Structure- and Cation-Dependent Mechanism of Interaction of Tricyclic Antidepressants with NMDA Receptor According to Molecular Modeling Data.

Some tricyclic antidepressants (TCAs), including amitriptyline (ATL), clomipramine (CLO), and desipramine (DES), are known to be effective for management of neuropathic pain. It was previously determined that ATL, CLO, and DES are capable of voltage-dependent blocking of NMDA receptors of glutamate (NMDAR), which play a key role in pathogenesis of neuropathic pain. Despite the similar structure of ATL, CLO, and DES, efficacy of their interaction with NMDAR varies significantly. In the study presented here, we applied molecular modeling methods to investigate the mechanism of binding of ATL, CLO, and DES to NMDAR and to identify structural features of the drugs that determine their inhibitory activity against NMDAR. Molecular docking of the studied TCAs into the NMDAR channel was performed. Conformational behavior of the obtained complexes in the lipid bilayer was simulated by the method of molecular dynamics (MD). A single binding site (upper) for the tertiary amines ATL and CLO and two binding sites (upper and lower) for the secondary amine DES were identified inside the NMDAR channel. The upper and lower binding sites are located along the channel axis at different distances from the extracellular side of the plasma membrane. MD simulation revealed that the position of DES in the lower site is stabilized only in the presence of sodium cation inside the NMDAR channel. DES binds more strongly to NMDAR compared to ATL and CLO due to simultaneous interaction of two hydrogen atoms of its cationic group with the asparagine residues of the ion pore of the receptor. This feature may be responsible for the stronger side effects of DES. It has been hypothesized that ATL binds to NMDAR less efficiently compared to DES and CLO due to its lower conformational mobility. The identified features of the structure- and cation-dependent mechanism of interaction between TCAs and NMDAR will help in the further development of effective and safe analgesic therapy.

Investigation of Bemethyl Biotransformation Pathways by Combination of LC-MS/HRMS and In Silico Methods.

Bemethyl is an actoprotector, an antihypoxant, and a moderate psychostimulant. Even though the therapeutic effectiveness of bemethyl is well documented, there is a gap in knowledge regarding its metabolic products and their quantitative and qualitative characteristics. Since 2018, bemethyl is included to the Monitoring Program of the World Anti-Doping Agency, which highlights the challenge of identifying its urinary metabolites. The objective of the study was to investigate the biotransformation pathways of bemethyl using a combination of liquid chromatography-high-resolution mass spectrometry and in silico studies. Metabolites were analyzed in a 24 h rat urine collected after oral administration of bemethyl at a single dose of 330 mg/kg. The urine samples were prepared for analysis by a procedure developed in the present work and analyzed by high performance liquid chromatography-tandem mass spectrometry. For the first time, nine metabolites of bemethyl with six molecular formulas were identified in rat urine. The most abundant metabolite was a benzimidazole-acetylcysteine conjugate; this biotransformation pathway is associated with the detoxification of xenobiotics. The BioTransformer and GLORY computational tools were used to predict bemethyl metabolites in silico. The molecular docking of bemethyl and its derivatives to the binding site of glutathione S-transferase has revealed the mechanism of bemethyl conjugation with glutathione. The findings will help to understand the pharmacokinetics and pharmacodynamics of actoprotectors and to improve antihypoxant and adaptogenic therapy.

Tozasertib Attenuates Neuropathic Pain by Interfering with Aurora Kinase and KIF11 Mediated Nociception.

Kinesins are the motor proteins that transport excitatory receptors to the synaptic membrane by forming a complex with receptor cargo leading to central sensitization causing neuropathic pain. Many regulatory proteins govern the transit of receptors by activating kinesin, and Aurora kinases are one of them. In this study, we have performed in silico molecular dynamics simulation to delineate the dynamic interaction of Aurora kinase A with its pharmacological inhibitor, tozasertib. The results from the molecular dynamics study shows that tozasertib-Aurora kinase A complex is stabilized through hydrogen bonding, polar interactions, and water bridges. Findings from the in vitro studies suggest that tozasertib treatment significantly attenuates lipopolysaccharide (LPS)-induced increase in oxidonitrosative stress and kif11 overexpression in C6 glial cell lines. Further, we investigated the regulation of kif11 and its modulation by tozasertib in an animal model of neuropathic pain. Two weeks post-CCI surgery we observed a significant increase in pain hypersensitivity and kif11 overexpression in DRG and spinal cord of nerve-injured rats. Tozasertib treatment significantly attenuates enhanced pain hypersensitivity along with the restoration of kif11 expression in DRG and spinal cord and oxidonitrosative stress in the sciatic nerve of injured rats. Our findings demonstrate the potential role of tozasertib for the management of neuropathic pain.

