The role of the nicotinic receptor β4 subunit in the antidepressant activity of N,6-dimethyltricy... more The role of the nicotinic receptor β4 subunit in the antidepressant activity of N,6-dimethyltricyclo[5.2.1.0(2,6)]decan-2-amine enantiomers was investigated using wild-type (β4+/+) and knockout (β4-/-) mice. Mice were injected (i.p.) with saline (control) or with either enantiomer (1.0mg/kg base drug) daily for the first two weeks. Forced swim tests (FST) were performed on Day 1 to determine the acute effect of each enantiomer, and on Day 7 and 14, to determine the chronic activity. To examine the remnant effects after drug treatment, a withdrawal period of two more weeks was continued with FSTs performed on Day 21 and 28. Our results indicate that: (1) the acute antidepressant effect elicited by the (S,S)-enantiomer is observed in β4+/+ mice from both sexes, whereas the effect elicited by the (R,R)-enantiomer is only observed in male β4+/+ mice. There is no antidepressant effect for both novel compounds on male and female β4-/- mice, (2) the chronic antidepressant effect elicited by both enantiomers is observed in β4+/+, but not in β4-/-, mice from both sexes, and (3) the residual antidepressant effect mediated by each enantiomer after withdrawal was observed only in female β4+/+ mice. Our results clearly indicate that β4-containing AChRs are targets for the antidepressant activity of these compounds, and that this activity is gender-dependent.
To determine the structural components underlying differences in affinity, potency, and selectivi... more To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca(2+) influx results established that: (a) varenicline activates (μM range) nAChR subtypes with the following rank sequence: hα7>hα4β4>hα4β2>hα3β4>hα1β1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4β2~hα4β4>hα3β4>hα7>Torpedo α1β1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and hα3β4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3β4 and hα4β2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4β2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4β2 nAChRs, hα4β4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3β4- and hα4β2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.
The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine recepto... more The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.
We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, ... more We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, and vinblastine, with that for the noncompetitive antagonist phencyclidine (PCP) at muscle nicotinic acetylcholine receptors (AChRs) in different conformational states. The results established that catharanthine alkaloids: (a) inhibit, in a noncompetitive manner, (+/-)-epibatidine-induced Ca(2+) influx in TE671-halpha1beta1gammadelta cells with similar potencies (IC(50)=17-25microM), (b) inhibit [(3)H]TCP binding to the desensitized Torpedo AChR with higher affinity compared to the resting AChR, and (c) enhance [(3)H]cytisine binding to resting but activatable Torpedo AChRs, suggesting desensitizing properties. Interestingly, PCP inhibits [(3)H]ibogaine binding to the AChR in a steric fashion. This is corroborated by additional docking experiments indicating that the amino groups of neutral ibogaine form hydrogen bonds with the serine ring (position 6'), a location shared with PCP. Since protonated ibogaine forms a salt bridge with one of the acidic residues at the outer ring (position 20'), this ligand could be first attracted to the entrance of the channel by electrostatic interactions. Our data indicate that the catharanthine moiety is a minimum structural requirement for AChR inhibition including, ion channel blocking and desensitization, and that ibogaine and PCP bind to overlapping sites in the desensitized AChR ion channel.
The interaction of tricyclic antidepressants with the human (h) alpha4beta2 nicotinic acetylcholi... more The interaction of tricyclic antidepressants with the human (h) alpha4beta2 nicotinic acetylcholine receptor in different conformational states was compared with that for the noncompetitive antagonist mecamylamine by using functional and structural approaches. The results established that: (a) [(3)H]imipramine binds to halpha4beta2 receptors with relatively high affinity (K(d)=0.83+/-0.08 microM), but imipramine does not differentiate between the desensitized and resting states, (b) although tricyclic antidepressants inhibit (+/-)-epibatidine-induced Ca(2+) influx in HEK293-halpha4beta2 cells with potencies that are in the same concentration range as that for (+/-)-mecamylamine, tricyclic antidepressants inhibit [(3)H]imipramine binding to halpha4beta2 receptors with affinities >100-fold higher than that for (+/-)-mecamylamine. This can be explained by our docking results where imipramine interacts with the leucine (position 9') and valine (position 13') rings by van der Waals contacts, whereas mecamylamine interacts electrostatically with the outer ring (position 20'), (c) van der Waals interactions are in agreement with the thermodynamic results, indicating that imipramine interacts with the desensitized and resting receptors by a combination of enthalpic and entropic components. However, the entropic component is more important in the desensitized state, suggesting local conformational changes. In conclusion, our data indicate that tricyclic antidepressants and mecamylamine efficiently inhibit the ion channel by interacting at different luminal sites. The high proportion of protonated mecamylamine calculated at physiological pH suggests that this drug can be attracted to the channel mouth before binding deeper within the receptor ion channel finally blocking ion flux.
9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhi... more 9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards α3β4 nicotinic acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range. Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with α3β4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of α3β4 nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds for the treatment of addiction.
