Defining the Putative Inhibitory Site for a Selective Negative Allosteric Modulator of Human α4β2 Neuronal Nicotinic Receptors
Abstract
Neuronal nicotinic receptors (nAChRs) have been implicated in several diseases and disorders such as autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, and nicotine addiction. To understand the role of nAChRs in these conditions, it would be beneficial to have selective molecules that target specific nAChRs in vitro and in vivo. Our laboratory has previously identified a novel allosteric site on human α4β2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human α4β2 nAChRs, a subtype implicated in nicotine addiction. This study characterizes the allosteric site via site-directed mutagenesis. Three amino acids (Phe118, Glu60, and Thr58) on the β2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human α4β2 nAChRs. SAR studies with KAB-18 analogues and various mutant α4β2 nAChRs also provided information concerning how different physiochemical features influence the inhibition of nAChRs through this allosteric site. Together, these studies identify the amino acids that contribute to the selective antagonism of human α4β2 nAChRs at this allosteric site. Finally, these studies define the physiochemical features of ligands that influence interaction with specific amino acids in this allosteric site.
Additional Information
© 2012 American Chemical Society. Published In Issue September 19, 2012; Article ASAP June 06, 2012; Just Accepted Manuscript May 25, 2012; Received: March 17, 2012; Accepted: May 25, 2012. All cDNAs for human nAChR subunits and the human α4β2 stably transfected nAChR cell line (used in the supplemental material) were kindly provided by Dr. Jon M. Lindstrom, Department of Neuroscience School of Medicine, University of Pennsylvania, Philadelphia, PA. We also thank Dr. Henry A. Lester and Dr. Dennis A. Dougherty at the California Institute of Technology for their suggestions in improving this manuscript. Funding: This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant DA029433]. Financial support for B.J.H. is from the National Institutes of Health National Institute on Drug Abuse Diversity Supplement. Financial support for REP is from the American Foundation for Pharmaceutical Education. A grant of computational resources was received from the Ohio Supercomputer Center. Author Contributions: H.B.J., B.R.T., and M.D.B. participated in research design; H.B.J., G.-C.T.F., Y.B., and P.R.E. conducted experiments; B.S.C., B.R.T., and L.C. contributed new reagents or analytic tools; H.B.J., G.-C.T.F., Y.B., and P.R.E performed data analysis; H.B.J., G.-C.T.F., B.R.T., and M.D.B. wrote or contributed to the writing of the manuscript.Attached Files
Published - cn300035f.pdf
Supplemental Material - cn300035f_si_001.pdf
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Additional details
- PMCID
- PMC3447398
- Eprint ID
- 35434
- Resolver ID
- CaltechAUTHORS:20121113-123242663
- DA029433
- NIH
- National Institute on Drug Abuse
- American Foundation for Pharmaceutical Education
- Ohio Supercomputer Center
- Created
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2012-11-14Created from EPrint's datestamp field
- Updated
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2021-11-09Created from EPrint's last_modified field