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.

Molecular Basis for the Involvement of Mammalian Serum Albumin in the AGE/RAGE Axis: A Comprehensive Computational Study.

In mammals, glycated serum albumin (gSA) contributes to the pathogenesis of many metabolic diseases by activating the receptors (RAGE) for advanced glycation end products (AGEs). Many aspects of the gSA-RAGE interaction remain unknown. The purpose of the present paper was to study the interaction of glycated human albumin (gHSA) with RAGE using molecular modeling methods. Ten models of gHSA modified with different lysine residues to carboxymethyl-lysines were prepared. Complexes of gHSA-RAGE were obtained by the macromolecular docking method with subsequent molecular dynamics simulation (MD). According to the MD, the RAGE complexes with gHSA glycated at Lys233, Lys64, Lys525, Lys262 and Lys378 are the strongest. Three-dimensional models of the RAGE dimers with gHSA were proposed. Additional computational experiments showed that the binding of fatty acids (FAs) to HSA does not affect the ability of Lys525 (the most reactive lysine) to be glycated. In contrast, modification of Lys525 reduces the affinity of albumin for FA. The interspecies differences in the molecular structure of albumin that may affect the mechanism of the gSA-RAGE interaction were discussed. The obtained results will help us to learn more about the molecular basis for the involvement of serum albumin in the AGE/RAGE axis and improve the methodology for studying cellular signaling pathways involving RAGE.

Immunological Profile and Markers of Endothelial Dysfunction in Elderly Patients with Cognitive Impairments.

The process of aging is accompanied by a dynamic restructuring of the immune response, a phenomenon known as immunosenescence. Further, damage to the endothelium can be both a cause and a consequence of many diseases, especially in elderly people. The purpose of this study was to carry out immunological and biochemical profiling of elderly people with acute ischemic stroke (AIS), chronic cerebral circulation insufficiency (CCCI), prediabetes or newly diagnosed type II diabetes mellitus (DM), and subcortical ischemic vascular dementia (SIVD). Socio-demographic, lifestyle, and cognitive data were obtained. Biochemical, hematological, and immunological analyses were carried out, and extracellular vesicles (EVs) with endothelial CD markers were assessed. The greatest number of significant deviations from conditionally healthy donors (HDs) of the same age were registered in the SIVD group, a total of 20, of which 12 were specific and six were non-specific but with maximal differences (as compared to the other three groups) from the HDs group. The non-specific deviations were for the MOCA (Montreal Cognitive Impairment Scale), the MMSE (Mini Mental State Examination) and life satisfaction self-assessment scores, a decrease of albumin levels, and ADAMTS13 (a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13) activity, and an increase of the VWF (von Willebrand factor) level. Considering the significant changes in immunological parameters (mostly Th17-like cells) and endothelial CD markers (CD144 and CD34), vascular repair was impaired to the greatest extent in the DM group. The AIS patients showed 12 significant deviations from the HD controls, including three specific to this group. These were high NEFAs (non-esterified fatty acids) and CD31 and CD147 markers of EVs. The lowest number of deviations were registered in the CCCI group, nine in total. There were significant changes from the HD controls with no specifics to this group, and just one non-specific with a maximal difference from the control parameters, which was α1-AGP (alpha 1 acid glycoprotein, orosomucoid). Besides the DM patients, impairments of vascular repair were also registered in the CCCI and AIS patients, with a complete absence of such in patients with dementia (SIVD group). On the other hand, microvascular damage seemed to be maximal in the latter group, considering the biochemical indicators VWF and ADAMTS13. In the DM patients, a maximum immune response was registered, mainly with Th17-like cells. In the CCCI group, the reaction was not as pronounced compared to other groups of patients, which may indicate the initial stages and/or compensatory nature of organic changes (remodeling). At the same time, immunological and biochemical deviations in SIVD patients indicated a persistent remodeling in microvessels, chronic inflammation, and a significant decrease in the anabolic function of the liver and other tissues. The data obtained support two interrelated assumptions. Taking into account the primary biochemical factors that trigger the pathological processes associated with vascular pathology and related diseases, the first assumption is that purine degradation in skeletal muscle may be a major factor in the production of uric acid, followed by its production by non-muscle cells, the main of which are endothelial cells. Another assumption is that therapeutic factors that increase the levels of endothelial progenitor cells may have a therapeutic effect in reducing the risk of cerebrovascular disease and related neurodegenerative diseases.

Searching for New Biomarkers to Assess COVID-19 Patients: A Pilot Study.