The role of the nicotinic receptor β4 subunit in the antidepressant activity of N,6-dimethyltricy... more The role of the nicotinic receptor β4 subunit in the antidepressant activity of N,6-dimethyltricyclo[5.2.1.0(2,6)]decan-2-amine enantiomers was investigated using wild-type (β4+/+) and knockout (β4-/-) mice. Mice were injected (i.p.) with saline (control) or with either enantiomer (1.0mg/kg base drug) daily for the first two weeks. Forced swim tests (FST) were performed on Day 1 to determine the acute effect of each enantiomer, and on Day 7 and 14, to determine the chronic activity. To examine the remnant effects after drug treatment, a withdrawal period of two more weeks was continued with FSTs performed on Day 21 and 28. Our results indicate that: (1) the acute antidepressant effect elicited by the (S,S)-enantiomer is observed in β4+/+ mice from both sexes, whereas the effect elicited by the (R,R)-enantiomer is only observed in male β4+/+ mice. There is no antidepressant effect for both novel compounds on male and female β4-/- mice, (2) the chronic antidepressant effect elicited by both enantiomers is observed in β4+/+, but not in β4-/-, mice from both sexes, and (3) the residual antidepressant effect mediated by each enantiomer after withdrawal was observed only in female β4+/+ mice. Our results clearly indicate that β4-containing AChRs are targets for the antidepressant activity of these compounds, and that this activity is gender-dependent.
To determine the structural components underlying differences in affinity, potency, and selectivi... more To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca(2+) influx results established that: (a) varenicline activates (μM range) nAChR subtypes with the following rank sequence: hα7>hα4β4>hα4β2>hα3β4>hα1β1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4β2~hα4β4>hα3β4>hα7>Torpedo α1β1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and hα3β4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3β4 and hα4β2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4β2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4β2 nAChRs, hα4β4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3β4- and hα4β2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.
The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine recepto... more The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.
We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, ... more We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, and vinblastine, with that for the noncompetitive antagonist phencyclidine (PCP) at muscle nicotinic acetylcholine receptors (AChRs) in different conformational states. The results established that catharanthine alkaloids: (a) inhibit, in a noncompetitive manner, (+/-)-epibatidine-induced Ca(2+) influx in TE671-halpha1beta1gammadelta cells with similar potencies (IC(50)=17-25microM), (b) inhibit [(3)H]TCP binding to the desensitized Torpedo AChR with higher affinity compared to the resting AChR, and (c) enhance [(3)H]cytisine binding to resting but activatable Torpedo AChRs, suggesting desensitizing properties. Interestingly, PCP inhibits [(3)H]ibogaine binding to the AChR in a steric fashion. This is corroborated by additional docking experiments indicating that the amino groups of neutral ibogaine form hydrogen bonds with the serine ring (position 6'), a location shared with PCP. Since protonated ibogaine forms a salt bridge with one of the acidic residues at the outer ring (position 20'), this ligand could be first attracted to the entrance of the channel by electrostatic interactions. Our data indicate that the catharanthine moiety is a minimum structural requirement for AChR inhibition including, ion channel blocking and desensitization, and that ibogaine and PCP bind to overlapping sites in the desensitized AChR ion channel.
The interaction of tricyclic antidepressants with the human (h) alpha4beta2 nicotinic acetylcholi... more The interaction of tricyclic antidepressants with the human (h) alpha4beta2 nicotinic acetylcholine receptor in different conformational states was compared with that for the noncompetitive antagonist mecamylamine by using functional and structural approaches. The results established that: (a) [(3)H]imipramine binds to halpha4beta2 receptors with relatively high affinity (K(d)=0.83+/-0.08 microM), but imipramine does not differentiate between the desensitized and resting states, (b) although tricyclic antidepressants inhibit (+/-)-epibatidine-induced Ca(2+) influx in HEK293-halpha4beta2 cells with potencies that are in the same concentration range as that for (+/-)-mecamylamine, tricyclic antidepressants inhibit [(3)H]imipramine binding to halpha4beta2 receptors with affinities >100-fold higher than that for (+/-)-mecamylamine. This can be explained by our docking results where imipramine interacts with the leucine (position 9') and valine (position 13') rings by van der Waals contacts, whereas mecamylamine interacts electrostatically with the outer ring (position 20'), (c) van der Waals interactions are in agreement with the thermodynamic results, indicating that imipramine interacts with the desensitized and resting receptors by a combination of enthalpic and entropic components. However, the entropic component is more important in the desensitized state, suggesting local conformational changes. In conclusion, our data indicate that tricyclic antidepressants and mecamylamine efficiently inhibit the ion channel by interacting at different luminal sites. The high proportion of protonated mecamylamine calculated at physiological pH suggests that this drug can be attracted to the channel mouth before binding deeper within the receptor ion channel finally blocking ion flux.
9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhi... more 9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards α3β4 nicotinic acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range. Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with α3β4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of α3β4 nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds for the treatment of addiction.
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Papers by Katarzyna Targowska-duda