Imipramine Binding Research Articles

Mechanism of EAG1 channel inhibition by imipramine binding to the PAS domain

Mechanism of EAG1 channel inhibition by imipramine binding to the PAS domain

Molecular mechanism of EAG1 channel inhibition by imipramine binding to the PAS domain

Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an N-terminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels.

The mechanism of a high-affinity allosteric inhibitor of the serotonin transporter

The serotonin transporter (SERT) terminates serotonin signaling by rapid presynaptic reuptake. SERT activity is modulated by antidepressants, e.g., S-citalopram and imipramine, to alleviate symptoms of depression and anxiety. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). In this study, our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. The SERT inhibition by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we reveal a S1-S2 coupling mechanism that will facilitate rational design of high-affinity SERT allosteric inhibitors.

Mechanistic Characterization of the Allosteric Communications between the Central Binding Site and the Extracellular Vestibule of the Serotonin Transporter

The serotonin transporter (SERT) is a primary target for antidepressants, e.g. S-citalopram and imipramine to alleviate symptoms of depression and anxiety, and functions by exploiting pre-existing ion gradients of Na+, Cl−, and K+ to translocate serotonin from the synaptic cleft into the presynaptic neuron. SERT crystal structures reveal that two S-citalopram molecules bind in both the central binding (S1) site and a site in the extracellular vestibule (S2 site). We investigated the allosteric communications between the S1 and S2 sites by carrying out extensive molecular dynamics (MD) simulations of hSERT in complex with various combinations of S1- and S2-bound ligands. The comparative analysis of the simulation results in combination with in vitro binding assays and mutagenesis reveal that the binding of S-citalopram or imipramine to the S1 site is allosterically coupled to the S-citalopram binding to S2 through the Thr497-Phe335 motif. The different configurations of this motif in turn affect the propensity of Glu494 in the S2 site to form a salt bridge with the S2-bound S-citalopram. In addition, our MD simulation results indicate that Lu AF60097, the first high-affinity S2-ligand, allosterically couples the ligand binding to S1 through a similar mechanism. In the presence of S1-bound imipramine but not S-citalopram, our combined in silico and in vitro analysis show that Lu AF60097 occupies a dynamically formed sub-pocket in the extracellular vestibule. We propose that the occupation of this cavity is responsible for the significantly improved affinity of Lu AF60097 in the presence of S1-bound imipramine. Together we reveal a coupling mechanism between the S1 and S2 sites that will facilitate rational development of high-affinity SERT allosteric inhibitors.

Tricyclic antidepressants inhibit hippocampal α7* and α9α10 nicotinic acetylcholine receptors by different mechanisms.

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca2+ influx in GH3-α7 cells with the following potency (IC50 in μM) rank: amitriptyline (2.7 ± 0.3) > doxepin (5.9 ± 1.1) ∼ imipramine (6.6 ± 1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ± 8.5 μM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ± 0.05 μM) in a competitive and voltage-independent manner, and (b) inhibit [3H]imipramine binding to resting α7 AChRs with the following affinity rank (IC50 in μM): imipramine (1.6 ± 0.2) > amitriptyline (2.4 ± 0.3) > doxepin (4.9 ± 0.6), whereas imipramine's affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action.

Studies of drug interactions with alpha1-acid glycoprotein by using on-line immunoextraction and high-performance affinity chromatography

A method that combined on-line immunoextraction with high-performance affinity chromatography was developed to examine the binding of drugs with α1-acid glycoprotein (AGP). Affinity microcolumns containing immobilized polyclonal anti-AGP antibodies were developed that had a capture efficiency of up to 98.4% for AGP and a binding capacity of 0.72nmol AGP when using a 20mm×2.1mm i.d. microcolumn. These microcolumns were employed in various formats to examine the binding of drugs to normal AGP and AGP that had been adsorbed from serum samples for patients with systemic lupus erythematosus (SLE). Drugs that were screened in zonal elution experiments for their overall binding to these types of AGP included chlorpromazine, disopyramide, imipramine, propranolol, and warfarin. Most of these drugs showed an increase in their binding to the AGP from SLE serum when compared to normal AGP (i.e., an increase of 13–76%); however, disopyramide gave a 21–25% decrease in retention when the same AGP samples were compared. Frontal analysis was used to further evaluate the binding of disopyramide and imipramine to these forms of AGP. Both drugs gave a good fit to a model that involved a combination of saturable and non-saturable interactions with AGP. Changes in the non-saturable interactions accounted for most of variations seen in the binding of disopyramide and imipramine with the AGP samples. The methods used in this study could be adapted for use in personalized medicine and the study of other proteins or drugs using aqueous mixtures or clinical samples.