During the initial diagnosis of urgent medical conditions, which include acute infectious diseases, it is important to assess the severity of the patient's clinical state as quickly as possible. Unlike individual biochemical or physiological indicators, derived indices make it possible to better characterize a complex syndrome as a set of symptoms, and therefore quickly take a set of adequate measures. Recently, we reported on novel diagnostic indices containing butyrylcholinesterase (BChE) activity, which is decreased in COVID-19 patients. Also, in these patients, the secretion of von Willebrand factor (vWF) increases, which leads to thrombosis in the microvascular bed. The objective of this study was the determination of the concentration and activity of vWF in patients with COVID-19, and the search for new diagnostic indices. One of the main objectives was to compare the prognostic values of some individual and newly derived indices. Patients with COVID-19 were retrospectively divided into two groups: survivors (n = 77) and deceased (n = 24). According to clinical symptoms and computed tomography (CT) results, the course of disease was predominantly moderate in severity. The first blood sample (first point) was taken upon admission to the hospital, the second sample (second point)-within 4-6 days after admission. Along with the standard spectrum of biochemical indicators, BChE activity (BChEa or BChEb for acetylthiocholin or butyrylthiocholin, respectively), malondialdehyde (MDA), and vWF analysis (its antigen level, AGFW, and its activity, ActWF) were determined and new diagnostic indices were derived. The pooled sensitivity, specificity, and area under the receiver operating curve (AUC), as well as Likelihood ratio (LR) and Odds ratio (OR) were calculated. The level of vWF antigen in the deceased group was 1.5-fold higher than the level in the group of survivors. Indices that include vWF antigen levels are superior to indices using vWF activity. It was found that the index [Urea] × [AGWF] × 1000/(BChEb × [ALB]) had the best discriminatory power to predict COVID-19 mortality (AUC = 0.91 [0.83, 1.00], p < 0.0001; OR = 72.0 [7.5, 689], p = 0.0002). In addition, [Urea] × 1000/(BChEb × [ALB]) was a good predictor of mortality (AUC = 0.95 [0.89, 1.00], p < 0.0001; OR = 31.5 [3.4, 293], p = 0.0024). The index [Urea] × [AGWF] × 1000/(BChEb × [ALB]) was the best predictor of mortality associated with COVID-19 infection, followed by [Urea] × 1000/(BChEb × [ALB]). After validation in a subsequent cohort, these two indices could be recommended for diagnostic laboratories.

Structure-Dependent Mechanism of Organophosphate Release from Albumin and Butyrylcholinesterase Adducts When Exposed to Fluoride Ion: A Comprehensive In Silico Study.

The most favorable targets for retrospectively determining human exposure to organophosphorus pesticides, insecticides, retardants, and other industrial organophosphates (OPs) are adducts of OPs with blood plasma butyrylcholinesterase (BChE) and human serum albumin (HSA). One of the methods for determining OP exposure is the reactivation of modified BChE using a concentrated solution of KF in an acidic medium. It is known that under the action of fluoride ion, OPs or their fluoroanhydrides can be released not only from BChE adducts but also from the adducts with albumin; however, the contribution of albumin to the total pool of released OPs after plasma treatment with KF has not yet been studied. The efficiency of OP release can be affected by many factors associated with the experimental technique, but first, the structure of the adduct must be taken into account. We report a comparative analysis of the structure and conformation of organophosphorus adducts on HSA and BChE using molecular modeling methods and the mechanism of OP release after fluoride ion exposure. The conformational analysis of the organophosphorus adducts on HSA and BChE was performed, and the interaction of fluoride ions with modified proteins was studied by molecular dynamics simulation. The geometric and energy characteristics of the studied adducts and their complexes with fluoride ion were calculated using molecular mechanics and semiempirical approaches. The structural features of modified HSA and BChE that can affect the efficiency of OP release after fluoride ion exposure were revealed. Using the proposed approach, the expediency of using KF for establishing exposure to different OPs, depending on their structure, can be assessed.

Serum Albumin in Health and Disease: From Comparative Biochemistry to Translational Medicine.

Being one of the main proteins in the human body and many animal species, albumin plays a decisive role in the transport of various ions, electrically neutral molecules and in maintaining the colloid osmotic pressure of the blood [...].

Anticholinesterase and Serotoninergic Evaluation of Benzimidazole-Carboxamides as Potential Multifunctional Agents for the Treatment of Alzheimer's Disease.

The etiology and pathogenesis of Alzheimer's disease are multifactorial, so one of the treatment strategies is the development of the drugs that affect several targets associated with the pathogenesis of the disease. Within this roadmap, we investigated the interaction of several substituted 1,3-dihydro-2-oxo-1H-benzimidazol-2-ones with their potential molecular targets: cholinesterases (ChE) and three types of the Gs-protein-coupled serotonin receptors (5-HTR) 5-HT6, 5-HT4 and 5-HT7 (5-HT4R, 5-HT6R and 5-HT7R, respectively). A microplate modification of the Ellman method was used for the biochemical analysis of the inhibitory ability of the drugs towards ChE. Molecular modeling methods, such as molecular docking and molecular dynamics (MD) simulation in water and the lipid bilayer, were used to study the interaction of the compounds with ChE and 5-HTR. In vitro experiments showed that the tested compounds had moderate anticholinesterase activity. With the help of molecular modeling methods, the mechanism of interaction of the tested compounds with ChE was investigated, the binding sites were described and the structural features of the drugs that determine the strength of their anticholinesterase activity were revealed. Primary in silico evaluation showed that benzimidazole-carboxamides effectively bind to 5-HT4R and 5-HT7R. The pool of the obtained data allows us to choose N-[2-(diethylamino)ethyl]-2-oxo-3-(tert-butyl)-2,3-dihydro-1H-benzimidazole-1-carboxamide hydrochloride (compound 13) as the most promising for further experimental development.

