Conformational mobility of immobilized alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nicotinic acetylcholine receptors
Anal Chem. 2005 Feb 1;77(3):895-901
Moaddel R, Jozwiak K, Whittington K, Wainer IW
Four affinity chromatography stationary phases have been developed based upon immobilized nicotinic acetylcholine receptor (nAChR) subtypes, the alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nAChRs. The stationary phases were created using membranes from cell lines expressing the subtypes and an immobilized artificial membrane stationary phase. The immobilized nAChRs were characterized using frontal chromatography with the agonist epibatidine as the marker. The observed binding affinities for the agonists epibatidine, nicotine, and cytisine were consistent with reported values, indicating that the nAChRs retained their ability to bind agonists. The noncompetitive inhibitors (NCIs) of the nAChR (R)- and (S)-mecamylamine, phencylcidine, dextromethoprphan, and levomethorphan were also chromatographed on the columns using nonlinear chromatography techniques. The studies were carried out before and after exposure of the columns to epibatidine. The NCI retention times increased after exposure to epibtatidine as did the enantioselective separation of mecamylamine and methorphan. The results indicate that the immobilized nAChRs retained their ability to undergo agonist-induced conformational change from the resting to the desensitized states. The columns provide a unique ability to study the interactions of NCIs with both of these conformational states.
Qualitative assessment of IC50 values of inhibitors of the neuronal nicotinic acetylcholine receptor using a single chromatographic experiment and multivariate cluster analysis
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 May 5;819(1):169-74
Jozwiak K, Moaddel R, Yamaguchi R, Ravichandran S, Collins JR, Wainer IW
It has been widely demonstrated that affinity chromatography can be used to derive binding affinities, and that these affinities can be correlated to data obtained using standard techniques such as membrane binding, ultrafiltration and equilibrium dialysis. The purpose of this study is to evaluate the use of immobilized nicotinic acetylcholine receptor stationary phase in chromatographic experiments to assess the functional activity of series of noncompetitive inhibitors (NCIs) as reflected in their IC50 values. Chromatographically determined retention values and computer generated molecular descriptors were obtained for 29 compounds and the data were analyzed by cluster analysis. The approach qualitatively ranked the test compounds as efficient NCIs (low IC50 values) or poor NCIs (high IC50 values). The data obtained with the 29 compounds used in this study demonstrate that the experimental approach had been able to place 25 of these compounds in the correct IC(50) clusters. To our knowledge, this is the first relationship established between chromatographic retention and IC50 for membrane-bound receptors. These results suggest that the chromatographic approach may be useful in development of lead drug candidates including the determination of off-target binding.
Direct chromatographic determination of dissociation rate constants of ligand-receptor complexes: assessment of the interaction of noncompetitive inhibitors with an immobilized nicotinic acetylcholine receptor-based liquid chromatography stationary phase
Anal Chem. 2005 Aug 15;77(16):5421-6
Moaddel R, Jozwiak K, Yamaguchi R, Wainer IW
A liquid chromatographic stationary phase containing immobilized membranes from a cell line expressing the alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) has been used to assess dissociation rate constants (kd) of 12 noncompetitive inhibitor-nAChR complexes. The pharmacological effects of the noncompetitive inhibitors, expressed as percent recovery of activity at 7 min and 4 h postexposure to the inhibitor, were also determined. The results demonstrate that the kd values correlated with the pharmacological effect and that this approach can be used to identify molecular structures associated with differences in kd values. The method can be adapted for use with membrane-bound receptors, ion channels, and transporters and represents a direct and facile technique for the assessment of dissociation rate constants (kd) of ligand-receptor complexes.