Coronaridine congeners inhibit human α3β4 nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites

To characterize the interaction of coronaridine congeners with human (h) α3β4 nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The Ca2+ influx results established that coronaridine congeners noncompetitively inhibit hα3β4 AChRs with the following potency (IC50's in μM) sequence: (−)-ibogamine (0.62±0.23)∼(+)-catharanthine (0.68±0.10)>(−)-ibogaine (0.95±0.10)>(±)-18-methoxycoronaridine [(±)-18-MC] (1.47±0.21)>(−)-voacangine (2.28±0.33)>(±)-18-methylaminocoronaridine (2.62±0.57μM)∼(±)-18-hydroxycoronaridine (2.81±0.54)>(−)-noribogaine (6.82±0.78). A good linear correlation (r2=0.771) between the calculated IC50 values and their polar surface area was found, suggesting that this is an important structural feature for its activity. The radioligand competition results indicate that (±)-18-MC and (−)-ibogaine partially inhibit [3H]imipramine binding by an allosteric mechanism. Molecular docking, molecular dynamics, and in silico mutation results suggest that protonated (−)-18-MC binds to luminal [i.e., β4-Phe255 (phenylalanine/valine ring; position 13′), and α3-Leu250 and β4-Leu251 (leucine ring; position 9′)], non-luminal, and intersubunit sites. The pharmacophore model suggests that nitrogens from the ibogamine core as well as methylamino, hydroxyl, and methoxyl moieties at position 18 form hydrogen bonds. Collectively our data indicate that coronaridine congeners inhibit hα3β4 AChRs by blocking the ion channel's lumen and probably by additional negative allosteric mechanisms by interacting with a series of non-luminal sites.

Novel 2-(substituted benzyl)quinuclidines inhibit human α7 and α4β2 nicotinic receptors by different mechanisms

This work presents the design and synthesis of a series of novel 2-benzylquinuclidine derivatives, comprising 12 methiodide and 11 hydrochloride salts, and their structural and pharmacological characterization at the human (h) α7 and α4β2 nicotinic receptors (nAChRs). The antagonistic potency of these compounds was tested by Ca2+ influx assays on cells expressing the hα7 or hα4β2 nAChR subtype. To determine the inhibitory mechanisms, additional radioligand binding experiments were performed. The results indicate that the methiodides present the highest affinities for the hα7 nAChR agonist sites, while the same compounds bind preferably to the hα4β2 nAChR ion channel domain. These results indicate that the methiodides are competitive antagonists of the hα7 nAChR but noncompetitive antagonists of the hα4β2 subtype. Docking and molecular dynamics simulations showed that the methiodide derivative 8d binds to the hα7 orthosteric binding sites by forming stable cation-π interactions between the quaternized quinulinuim moiety and the aromatic box in the receptor, whereas compounds 7j and 8j block the hα4β2 AChR ion channel by interacting with a luminal domain formed between the serine (position 6′) and valine (position 13′) rings that overlaps the imipramine binding site.

A way forward for research on biomarkers for psychiatric disorders

Webster’s New World Medical Dictionary provides a simple definition of a biomarker as “a biologic feature that can be used to measure the presence or progress of disease or the effects of treatment.” In the research literature, the term “candidate biomarker” is often loosely applied to any biological feature associated with a disorder. The search for biomarkers for psychiatric disorders has a long history, with earlier studies investigating molecular markers, like platelet imipramine binding or cerebrospinal 5-hydroxyindoleacetic acid (5-HIAA) in people with depression, or behavioural markers, such as smooth pursuit eye movements in people with schizophrenia. More recent searches continue to look for blood biomarkers (e.g., brain-derived neurotrophic factor and cytokines in people with schizophrenia or depression) and behavioural biomarkers (e.g., fear extinction in people with anxiety), and are progressing on to more high-tech searches, such as the use of proteomics to define signatures of proteins altered in blood or the use of support vector machine analysis to define informative neuroimaging patterns or panels of immunoassays altered in specific disorders.

