Screening of tobacco smoke condensate for nicotinic acetylcholine receptor ligands using cellular membrane affinity chromatography columns and missing peak chromatography.

This manuscript reports an approach to the screening of natural product extracts for compounds which are active at membrane-bound receptors, ion channels and transporters. The technique is based upon cellular membrane affinity chromatography (CMAC) columns created through the immobilization of cellular membrane fragments on liquid chromatography stationary phases. In this study a CMAC(nAChR(+)) column was created out of membranes from a transfected cell line expressing the alpha3beta4 neuronal nicotinic acetylcholine receptor (nAChR) and the column was used to screen tobacco smoke condensates. A strategy involving parallel screening with a CMAC column created from a non-transfected form of the same cell line, CMAC(nAChR(-)) was adopted. The condensate was chromatographed on both columns, timed fractions collected and concentrated. Each fraction was analyzed on a C18 column in order to establish a chromatographic fingerprint of each fraction and a differential elution profile of each compound. Comparison of the elution profiles from the CMAC(nAChR(+)) and CMAC(nAChR(-)) columns identified patterns that could be associated with high affinity ligands and with low-affinity/non-binding compounds. Known strong ligands ((S)-nicotine, (R,S)-anatabine, N'-nitrosonornicotine), weak ligands ((R,S)-nornicotine, anabasine) as well as known non-ligands (N-methyl-gamma-oxo-3-pyridinebutanamide, (1'S,2'S)-nicotine 1'-oxide) have been identified in the complex extract. The results demonstrate that CMAC-based screens can be used in the identification of compounds within natural product extracts that bind to membrane-based targets.