the utilization of immobilized cellular and tissue fragments to characterize multiple transmembrane proteins co-immobilized onto a stationary phase. This approach will be illustrated by demonstrating that multiple transmembrane proteins were immobilized from cell lines and tissue fragments. In addition, the immobilization of individual compartments/organelles within a cell will be discussed and the changes in the proteins binding/kinetics based on their location. [ABSTRACT FROM AUTHOR]

Highlights: [•] ABC columns. [•] Screening against BCRP. [•] Difference between cellular and nuclear BCRP. [Copyright &y& Elsevier]

Abstract: The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening toward PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of frontal affinity chromatography coupled to mass spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and K d experiments toward new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the K d values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes. [Copyright &y& Elsevier]

Abstract: BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC–MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized. [Copyright &y& Elsevier]

Abstract: This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [3H]-adefovir for the hOAT1 and [14C]-p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (K d) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r 2 =0.688 (p <0.05) and r 2 =0.9967 (p <0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented. [Copyright &y& Elsevier]