Abstract
Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. We have previously reported on the pharmacological activity of several compounds that act as negative allosteric modulators (NAMs) of nAChRs. In the following studies, the effects of 30 NAMs from our small chemical library on both human α4β2 (Hα4β2) and human α3β4 (Hα3β4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC50 values ranging from 2.4 μM to more than 100 μM. Several NAMs showed relative selectivity for Hα4β2 nAChRs with IC50 values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for Hα4β2 nAChRs. This molecule contains three phenyl rings, one piperidine ring, and one ester bond linkage. Structure–activity relationship (SAR) analyses of our data revealed three regions of KAB-18 that contribute to its relative selectivity. Predictive three-dimensional quantitative SAR (comparative molecular field analysis and comparative molecular similarity indices analysis) models were generated from these data, and a pharmacophore model was constructed to determine the chemical features that are important for biological activity. Using docking approaches and molecular dynamics on a Hα4β2 nAChR homology model, a binding mode for KAB-18 at the α/β subunit interface that corresponds to the predicted pharmacophore is described. This binding mode was supported by mutagenesis studies. In summary, these studies highlight the importance of SAR, computational, and molecular biology approaches for the design and synthesis of potent and selective antagonists targeting specific nAChR subtypes.
Footnotes
This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant DA029433]. B.J.H. was supported by the National Institutes of Health National Institute on Drug Abuse Diversity Supplement. R.E.P. was supported by the American Foundation for Pharmaceutical Education. A grant of computational resources was received from the Ohio Supercomputer Center.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.168211.
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ABBREVIATIONS:
- nAChR
- neuronal nicotinic acetylcholine receptor
- Hα4β2
- human α4β2
- Hα3β4
- human α3β4
- NAM
- negative allosteric modulator
- LBD
- ligand binding domain
- MD
- molecular dynamics
- CoMFA
- comparative molecular field analysis
- CoMSIA
- comparative molecular similarity indices analysis
- SAR
- structure–activity relationship
- QSAR
- quantitative SAR
- GASP
- genetic algorithm similarity program
- HBK
- HEPES-buffered Krebs
- HEK
- human embryonic kidney
- fluo-4-AM
- fluo-4-acetoxymethyl ester
- DMSO
- dimethyl sulfoxide
- HBA
- hydrogen bond acceptor.
- Received April 2, 2010.
- Accepted June 14, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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