N,6-dimethyltricyclo[5.2.1.02,6]decan-2-amine
Enantiomers Interact with Luminal and Non-luminal Domains at the Human 34 Nicotinic
Acetylcholine Receptor
Hugo
R. Arias, Katarzyna M. Targowska-Duda, Krzysztof Jozwiak
Hugo R. Arias, Department of Medical Education,
College of Medicine, California Northstate University, Elk Grove, CA 95757, USA
Katarzyna M.
Targowska-Duda, Krzysztof Jozwiak, Department of Chemistry, Laboratory of Medicinal Chemistry
and Neuroengineering, Medical University of Lublin, 20-093 Lublin, Poland
Correspondence to: Hugo R. Arias, Department of Medical Education,
California Northstate University College of Medicine, 9700 W. Taron Dr., Elk
Grove, CA 95757, USA.
Email: hugo.arias@cnsu.edu
Telephone: +1-916-647-0458
Fax:
+1-916-686-7310
Received: November 22,
2014 Revised: January 20, 2015
Accepted: January 25, 2015
Published online: March 30, 2015
ABSTRACT
The objective of
current study is to characterize the interaction of (S,S)- and (R,R)-N,6-dimethyltricyclo[5.2.1.02,6]decan-2-amine
enantiomers with human (h) ¦Á3¦Â4 nicotinic acetylcholine receptors (AChRs) in
different conformational states by using pharmacological and structural
approaches. The pharmacological results established that both enantiomers bind
with very low affinity to the [3H]imipramine binding sites at h¦Á3¦Â4
AChRs in the resting and desensitized states (apparent Ki~0.5-0.8
mM). The obtained nH values are lower than unity (~0.5) indicating
negative cooperative interactions between imipramine and each enantiomer. Based
on these results, we suggest that these interactions are allosteric in nature,
and consequently, imipramine and each enantiomer bind to non-overlapping sites.
This possibility is supported by molecular docking and dynamics results,
indicating that the enantiomers, in the protonated and neutral states, interact
with mixed luminal/non-luminal and intersubunit (i.e., non-luminal) domains,
both formed between the ¦Á3 and ¦Â4 transmembrane segments. Based on our finding,
we suggest that the previously observed antidepressant-like activity elicited
by these enantiomers (Targowska-Duda et al, 2013; Neurosci. Lett. 553,
186-190) may be mediated by a novel negative allosteric modulatory mechanism at
the h¦Á3¦Â4 AChR.
© 2015 ACT. All
rights reserved.
Key words: Nicotinic acetylcholine receptors; Antidepressants;
Allosteric modulation; Conformational states; Molecular modeling; Molecular
dynamics
Arias HR,
Targowska-Duda KM, Jozwiak K. N,6-dimethyltricyclo [5.2.1.02,6]decan-2-amine
Enantiomers Interact with Luminal and Non-luminal Domains at the Human ¦Á3¦Â4 Nicotinic
Acetylcholine Receptor. Journal of
Biochemistry and Molecular Biology Research 2015; 1(1): 19-24 Available from: URL:
http://www.ghrnet.org/index.php/jbmbr/article/view/929
INTRODUCTION
An increasing amount of data supports the concept that nicotinic
acetylcholine receptors (AChRs) are targets for the clinical activity of
different antidepressants[1,2]. Structurally and functionally
different antidepressants inhibit various neuronal AChRs by noncompetitive
mechanisms[1-5]. In particular, ¦Á3¦Â4 AChRs can be inhibited by
different antidepressant-like compounds, including tricyclic antidepressants
(TCAs) and mecamylamine[6], serotonin selective reuptake inhibitors
(SSRIs)[7], and bupropion[8].
Recent studies
demonstrated that the N,6-dimethyltricyclo [5.2.1.02,6]decan-2-amine
enantiomers show antidepressant-like activity[9], and interact with
luminal and non-luminal binding sites at the human (h) ¦Á4¦Â2 AChR[10].