Liposomal binding of imipramine in human red cell/albumin solution with simulated plasmapharesis

Abstract There has been much recent interest in the use of intravenous lipid based products inthe treatment of tricyclic ntidepressant toxicity. High affinity liposomal carriers have been designed which may be more effective binders of

Synthesis of 3,7‐Disubstituted Imipramines by Palladium‐Catalysed Amination/Cyclisation and Evaluation of Their Inhibition of Monoamine Transporters

We describe a novel approach for the synthesis of a series of 3,7-difunctionalised symmetric and unsymmetrical analogues of the tricyclic antidepressant (TCA) imipramine, which uses a key palladium-catalysed amination/cyclisation of an ester-functionalised dibromide. Of the ester, methyl, hydroxymethyl and methoxymethyl disubstituted compounds prepared, 3,7-dimethyl-imipramine was found to be the most potent against the human serotonin transporter (hSERT). The inhibitory potency of 3,7-dimethyl imipramine was found to be at least as high as the parent imipramine. This novel TCA also exhibits an increased selectivity (relative to imipramine) in binding to hSERT versus the human norepinephrine transporter (hNET). Even higher selectivity could be obtained with 3,7-dihydroxymethyl imipramine, which was found to be 167-fold more selective for hSERT over hNET, representative of a 120-fold gain in selectivity relative to the parent imipramine. These results further validate our previous model for the binding of imipramine and high-affinity analogues of imipramine to the central binding site of hSERT.

3H]8-OH-DPAT labels the 5-Hydroxytryptamine uptake recognition site and the 5-HT1A binding site in the rat striatum

The binding of [3H]8-hydroxy-2-(di-N-propylamino)-tetralin ([ 3H]8-OH-DPAT) to rat hippocampal and striatal membranes has been compared. In the hippocampus, low concentrations of [3H]8-OH-DPAT bound to a single, high affinity site which was sensitive to inhibition by spiperone, buspirone and ergotamine but not by mianserin, quipazine or (-)-propranolol. This is consistent with a selective labeling of the 5-HT1A receptor. In the striatum, [3H]8-OH-DPAT bound to two sites with high and low affinity (KD's 1.18 and 109 nM). The high affinity component was blocked by low concentrations of buspirone, spiperone and ergotamine. The low affinity component was blocked only by high concentrations of buspirone and spiperone, and was not displaced by ergotamine at concentrations up to 1 microM. The ergotamine-resistant component of striatal [3H]8-OH-DPAT binding was blocked by low concentrations of the 5-HT uptake inhibitors fluvoxamine and paroxetine, and by relatively low concentrations of 5-HT itself. Thus [3H]8-OH-DPAT labels the 5-HT transporter in the rat striatum. Unlike [3H]imipramine binding, the binding of [3H]8-OH-DPAT to the 5-HT transporter was independent of external sodium ions. It is therefore suggested that 8-OH-DPAT acts as substrate for the 5-HT transporter and labels the 5-HT recognition site of the transporter complex.

Psychotropic drug competition for [3H]imipramine binding further indicates the presence of only one high-affinity drug binding site on human alpha 1-acid glycoprotein.

Psychotropic drug competition for [3H]imipramine binding further indicates the presence of only one high-affinity drug binding site on human alpha 1-acid glycoprotein.

Binding of chlorpromazine and imipramine to red cells, albumin, lipoproteins and other blood components

The binding of model drugs to human blood and individual blood components has been determined by equilibrium dialysis and expressed in terms of classes of binding sites, association constants and binding capacities. Chlorpromazine and imipramine are bound to three major components: membranes of red cells, albumin, and lipoproteins. The affinity and capacity of lipoprotein binding is at least as high as that of albumin and is equally distributed on HDL, LDL, VLDL, and on the chylomicrons. White blood cells and platelets are of minor importance in terms of binding capacity. No binding was detected with gamma-globulins or alpha- or beta-globulins other than lipoproteins. In contrast, salicylic acid was not bound to red cells or lipoproteins.

A method to estimate binding constants at variable protein concentrations.

The association constants of the binding of chlorpromazine and imipramine to serum albumin at low saturation of the protein were determined by a new experimental approach with the protein concentration rather than the ligand concentration being varied. This approach is suitable for estimating binding constants in systems with one class of binding sites. In addition, the method is proposed to complement conventional binding studies of systems with two classes of binding constant with higher accuracy.

The binding of imipramine to the outer membrane of blood platelets.

The binding of imipramine to the outer membrane of blood platelets.

