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Defining the putative inhibitory site for a selective negative allosteric modulator of human ?4?2 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 ?4?2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human ?4?2 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 ?2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human ?4?2 nAChRs. SAR studies with KAB-18 analogues and various mutant ?4?2 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 ?4?2 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.

SUBMITTER: Henderson BJ

PROVIDER: S-EPMC3447398 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Defining the putative inhibitory site for a selective negative allosteric modulator of human α4β2 neuronal nicotinic receptors.

Henderson Brandon J BJ González-Cestari Tatiana F TF Yi Bitna B Pavlovicz Ryan E RE Boyd R Thomas RT Li Chenglong C Bergmeier Stephen C SC McKay Dennis B DB

ACS chemical neuroscience 20120525 9

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 ...[more]

PMID: 23019495

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Project description:Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human ?4?2 and ?3?4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the ?2 subunit were independently mutated to the corresponding residues found on the ?4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human ?4?2 over ?3?4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human ?4?2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders.

Dataset Information

Defining the putative inhibitory site for a selective negative allosteric modulator of human ?4?2 neuronal nicotinic receptors.

Publications

Defining the putative inhibitory site for a selective negative allosteric modulator of human α4β2 neuronal nicotinic receptors.

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