These novel compounds inhibit ¦Á4¦Â2 and ¦Á3¦Â4 AChRs in the submicromolar
concentration range (unpublished data, Targacept, Inc., Winston-Salem, NC,
USA). However, we do not have a clear picture of how these compounds interact
with ¦Á3¦Â4 AChRs and what are the structural components of its binding site(s).
AChRs
containing the ¦Â4-subunit (e.g., ¦Á3¦Â4 and ¦Á3¦Â3¦Â4) are expressed in the
habenulo-interpeduncular pathway in high amounts[11]. These AChR
subtypes are involved in drug addiction and mood disorders, among other
pathological states[2]. Interestingly, these receptors modulate the
antidepressant-like activity of bupropion[12] and nicotine[13].
Thus, to characterize the interaction of N,6-dimethyltricyclo[5.2.1.02,6]decan-2-amine
enantiomers (Figure 1) with h¦Á3¦Â4 AChRs in different conformational states,
structural and functional approaches are applied, including radioligand
competition binding assays using [3H]imipramine as a probe for the
antidepressant sites[6], as well as molecular docking and molecular
dynamics (MD) studies.
Materials
and Methods
Materials
[3H]Imipramine (47.5 Ci/mmol) was obtained from PerkinElmer
Life Sciences Products, Inc. (Boston, MA, USA) and stored in ethanol at -20¡æ. The N,6-dimethyltricyclo[5.2.1.02,6]decan-2-amine
enantiomers were obtained from Targacept, Inc. (Winston-Salem, NC, USA).
Imipramine hydrochloride, polyethylenimine, and trypsin/EDTA were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). (¡À)-Epibatidine hydrochloride was
obtained from Tocris Bioscience (Ellisville, MO, USA). -Bungarotoxin (-BTx) was
obtained from Biotoxins Incorporated (St. Cloud, FL, USA). Fetal bovine serum
(FBS) was obtained from Aleken Biologicals (Nash, TX, USA). Salts were of
analytical grade.
[3H]Imipramine
competition binding experiments using h¦Á3¦Â4 AChRs in different conformational
states
The effect of (S,S)- and (R,R)-N,6-dimethyltricyclo[5.2.1.02,6]decan-2-amine
on [3H]imipramine binding to h¦Á3¦Â4 AChRs in different conformational
states was determined using membranes prepared from HEK293-h¦Á3¦Â4 cells as
described previously[6]. In this regard, HEK293-h¦Á3¦Â4 cells,
cultured as described previously[6], express predominantly AChRs
with the (¦Á3)3(¦Â4)2 stoichiometry[14]. AChR
membranes (1.5 mg/mL), prepared from these cells, were suspended in binding
saline buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1
mM MgCl2, pH 7.4) with 20 nM [3H]imipramine, in the
presence of 0.1 ¦ÌM -BTx (resting/-BTx-bound state) or 0.1 ¦ÌM (¡À)-epibatidine
(desensitized/epibatidine-bound state), and preincubated for 30 min at room
temperature (RT). Bungarotoxins such as -BTx are competitive antagonists
that maintain the AChRs in the resting (closed) state[15]. The
nonspecific binding was determined in the presence of 100 µM imipramine. The
total volume was divided into aliquots, and increasing concentrations of each
enantiomer were added to each tube and incubated for 90 min at RT. AChR-bound
radioligand was then separated from free ligand by a filtration assay using a
48-sample harvester system with GF/B Whatman filters (Brandel Inc.,
Gaithersburg, MD, USA), previously soaked with 0.5% polyethylenimine for 30
min. The membrane-containing filters were transferred to scintillation vials
with 3 mL of Bio-Safe II (Research Product International Corp, Mount Prospect,
IL, USA), and the radioactivity was determined using a Beckman LS6500
scintillation counter (Beckman Coulter, Inc., Fullerton, CA, USA).
