Search

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [[sup.3]H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric [alpha]7 nicotinic acetylcholine receptors ([[alpha].sub.7] nAChR) and heteromeric nicotinic acetylcholine receptors ([[alpha].sub.x][[beta].sub.y] nAChRs), which was confirmed by the addition of subtype-specific inhibitors, [alpha]-bungarotoxin ([[alpha].sub.7] nAChR) and [k.sub.]-bungarotoxin ([[alpha].sub.x][[beta].sub.y] nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), [gamma]-aminobutyric acid (GAB[A.sub.A]) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GAB[A.sub.A] receptor) and MK801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flowcytometry. The results indicate that the CMAC (1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.

Heat shock protein 90[alpha] (Hsp90[alpha]) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90[alpha] has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90[alpha] ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90[alpha] coated magnetic beads can be used to 'fish' from complex chemical and biological mixtures for new lead drug candidates and client proteins.

Human serum albumin, HSA, was immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known HSA ligands from a mixture containing ligands and nonligands. The separation was accomplished manually and was also automated. The results indicate that an automated 'ligand-fishing' technique can be developed using magnetic beads containing an immobilized protein.

Heat shock protein 90α (Hsp90α) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90α has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90α ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90α coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins. [ABSTRACT FROM AUTHOR]

Background/aims: Tobacco extract and smoke condensate are complex natural mixtures showing biological activities. Nicotinic receptors are known to be involved in the self-medication behaviour observed in schizophrenia patients. We use the bioaffinity screening technology, i.e. nicotinic receptors immobilized on stationary phases, to screen tobacco smoke for unknown ligands.Methods: Membrane α3β4-subtype nicotinic receptors are expressed in HEK-293 cells, partly purified and immobilized on artificial membranes silica-based stationary phase (IAM-PCDD2, Regis Technologies®). Binding properties of columns are characterized by frontal chromatography. Tobacco extracts are injected and retained compounds are directed to a second analytical column, connected to a suitable detector for structural identification.Results: Preliminary results show that columns effectively bind nicotinic ligands. HPLC fingerprinting of the hydrophilic/cationic fraction of smoke condensate show a large proportion of nicotine that can flaw binding of minor ligands. Nicotine imprinted chromatography has been tested to solve this problem by selectively removing the nicotine of the extract. Binding capacity of the columns is of critical importance.Conclusions: The ability of bioaffinity chromatography to identify ligands in a mixture by an on-line process is promising for the drug discovery of drugs acting as receptor agonists, competitive and non-competitive antagonists.Clinical Pharmacology & Therapeutics (2005) 79, P45–P45; doi: 10.1016/j.clpt.2005.12.160 [ABSTRACT FROM AUTHOR]

Background/aims: To compare a new column based method for characterization of drug-hOCT1 protein interaction.Method: We have immobilized the hOCT1 protein onto an immobilized artificial membrane stationary phase (IAM), resulting in the hOCT1 (+) IAM column. Initial characterization was carried out on the radio flow detector using frontal displacement chromatography techniques.Results: The binding affinities of nineteen ligands were determined in this manner and compared to reported IC50/Ki values when available and resulted in a significant correlation with and r2 of 0.9364 and a P value of 0.0015. The sample compounds included 6 chiral compounds and the method was able to identify enantioselective binding interactions.Conclusion: The successful immobilization of the hOCT1 transporter on the IAM stationary phase has been clearly demonstrated. This method allowed the identification of a high affinity binding site for MPP for the first time. We have also shown that the transporter is enantioselective. Future studies will be conducted to determine the effect of certain mutations on binding to the hOCT1 transporter. This method can be easily adapted to other members of this family including but not limited to hOCT2, hOCT3, hOCTN1, hOCTN2 and OAT.Clinical Pharmacology & Therapeutics (2005) 79, P76–P76; doi: 10.1016/j.clpt.2005.12.274 [ABSTRACT FROM AUTHOR]

Purpose: To develop a liquid chromatographic stationary phase containing immobilized membranes that express the alpha7 nicotinic receptor and to validate its use in the determination of substrate binding affinities.Methods: The membranes were obtained from a transfected SHEP-1 cell line that expressed alpha7[alpha7(+)] and non-transfected SHEP-1 cells[alpha7(−)]. The alpha7 (+/−) cells were homogenized, solubilized, and subsequently mixed with 200 mg of immobilized artificial membrane (IAM) stationary phase and the resulting mixture was dialyzed for 2 days. The resulting stationary phase mixture was packed into a HR 5/2 glass column. The column was placed in a HPLC system containing a scintillation detector. Nicotine, methyllycaconitine and epibatidine were run with a radiolabeled marker ligand through the alpha7-IAM column to obtain binding affinities. The presence of the alpha7 nicotinic receptors was also shown on the alpha7 (+) cells by confocal microscopy.Results: Frontal displacement chromatography was used to determine the binding affinities (Kd) of the nicotine, methyllycaconitine and epibatidine ligands and the data was compared to literature values. For example, the calculated Kd for epibatidine was 670 pM (versus 640 pM literature).Conclusions: An immobilized alpha7 column has been developed. The data demonstrate that the column can be used for the on-line identification of alpha7 ligands.Clinical Pharmacology & Therapeutics (2005) 79, P81–P81; doi: 10.1016/j.clpt.2005.12.290 [ABSTRACT FROM AUTHOR]

Aims: To compare a new column-based method for characterization of drug-Pgp interactions with a more conventional model, monolayers of Caco-2 cells.Methods: A chromatographic approach was developed for the screening of compounds for their interactions with Pgp by immobilizing membranes from cells expressing and not expressing Pgp. The comparison of these columns can be used to sort compounds with/without affinity for Pgp. The change in retention time (rt) between these columns is proportional to the strength of the interaction between the compound and Pgp. These results were compared with the behavior of the same compounds in Caco-2 monolayers, where they were sorted by calculating the coefficient of permeability in the apical-to-basolateral direction (PappA-B) and the ratio PappB-A/PappA-B.Results: 14 compounds were studied using the chromatographic and Caco-2 methods. In the parallel chromatographic screen, the value of rt varied from 0.11 min (nicardipine) to 21.53 min (domperidone). The value of PappB-A/PappA-B among the same set of compounds ranged from 1.4 to 45.0, respectively. In almost every case, when rt exceeded 0.25 min, the value of PappB-A/PappA-B was greater than 2.0, indicating a Pgp substrate. A positive correlation between the two data sets was seen (r2 = 0.95).Conclusions: The results of this study suggest that the parallel screening of new pharmaceutical compounds on Pgp (+) and Pgp (−) open tubular columns can be interchanged with the conventional and widely used Caco-2 model.Clinical Pharmacology & Therapeutics (2005) 79, P7–P7; doi: 10.1016/j.clpt.2005.12.022 [ABSTRACT FROM AUTHOR]