Albumin Is a Component of the Esterase Status of Human Blood Plasma.

The esterase status of blood plasma can claim to be one of the universal markers of various diseases; therefore, it deserves attention when searching for markers of the severity of COVID-19 and other infectious and non-infectious pathologies. When analyzing the esterase status of blood plasma, the esterase activity of serum albumin, which is the major protein in the blood of mammals, should not be ignored. The purpose of this study is to expand understanding of the esterase status of blood plasma and to evaluate the relationship of the esterase status, which includes information on the amount and enzymatic activity of human serum albumin (HSA), with other biochemical parameters of human blood, using the example of surviving and deceased patients with confirmed COVID-19. In experiments in vitro and in silico, the activity of human plasma and pure HSA towards various substrates was studied, and the effect of various inhibitors on this activity was tested. Then, a comparative analysis of the esterase status and a number of basic biochemical parameters of the blood plasma of healthy subjects and patients with confirmed COVID-19 was performed. Statistically significant differences have been found in esterase status and biochemical indices (including albumin levels) between healthy subjects and patients with COVID-19, as well as between surviving and deceased patients. Additional evidence has been obtained for the importance of albumin as a diagnostic marker. Of particular interest is a new index, [Urea] × [MDA] × 1000/(BChEb × [ALB]), which in the group of deceased patients was 10 times higher than in the group of survivors and 26 times higher than the value in the group of apparently healthy elderly subjects.

Serum Albumin in Health and Disease: Esterase, Antioxidant, Transporting and Signaling Properties.

Being one of the main proteins in the human body and many animal species, albumin plays a decisive role in the transport of various ions-electrically neutral and charged molecules-and in maintaining the colloidal osmotic pressure of the blood. Albumin is able to bind to almost all known drugs, as well as many nutraceuticals and toxic substances, largely determining their pharmaco- and toxicokinetics. Albumin of humans and respective representatives in cattle and rodents have their own structural features that determine species differences in functional properties. However, albumin is not only passive, but also an active participant of pharmacokinetic and toxicokinetic processes, possessing a number of enzymatic activities. Numerous experiments have shown esterase or pseudoesterase activity of albumin towards a number of endogeneous and exogeneous esters. Due to the free thiol group of Cys34, albumin can serve as a trap for reactive oxygen and nitrogen species, thus participating in redox processes. Glycated albumin makes a significant contribution to the pathogenesis of diabetes and other diseases. The interaction of albumin with blood cells, blood vessels and tissue cells outside the vascular bed is of great importance. Interactions with endothelial glycocalyx and vascular endothelial cells largely determine the integrative role of albumin. This review considers the esterase, antioxidant, transporting and signaling properties of albumin, as well as its structural and functional modifications and their significance in the pathogenesis of certain diseases.

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling.

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (1H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.

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.

The Universal Soldier: Enzymatic and Non-Enzymatic Antioxidant Functions of Serum Albumin.

As a carrier of many biologically active compounds, blood is exposed to oxidants to a greater extent than the intracellular environment. Serum albumin plays a key role in antioxidant defence under both normal and oxidative stress conditions. This review evaluates data published in the literature and from our own research on the mechanisms of the enzymatic and non-enzymatic activities of albumin that determine its participation in redox modulation of plasma and intercellular fluid. For the first time, the results of numerous clinical, biochemical, spectroscopic and computational experiments devoted to the study of allosteric modulation of the functional properties of the protein associated with its participation in antioxidant defence are analysed. It has been concluded that it is fundamentally possible to regulate the antioxidant properties of albumin with various ligands, and the binding and/or enzymatic features of the protein by changing its redox status. The perspectives for using the antioxidant properties of albumin in practice are discussed.

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.

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.

Rational in silico design of aptamers for organophosphates based on the example of paraoxon.

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed.

Serum Albumin Binding and Esterase Activity: Mechanistic Interactions with Organophosphates.

The albumin molecule, in contrast to many other plasma proteins, is not covered with a carbohydrate moiety and can bind and transport various molecules of endogenous and exogenous origin. The enzymatic activity of albumin, the existence of which many scientists perceive skeptically, is much less studied. In toxicology, understanding the mechanistic interactions of organophosphates with albumin is a special problem, and its solution could help in the development of new types of antidotes. In the present work, the history of the issue is briefly examined, then our in silico data on the interaction of human serum albumin with soman, as well as comparative in silico data of human and bovine serum albumin activities in relation to paraoxon, are presented. Information is given on the substrate specificity of albumin and we consider the possibility of its affiliation to certain classes in the nomenclature of enzymes.