The activity of 25 paroxetine/femoxetine structure variants in various reactions, assumed to be important for the effect of antidepressants.

Structure-activity relationships for 25 structural variants around the 5-hydroxytryptamine (5-HT) uptake inhibitors paroxetine and femoxetine have been investigated. Three parameters related to the 5-HT system were investigated: (i) The inhibition of [3H]5-HT uptake into rat brain synaptosomes, (ii) the inhibition of [3H]paroxetine binding to rat neuronal membranes and (iii) the effect of the compounds on the affinity of [3H]imipramine for the human platelet membrane binding site, measured as the dissociation rate of the [3H]imipramine human platelet membrane binding site complex. A highly significant correlation was found for 5-HT uptake inhibition and inhibition of [3H]paroxetine binding for the different substances, indicating that the two parameters are closely connected. However the slope of the regression line was only 0.6 and not 1.0; this may indicate that [3H]paroxetine binding is necessary, but not sufficient for 5-HT uptake inhibition. No correlation was found between the inhibition of [3H]paroxetine binding and the affinity of the compounds for the [3H]imipramine binding site complex. The two binding sites are therefore probably situated on different parts of the 5-HT transport system, the [3H]paroxetine binding site being part of the 5-HT transport mechanism whereas the [3H]imipramine binding site may represent a site modulating the activity of, and affinity for, 5-HT in the 5-HT transport mechanism. Structure-activity relationships among the substances showed that stereochemical changes from (-)- to (+)-trans changed the activity towards both 5-HT uptake inhibition and [3H]paroxetine displacement for most of the (-)-/(+)-pairs. The substitution of -H with -F or -CH3 also affected the activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of Tricyclic Antidepressants with the Hα3β4 Nicotinic Receptor

The interaction of tricyclic antidepressants (TCAs) with human (h)α3β4 nicotinic acetylcholine receptor (AChR) was compared to that for mecamylamine. The results established that: (a) [3H]imipramine binds to a single site with high affinity (Kd = 0.41 ± 0.04 μM), (b) imipramine inhibits [3H]imipramine binding to resting AChR with the same affinity as to desensitized AChR, suggesting that TCAs do not distinguish between these conformational states, (c) imipramine inhibits [3H]ibogaine binding, suggesting mutual interaction between both compounds, (d) both TCAs and mecamylamine inhibit (±)-epibatidine-induced Ca2+ influx with potencies in the same concentration range, but only TCAs inhibit [3H]imipramine binding to hα3β4 AChRs. This is supported by our molecular docking results, where imipramine, in the neutral and protonated states, interacts with the leucine (position 9’) and valine/phenylalanine (position 13’) rings, while protonated mecamylamine interacts with the outer ring (position 20’). Our data suggest that TCAs and ibogaine bind to overlapping sites located between the serine and valine/phenylalanine rings in the hα3β4 AChR ion channel, while protonated mecamylamine can be attracted to the channel mouth before blocking ion flux by interacting with a luminal site in its neutral state.

Regulation of Serotonin Transport in Human Platelets by Tyrosine Kinase Syk

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the regulation of numerous neuro-physiological processes. The circulating level of 5-HT is regulated by the membrane transporter SERT present both in the presynaptic nerve terminals and blood platelets. 5-HT transport is a process tightly regulated by a variety of factors including protein phosphorylation. Aim of this study was to ascertain if also the SERT Tyr-phosphorylation mediated by Syk-kinase concurs to the regulation of SERT activity. Indeed we found that 5-HT uptake decreased upon platelet exposure to piceatannol or Syk-inhibitor II, two structurally unrelated inhibitors of the tyrosine-kinase Syk. Tyr-phosphorylation of anti-SERT-immuno-stained proteins in membrane extracts and in anti-SERT-immuno-precipitates, decreased upon platelet treatment with piceatannol, in parallel with a reduction of Syk-activity. Syk was immuno-revealed in the anti-SERT immuno-precipitates, which displayed a piceatannol-sensitive kinase activity towards SERT itself and the Syk-substrate α-sinuclein. Syk inhibitors also caused a decrease of the monensin-induced 5-HT-efflux from platelets and of imipramine binding to them. It is concluded that, in addition to the phosphorylation of SERT mediated by various other kinases, also that catalyzed by Syk might play an important role in the 5-HT transport, likely favoring the transporter conformation exposing the neurotransmitter binding sites.

The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants.