Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening

Abstract

Cannabinoid Receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, (KU-812), were immobilized onto the surface of an open tubular capillary to create a CB1/CB2-OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2-OT column can be used to determine the binding affinities (K i values) for a single compound and to screen individual or a mixture of multiple compounds. The CB1/ CB2-OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high affinity phytocannabinoid.

Cellular membrane affinity chromatography (CMAC) in drug discovery from complex natural matrices

“…Since the successful immobilization of nAChRs on the IAM stationary phase, multiple transmembrane proteins have been used to prepare CMACs with IAM.PC (immobilized artificial membrane phosphatidylcholine). The examples of transmembrane proteins immobilized on IAM.PC particles include: ligand-gated ion channels ( x  y nAChR x = 3,4,7; y = 2,3,4; GABA A , NMDA, combination of nAChR, NMDA) [11,13,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30], G-protein coupled receptors (opioid, -adrenergic, P2Y1, Histamine 1-4, CB 1 , CB 2 ) [31][32][33][34][35] and drug transporters (ABC: Pgp, MRP1, MRP2, BCRP; SLC: hOCT and hOCT SNPs, hOAT) [36][37][38][39]. IAM.PC mimics the cell membrane environment and keeps the experimental conditions close to the physiological state.…”

“…Lipophilic compounds have been found to be strongly retained on IAM.PC particles and an alternative approach has been proposed to study such ligands: immobilization of cell membrane fragments onto the surface of an open tubular capillary (OT columns) [7]. For example, OT columns were successfully used to immobilize cell membrane fragments with CB1/CB2 receptors and further for the identification of endocannabinoid receptor ligands from the extract of Zanthoxylum clava-herculis L. [34,47]. The development and the use of OT columns remain beyond the scope of this review and interested readers are encouraged to refer to other publications [7].…”

Affinity Chromatography as a Tool for Quantification of Interactions Between Drug Molecules and Their Protein Targets

“…For this purpose, fragments of cell membrane containing membrane protein bind non-covalently with a capillary using the avidin-biotin pair. Capillaries with immobilized P-glycoprotein (Moaddel et al, 2004), and recently with immobilized cannabinoid receptors (CB1/CB2) (Moaddel et al, 2011) were developed using this approach.…”

Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts

“…These methods have been successfully used but predominantly to identify the most abundant compounds and not necessarily the most active. We have previously demonstrated that these issues can be addressed using bioaffinity chromatography and that this approach can be used for the isolation of active components from complex mixtures including tobacco smoke condensates and plant extracts [10–14]. The previous studies have utilized cellular membrane affinity chromatography (CMAC) including CMAC columns containing single or multiple nAChR subtypes [15, 16].…”