Tricyclic Antidepressant Structure-Related Alterations in Calcium-Dependent Inhibition and Open-Channel Block of NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are an essential target for the analgetic action of tricyclic antidepressants (TCAs). Their therapeutic blood concentrations achieve 0.5-1.5 µM, which, however, are insufficient to cause in vitro the open-channel block known as the only effect of TCAs on NMDARs. Whereas structures of amitriptyline (ATL), desipramine (DES), and clomipramine (CLO) are rather similar these compounds manifest different therapeutic profiles and side effects. To study structure-activity relationships of DES and CLO on NMDARs, we measured IC50s as a function of extracellular calcium ([Ca2+]) and membrane voltage (Vm) of NMDAR currents recorded in cortical neurons. Here two components of TCA action on NMDARs are described, which could be characterized as the Ca2+-dependent inhibition and the open-channel block. DES demonstrated a profound Ca2+-dependent inhibition of NMDARs, while the CLO effect was weak. DES IC50 exhibited an e-fold change with a [Ca2+] shift of 0.59 mM, which is consistent with ATL. The Ca2+ dependence of NMDAR inhibition by DES disappeared in BAPTA loaded neurons, suggesting that Ca2+ acts from the inside. Since CLO differs from DES and ATL by the presence of Cl-atom in the structure, most likely, this is the atom which is responsible for the loss of pronounced [Ca2+] dependence. As for the NMDAR open-channel block, both DES and CLO were about 5-folds more potent than ATL due to their slow rates of dissociation either from open and closed states. DES demonstrated stronger Vm-dependence than CLO, suggesting a deeper location of the DES binding site within the ion pore. Because DES and CLO differ from ATL by the nitrogen-containing tricycle, presumably this moiety of the molecules determines their high-affinity binding with the NMDAR channel, while the aliphatic chain mono-methyl amino-group of DES allows a deep permeation in the channel. Thus, different structure-activity relationships of the Ca2+-dependent inhibition and Vm-dependent open-channel block of NMDARs by DES and CLO suggest that these processes are independent and most likely may represent an action on different molecular targets. The proposed model of TCA action on NMDARs predicts well the experimental values of IC50s at physiological [Ca2+] and within a wide range of Vms.

Adenosine receptor signalling: Probing the potential pathways for the ministration of neuropathic pain.

Neuropathic pain is a critical burdensome problem due to the complex interplay of several pathological mechanisms and lack of availability of effective therapeutic interventions. The available therapeutic options are associated with a variety of limitations, including severe side effects, and unmet medical needs, warranting further research to identify and validate potential targets. Adenosine receptors system is a widely studied target, which evidently was successful in alleviation of neuropathic pain in several experimental paradigms, and researchers are putting efforts in building its clinical roadmap. The adenosine receptors act by different mechanisms and targeting adenosine receptors for neuropathic pain includes several important pathways such as p38-mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK), brain-derived neurotrophic factor (BDNF) signalling, γ-aminobutyric acid (GABA) as well as the ion channel modulations. Various studies have also shown the relevance of targeting adenosine receptors in chemotherapy-induced neuropathic pain and diabetic neuropathy. Several drugs acting on adenosine receptors have undergone clinical trials for management of neuropathic pain, whereas many other drugs are yet to be studied to find a potential anti-nociceptive agent. In this review, we have discussed the roadmap of adenosine receptors as a potential target for the treatment of neuropathic pain.

Dual action of amitriptyline on NMDA receptors: enhancement of Ca-dependent desensitization and trapping channel block.

Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca2+-dependent desensitization and 2) trapping channel block. Inhibition of NMDARs by ATL was found to be dependent upon external Ca2+ concentration ([Ca2+]) in a voltage-independent manner, with an IC50 of 0.72 µM in 4 mM [Ca2+]. The ATL IC50 value increased exponentially with decreasing [Ca2+], with an e-fold change observed per 0.69 mM decrease in [Ca2+]. Loading neurons with BAPTA abolished Ca2+-dependent inhibition, suggesting that Ca2+ affects NMDARs from the cytosol. Since there is one known Ca2+-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca2+-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC50 of 220 µM at 0 mV. An e-fold change in ATL IC50 was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca2+]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca2+], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.

Psychotropic Drugs for the Management of Chronic Pain and Itch.

Clinical observations have shown that patients with chronic neuropathic pain or itch exhibit symptoms of increased anxiety, depression and cognitive impairment. Such patients need corrective therapy with antidepressants, antipsychotics or anticonvulsants. It is known that some psychotropic drugs are also effective for the treatment of neuropathic pain and pruritus syndromes due to interaction with the secondary molecular targets. Our own clinical studies have identified antipruritic and/or analgesic efficacy of the following compounds: tianeptine (atypical tricyclic antidepressant), citalopram (selective serotonin reuptake inhibitor), mianserin (tetracyclic antidepressant), carbamazepine (anticonvulsant), trazodone (serotonin antagonist and reuptake inhibitor), and chlorprothixene (antipsychotic). Venlafaxine (serotonin-norepinephrine reuptake inhibitor) is known to have an analgesic effect too. The mechanism of such effect of these drugs is not fully understood. Herein we review and correlate the literature data on analgesic/antipruritic activity with pharmacological profile of these compounds.

Inhibition of the NMDA and AMPA receptor channels by antidepressants and antipsychotics.

It is known that some antidepressants and antipsychotics directly inhibit NMDA-type ionotropic glutamate receptors. In this study we systematically studied action of seven drugs (Fluoxetine, Citalopram, Desipramine, Amitriptyline, Atomoxetine, Chlorpromazine, and Clozapine) on NMDA receptors and Ca2+-permeable and -impermeable AMPA receptors in rat brain neurons by whole-cell patch-clamp technique. Except for weak effect of fluoxetine, all drugs were virtually inactive against Ca2+-impermeable AMPA receptors. Fluoxetine and desipramine significantly inhibited Ca2+-permeable AMPA receptors (IC50=43±7 and 105±12µM, respectively). Desipramine, atomoxetine and chlorpromazine inhibited NMDA receptors in clinically relevant low micromolar concentrations, while citalopram had only weak effect. All tested medicines have been clustered into two groups by their action on NMDA receptors: desipramine, amitriptyline, chlorpromazine, and atomoxetine display voltage- and magnesium-dependent open channel blocking mechanism. Action of fluoxetine and clozapine was found to be voltage- and magnesium-independent. All voltage-dependent compounds could be trapped in closed NMDA receptor channels. Possible contribution of NMDA receptor inhibition by certain antidepressants and antipsychotics to their analgesic effects in neuropathic pain is discussed.