The
concentration¨Cresponse data were curve-fitted by nonlinear least squares
analysis using the Prism software (GraphPad Software, San Diego, CA). The
calculated IC50 and Hill coefficient (nH) values were
calculated using the following equation. The observed IC50 values
from the competition experiments described above were transformed into
inhibition constant (Ki) values using the Cheng-Prusoff
relationship[16]: Ki = IC50/{1+([[3H]imipramine]/Kdimipramine)}
(1)
where [[3H]imipramine] is the initial concentration of [3H]imipramine,
and Kdimipramine is the [3H]imipramine
dissociation constant for the h¦Á3¦Â4 AChR (0.41 µM;[6]). The apparent
Ki and nH values were summarized in table 1.
Homology
modeling and molecular docking
Sequence alignment of the Torpedo AChR subunits and the h¦Á3 and
h¦Â4 subunits was first performed using the ClustalW2 server
(www.ebi.ac.uk/Tools/msa/clustalw2). The residue numbering of this alignment
corresponds to the h¦Á3 and h¦Â4 sequence from the Protein knowledgebase (i.e.,
UniProtKB) implemented in the ExPASy Molecular Biology Server[17].
The cryo-electron microscopy structure of the Torpedo AChR determined at
~4 Å resolution (PDB ID: 2BG9)[18] was used as target for molecular
modeling. Models of the h¦Á3¦Â4 AChR were subsequently constructed by homology
modeling methods using the Torpedo AChR as template, as previously
described[6,10,13,19-21]. Modeller 9.9 was used to obtain 100
homology models, and subsequently, their Discrete Optimized Protein Energy profiles[22]
were assessed. The best model was subjected to model quality assessment, using
the web-based tools of Verify3D[23] and ProCheck[24].
For the
molecular docking procedure, both (S,S)- and (R,R)-enantiomers,
in the protonated and neutral forms, were first modeled using HyperChem 6.03
(HyperCube Inc., Gainesville, FL, USA), optimized using the semi-empirical
method AM1, and then transferred for the docking simulation as previously
described[6,10,13,19-21]. Molegro Virtual Docker (MVD, v 5.0.0, Molegro
ApS Aarhus, Denmark) was used for docking simulations of flexible ligands into
the rigid target of the AChR model. The docking space was defined to cover all
transmembrane domains (TMDs) (i.e., M1-M4), the ion channel lumen, and the
extracellular (EXD)-TMD junction. The subsequent docking simulations were
performed using the settings previously described[6,10,13,19-21].
The lower energy conformations [i.e., representing the lowest value of the
scoring function (MolDockScore)] were selected from each cluster of superposed
poses.
Molecular
dynamics
To test the stability of each enantiomer in its predicted docking
sites, 15-ns MD simulations were performed using the Yasara package (v 11.11.2,
Yasara Biosciences, Graz, Austria) and the settings described previously[1,10,13,21].
The root mean
square deviation (RMSD) (see legend from figure 4) values were estimated using
the Yasara software, as previously described[10,13]. These values
represent the inter-molecular conformational changes and the translation of the
whole molecule in the binding site. Poses obtained every 25-ps simulation
snapshot were extracted from the simulation trajectory of 15-ns total time and
compared to its reference position obtained by docking.
Results
[3H]Imipramine
competition binding experiments using h¦Á3¦Â4 AChRs in different conformational
states
The binding affinity of each (S,S)- and (R,R)-enantiomer
for the imipramine binding sites at the h¦Á3¦Â4 AChR was determined by [3H]imipramine
competition binding experiments, when the receptor is in the resting (-BTx-bound) or
desensitized (epibatidine-bound) state (Figure 2). The enantiomers displace
only ~50% [3H]imipramine binding at the highest concentrations used.
The apparent Ki values suggest that both enantiomers bind with very
low affinity to the [3H]imipramine binding sites at h¦Á3¦Â4 AChRs in
either the resting or desensitized state (Table 1). The observed nH
values are lower than unity (close to 0.5) (Table 1), suggesting a negative
cooperative interaction between imipramine and each compound.
Molecular
docking results
The (S,S)- and (R,R)-enantiomer in the protonated state
interacts with mixed luminal/nonluminal and non-luminal binding domains (i.e.,
intersubunit site) (see 2D and 3D models in figure 3). Since the docking
results in the neutral state are the same as that for the protonated state, the
results in the neutral state are not shown. The docking results indicate that
the mixed luminal/non-luminal (Figure 3C) and intersubunit sites (Figure 3D)
for each enantiomer do not overlap the imipramine binding sites. In the mixed
luminal/non-luminal site, the enantiomers mainly interact by van der Waals
contacts with luminal residues, including 3-Val258 and 4-Leu259 (position
17¡¯; non-polar ring), 4-Phe255 and 3-Val254 (position 13¡¯; valine ring), as well as with non-luminal
residues, comprising 4-Ile263 (position 21¡¯), 3-Thr260 (position 19¡¯), 4-Ile260 (position 18¡¯), 3-Leu257 (position 16¡¯), and 4-Phe256 (position 14¡¯) (Table 2).
In addition, the molecule is stabilized by electrostatic interactions with 3-Glu261
(position 20¡¯) at the outer ring.
In the
intersubunit site, each enantiomer interacts predominantly by van der Waals
contacts with nonpolar residues at M1 (i.e., 3-Ile219, 3-Pro220, 3-Leu223, 3-Ile224, and 3-Leu227), M2
[i.e., 4-Leu250
(position 8¡¯), 4-Thr254 (position 12¡¯), 4-Leu258 (position 16¡¯), 3-Leu252 (position 10¡¯), 3-Phe255 (position 14¡¯), 3-Leu259 (position 18¡¯)], and M3 (i.e., 4-Ile289) (Table 2).
Molecular
dynamics results
The MD results for each protonated enantiomer indicate that the
interactions with either the mixed luminal/non-luminal or intersubunit binding
site at the h34 AChR are stable
during the 15-ns MD simulations (Figure 4). The RMSD values that represent the
inter-molecular conformational changes and the translation of the ligand within
the binding site were calculated for each studied enantiomer. The higher RMSD
value for the (S,S)-enantiomer (Figure 4A) at the mixed
luminal/non-luminal binding pocket than that for the (R,R)-enantiomer
(Figure 4B) suggests that the (S,S)-enantiomer oscillates within the
binding pocket more than the (R,R)-enantiomer. Despite the observed
molecular oscillation, each molecule remains stable within the binding site
during the 15-ns MD simulation.
Discussion
In this work, we determined the binding affinity of novel
N,6-dimethyltricyclo[5.2.1.02,6]decan-2-amine enantiomers for the
imipramine sites and the most important structural components of their binding sites at the h¦Á34 AChR.
The
radioligand binding competition results indicate that both enantiomers bind
with very low affinity (apparent Ki ~0.5-0.8 mM) to the [3H]imipramine
sites, but each enantiomer has opposite preference for the resting or
desensitized AChR (Table 1). The observed low affinity is similar to that
obtained for the h¦Á4¦Â2 AChR[10] and for mecamylamine-induced
inhibition of [3H]imipramine binding to resting and desensitized h¦Á3¦Â4
AChRs, where IC50 ~0.2 mM and nH ~0.5 values were also
observed[6]. These results support the notion that the enantiomers
interact with the imipramine sites mainly by allosteric mechanisms. Our docking
results (Figure 3) are in agreement with the [3H]imipramine binding
results showing that the sites for the enantiomers are different to that for
imipramine.
We previously
characterized the imipramine and mecamylamine binding sites in the h34 AChR by
pharmacologic and molecular docking methods[6]. In that work, we
used a docking space centered on the ion channel that ensured covering only the
M2 segments from each subunit, including the ion channel. However, increasing
evidence indicates that the AChR TMD might be involved in the interaction and function
of several allosteric modulators[4,10,13,25-30]. Based on these
reports, we used the whole h¦Á3¦Â4-TMD for the searching of potential binding
sites for our novel enantiomers. The molecular docking and MD results suggest
that the (S,S)- (Figure 3) and (R,R)-enantiomer (data not shown)
interacts with two non-overlapping binding sites, the mixed luminal/non-luminal
and intersubunit sites. These sites coincide with that previously described at
other ion channels. For example, the intersubunit site for the enantiomers
overlaps the intrasubunit pocket for desflurane and propofol at the
proton-activated ion channel from the bacteria Gleobacter violaceus
(GLIC) (i.e., GLIC-Ile202 corresponding to 3-Ile218)[31], two
intrasubunit sites for halothane at the ¦Á4¦Â2 AChR (i.e., ¦Â2-Leu226 and ¦Â2-Ile261
corresponding to ¦Á3-Leu227 and ¦Â4-Ile263, respectively)[26], the
non-luminal site 2 described for (S)-(+)-mecamylamine at the muscle AChR (i.e.,
¦Á1-Ile219 corresponding to ¦Á3-Ile219)[13], and the ketamine and
halothane binding sites determined at h¦Á7 AChRs (i.e., ¦Á7-Ile217 corresponding
to ¦Á3-Ile219)[27]. On the other hand, the mixed luminal/non-luminal
site for the enantiomers overlaps the luminal site 2 determined for
(S)-(+)-mecamylamine at the h(¦Á4)2(¦Â2)3-TMDs (i.e., ¦Â2-Lys260
corresponding to ¦Á3-Glu261 at position 20¡¯)[28].
Recent studies
demonstrated that the N,6-dimethyltricyclo [5.2.1.02,6]decan-2-amine
enantiomers produce antidepressant-like activity in mice in a gender-dependent
manner[10], and interact with luminal and non-luminal binding sites
at the h¦Á4¦Â2 AChR[10]. The present work supports two hypotheses: (1)
that ¦Â4-containing AChRs are involved in the antidepressant-like activity
mediated by the (S,S)- and (R,R)-enantiomers[9], and
(2) that, as previously suggested for the h¦Á4¦Â2 AChR[10], the
interaction of these enantiomers with the intersubunit locus may impede the
rotation of the M2 segment, blocking or disrupting the switching of the
hydrophobic residues located along the closed ion channel, finally maintaining
the receptor in a nonconducting conformation state. In this regard, the
antidepressant-like activity elicited by both enantiomers might be associated
with novel negative allosteric modulatory mechanisms, where mixed
luminal/non-luminal and intersubunit domains play an important role.
Abbreviations: AChR, nicotinic
acetylcholine receptor; EXD, extracellular domain; NCA, noncompetitive
antagonist; RMSD, root mean square deviation; MD, molecular dynamics; TCAs,
tricyclic antidepressants; TMD, transmembrane domain; SSRIs, serotonin
selective reuptake inhibitors; ¦Ê-BTx, ¦Ê-bungarotoxin; RT, room temperature; BS,
binding saline; Ki, inhibition constant; Kd, dissociation constant; IC50,
ligand concentration that produces 50% inhibition of binding; nH, Hill
coefficient; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum.
ACKNOWLEDGMENTS
This work was supported the TEAM research subsidy from the Foundation
for Polish Science (to K.J.), and from the National Science Center, Poland
(SONATA funding, UMO-2013/09/D/NZ7/04549) [to K.T-D. (PI) and H.R.A. (Co-PI)].
Dr. Targowska-Duda was supported by the TEAM program to work at Dr. Arias¡¯
laboratory.
CONFLICT OF INTERESTS
The Author has no conflicts of interest to declare.
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Peer reviewer: Chau
H. Nguyen, PhD, Assistant Professor, Department of Pharmaceutical Sciences,
D'Youville College School of Pharmacy, DAC 440, 320 Porter Avenue, Buffalo, NY,
14201, USA.
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