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

Abstract

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 α 3 β 4 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 C 18 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-γ-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.

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

“…However, CMAC columns can be successfully applied in the process of identification of pharmacologically active compounds present in complex natural matrices targeting transmembrane receptors [7,46]. There are several issues that need to be taken into consideration when applying CMACs in drug discovery from natural samples.…”

“…The identification process of individual secondary metabolites responsible for the observed displacement of the marker ligand is performed using the "missing peak chromatography" approach [46]. One of the greatest challenges in drug discovery from plant extracts, using CMAC columns, is discerning specific interactions (phytochemicals interacting with the immobilized transmembrane target) from nonspecific interactions (compounds interacting with IAM particles or other transmembrane proteins coimmobilized with the studied targeted receptor).…”

“…For example, in the case of nAChR CMAC columns, HEK cells overexpressing desired nAChR subtypes were used to prepare CMAC(+), while HEK 293 cells were utilized to develop CMAC(-) [30]. In the missing peak chromatography technique, plant extracts are chromatographed on CMAC(+) and CMAC(-) columns using ammonium acetate buffer (10 mM, pH 7.4) delivered at 0.2 ml/min as mobile phase [46]. Fractions are collected based on the observed chromatograms.…”

“…The difference between the CMAC(+) and CMAC(−) columns is in the availability of the immobilized protein target. The differences in the retention of a compound on the CMAC(+) column relative to the CMAC(−) column is due to specific interactions with the target [7,46]. A peak that is missing in the fingerprint of a CMAC(+) early fraction, but present in the control and corresponding CMAC(−) early fraction, is considered to bind to the immobilized transmembrane protein target on the CMAC(+) column [46].…”

“…The differences in the retention of a compound on the CMAC(+) column relative to the CMAC(−) column is due to specific interactions with the target [7,46]. A peak that is missing in the fingerprint of a CMAC(+) early fraction, but present in the control and corresponding CMAC(−) early fraction, is considered to bind to the immobilized transmembrane protein target on the CMAC(+) column [46]. Elution of this particular compound in a later fraction of CMAC(+), along with its absence in the corresponding fraction in CMAC(−), is a further confirmation of the affinity of this compound for the targeted transmembrane receptor in CMAC(+) [46].…”