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

Abstract

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.

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

“…The inflexion point depends on the ligand concentration, the number of available binding sites and the compound's binding affinity [45]. Typical chromatograms using FAC obtained for orthosteric (a) and allosteric (b) nAChR binding sites are presented in Figure 5 [30].…”

“…Using frontal affinity chromatography with CMAC columns was proved not only to be useful in characterization and studies involving the orthosteric but also with the allosteric binding sites of the immobilized transmembrane protein [30]. Frontal displacement chromatography can be applied to determine the binding affinity of unknown ligand binding to the same site as the marker ligand.…”

“…With permission from [30] This approach was for example used to determine K d values of several alkaloids binding to  3  4 and  3  4  5 nicotinic receptors [30].…”

“…by a high concentration of the displacer; K dDis -dissociation constant of the displacer; P -product of the B max (the number of the available binding sites) and (K dDis /K dmarker ), K dmarker -dissociation constant of the marker ligand [11]. [30].…”

“…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.…”