An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

Marotta, Christopher B.; Lester, Henry A.; Dougherty, Dennis A.

doi:10.1016/j.chembiol.2015.06.018

Cited by 7 publications

(11 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This saturating concentration was chosen on the basis of previously published data [ 33 , 37 ]. This modulator binds to a specific intrasubunit transmembrane allosteric site of α7 nAChR [ 47 – 50 ], so that the mutations inserted in the orthosteric ligand-binding site of α7 nAChR should not affect binding of PNU120596 to the receptor.…”

Section: Resultsmentioning

confidence: 99%

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

et al. 2017

View full text Add to dashboard Cite

Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117–119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.

show abstract

Section: Resultsmentioning

confidence: 99%

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

et al. 2017

View full text Add to dashboard Cite

show abstract

“…3; Table 1). The slope of the fluorination plot (20.16 6 0.03) is similar to that of acetylcholine (20.15 6 0.02) at TyrA for a7 (Marotta et al, 2015). This suggests a similar strength of interaction, despite the increased bulkiness of the ligand.…”

Section: Resultsmentioning

confidence: 57%

“…If the former mechanism were operative, it would complicate analysis of a silent agonist. However, we have previously shown that in a7 the type 2 PAM PNU-120596 does not alter the orthosteric binding site, as determined by detailed interactions with the native agonist ACh (Marotta et al, 2015). This was done by comparing the impact of a mutation on receptor function with or without the coapplication of the PAM.…”

Section: Resultsmentioning

confidence: 99%

“…This study uses noncanonical amino acid mutagenesis ( Fig. 2) to probe the binding of the silent agonist NS6740 at the orthosteric site of the a7 nAChR (Dougherty, 2013, Van Arnam and and compares the results to previous work from our laboratory on the binding of typical agonists such as acetylcholine (ACh) and nicotine (Marotta et al, 2015). We find that the silent agonist NS6740 makes a cation-p interaction to TyrA and a never before characterized, functionally significant hydrogen bond with TyrA.…”

Section: Introductionmentioning

confidence: 85%

See 1 more Smart Citation

Binding Interactions of NS6740, a Silent Agonist of the α7 Nicotinic Acetylcholine Receptor

Blunt

Dougherty

2019

Mol Pharmacol

View full text Add to dashboard Cite

The a7 nicotinic acetylcholine receptor (nAChR) is a potential drug target for the treatment of a number of neurologic and inflammatory disorders. Silent agonists are an emerging class of drugs that bind to the receptor but do not open the channel. Instead they shift the receptor to a desensitized state. Silent agonists may be able to target a subset of a7 nAChR-mediated signaling processes. Here we use noncanonical amino acid mutagenesis to characterize the binding to a7 by the silent agonist 1,4-diazabicyclo[3.2.2]nonan-4-yl(5-(3-(trifluoromethyl)phenyl)furan-2-yl)methanone (NS6740). We find that, like a7 agonists, NS6740 forms a cation-p interaction with Y115 (TyrA). We also showed that NS6740 makes a novel hydrogen bond to TyrA. This interaction is necessary for the silent agonist activity of NS6740; when the hydrogen bond is blocked, silent agonist NS6740 converts to a conventional partial agonist and appreciably opens the channel in the absence of a positive allosteric modulator (EC 50 150 nM). SIGNIFICANCE STATEMENT Noncanonical amino acids were used to show that a hydrogen bond to tyrosine (Y115) is required for silent agonist activity of NS6740 at the a7 nicotinic acetylcholine receptor.

show abstract

“…Binding interaction between nAChRs and some ligands were investigated experimentally [ 65 , 72 ]. Apart from developing the CDM, this study also investigated potential key binding interactions between human α7 nAChR-LBD and the ligands in the training set.…”

Section: Discussionmentioning

confidence: 99%

Competitive docking model for prediction of the human nicotinic acetylcholine receptor α7 binding of tobacco constituents

Leggett

Sakkiah

et al. 2018

Oncotarget

View full text Add to dashboard Cite

The detrimental health effects associated with tobacco use constitute a major public health concern. The addiction associated with nicotine found in tobacco products has led to difficulty in quitting among users. Nicotinic acetylcholine receptors (nAChRs) are the targets of nicotine and are responsible for addiction to tobacco products. However, it is unknown if the other >8000 tobacco constituents are addictive. Since it is time-consuming and costly to experimentally assess addictive potential of such larger number of chemicals, computationally predicting human nAChRs binding is important for in silico evaluation of addiction potential of tobacco constituents and needs structures of human nAChRs. Therefore, we constructed three-dimensional structures of the ligand binding domain of human nAChR α7 subtype and then developed a predictive model based on the constructed structures to predict human nAChR α7 binding activity of tobacco constituents. The predictive model correctly predicted 11 out of 12 test compounds to be binders of nAChR α7. The model is a useful tool for high-throughput screening of potential addictive tobacco constituents. These results could inform regulatory science research by providing a new validated predictive tool using cutting-edge computational methodology to high-throughput screen tobacco additives and constituents for their binding interaction with the human α7 nicotinic receptor. The tool represents a prediction model capable of screening thousands of chemicals found in tobacco products for addiction potential, which improves the understanding of the potential effects of additives.

show abstract

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

Cited by 7 publications

References 50 publications

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

Binding Interactions of NS6740, a Silent Agonist of the α7 Nicotinic Acetylcholine Receptor

Competitive docking model for prediction of the human nicotinic acetylcholine receptor α7 binding of tobacco constituents

Contact Info

Product

Resources

About