Interaction of Noncompetitive Inhibitors with the r32 Nicotinic Acetylcholine Receptor Investigated by Affinity Chromatography and Molecular Docking Krzysztof Jozwiak,* ,² Sarangan Ravichandran, ‡ Jack R. Collins, ‡ Ruin Moaddel, § and Irving W. Wainer § Department of Chemistry, Medical UniVersity of Lublin, Staszica 6, 20-081 Lublin, Poland, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock DriVe, Baltimore, Maryland 21224, and AdVanced Biomedical Computing Center, National Cancer Institute-Frederick/SAIC, Post Office Box B, Building 430, Miller DriVe, Frederick, Maryland 21702 ReceiVed July 1, 2007 A molecular model of the R32 nAChR lumen channel was constructed and hydrophobic clefts were observed near the receptor gate. Docking simulations indicated that ligand-nAChR complexes were formed by hydrophobic interactions with the cleft and hydrogen bond interactions. The equilibrium constants and association and dissociation constant rates associated with the binding interactions were determined using nonlinear chromatography on an immobilized R32 nAChR column. The computational-chromatography approach can be used to predict and describe ligand-nAChR interactions. Introduction Neuronal nicotinic acetylcholine receptors (nAChRs a ) are ligand-gated ion channels composed of five transmembrane subunits oriented around a central pore. To date, 12 different neuronal subunits have been identified, 9 R-subunits (R2-R10), and 3 -subunits (2-4), and these subunits combine to form a wide variety of homomeric and heteromeric subtypes. 1 Over 50 marketed drugs have been identified as noncompetitive inhibitors (NCIs) of nAChRs, and this property has been identified as the source of unwanted off-target clinical effects, as well as a possible new therapeutic approach. 2 The majority of known NCIs bind to the central lumen of the nAChR, at ring 15, and the ability to predict and describe binding at this site would be an aid in drug development. We have previously reported a model of the lumen of the R34 nAChR (PDB id: 2ASG), which included a defined hydrophobic cleft at ring 15 produced by steric interaction of the two isopropyl and three phenyl moieties of the V (R3) and F(4) residues that constitute ring 15V/F. Binding at this cleft fixes the position of the nonpolar part of a ligand, while the polar moieties form hydrogen bonds with vicinal serine residues in the 8S ring. However, this model is only applicable to nAChR subtypes containing 4 subunits, as these are the only nAChRs that incorporate F residues into ring 15. All of the other subunits contain V in this position and, consequently, five isopropyl moieties (ring 15V). There are pharmacological differences in the responses to NCI between nAChRs containing 4 and those containing 2 or 3. 3 Thus, the objective of the present work was to construct a model of the central lumen of the non-4-subtype of nAChR, to examine the binding of NCIs and to compare the results to data obtained with the R34 nAChR. The R32 nAChR was chosen as it introduced the smallest number of changes in the amino acid composition of the lumen relative to the R34 nAChR. Methods Chromatographic Experiments. The study utilized 18 com- mercially available compounds, Table 1, whose structures and sources have been previously described. 4 The chromatographic studies were carried out as previously described, 4 and a column containing membrane fragments obtained from a HEK293 cell line expressing recombinant R32 nAChR (R32 nAChR-IAM column) was prepared as previously reported. 5 Nonlinear Affinity Chromatography. A detailed description of nonlinear chromatography on an R34-nAChR affinity column and its application in the investigation of NCIs of the nAChR has been previously reported. 6,7 Four parameters (k′, k off , k on , and K) were derived using the Impulse Input Solution approach as described previously. 6 Molecular Modeling. The molecular model of the R32 nAChR binding domain for luminal NCIs was developed based on a previously generated model of the respective domain of the R34 subtype of this receptor. 4 The only differences in these models are associated with sequence polymorphisms in position 15 of the respective  subunits, where the 4 subunit contained an F residue and the 2 subunit contained a V residue. The modification of the R34 model by replacing this residue in each 4 M2 helix was performed with SYBYL 6.8 (Tripos Inc., St. Louis, MO). The resulted heteropentameric channel with R3, 2, R3, 2, and 2 helices, respectively, was further refined by energy minimization and employed in docking simulations using AutoDock (3.0.5.), where all settings and procedures were kept the same as those used in the study of the R34 nAChR. 4 Results Chromatographic Data. The chromatographic retentions (k′) and peak profiles of the 18 test compounds were determined on the R32 nAChR-IAM column. The observed k′ values ranged from 4.6 (5) to 44.1 (9) and 66.3 (3), Table 1. In the test set, 17 of the 18 compounds have been identified as interacting with the central lumen of the nAChR. Ethidium, compound 3, has been shown to inhibit nAChR activity through binding at a site located in the outer vestibule of the receptor and not within the central lumen. 8 This was reflected in a retention profile that substantially differed from the other NCIs used in this study. This result was consistent with data from the previous study of binding to the R34 nAChR central lumen, 4 and, as in the previous study, the data obtained using 3 was not utilized in the further analyses. The remaining set of 17 compounds had also been previously chromatographed on a column containing membrane fragments * To whom correspondence should be addressed. Krzysztof Jozwiak, PhD, Department of Chemistry, Medical University of Lublin, Staszica 6; 20-081 Lublin, Poland. Tel.: +4881 5324875. Fax: +4881 5320413. E-mail: krzysztof.jozwiak@am.lublin.pl. ² Medical University of Lublin. ‡ National Cancer Institute-Frederick/SAIC. § National Institute on Aging, National Institutes of Health. a Abbreviations: nAChR, nicotinic acetylcholine receptor; NCI , non- competitive inhibitors; IAM, immobilized artificial membrane stationary phase. 6279 J. Med. Chem. 2007, 50, 6279-6283 10.1021/jm070784s CCC: $37.00 © 2007 American Chemical Society Published on Web 10/31/2007