From RNA sequence to its three-dimensional structure: geometrical structure, stability and dynamics of selected fragments of SARS-CoV-2 RNA.

In this computational study, we explore the folding of a particular sequence using various computational tools to produce two-dimensional structures, which are then transformed into three-dimensional structures. We then study the geometry, energetics and dynamics of these structures using full electron quantum-chemical and classical molecular dynamics calculations. Our study focuses on the SARS-CoV-2 RNA fragment GGaGGaGGuguugcaGG and its various structures, including a G-quadruplex and five different hairpins. We examine the impact of two types of counterions (K+ and Na+) and flanking nucleotides on their geometrical characteristics, relative stability and dynamic properties. Our results show that the G-quadruplex structure is the most stable among the constructed hairpins. We confirm its topological stability through molecular dynamics simulations. Furthermore, we observe that the nucleotide loop consisting of seven nucleotides is the most flexible part of the RNA fragment. Additionally, we find that RNA networks of intermolecular hydrogen bonds are highly sensitive to the surrounding environment. Our findings reveal the loss of 79 old hydrogen bonds and the formation of 91 new ones in the case when the G-quadruplex containing flanking nucleotides is additionally stabilized by Na+ counterions.

4-(Azolyl)-Benzamidines as a Novel Chemotype for ASIC1a Inhibitors.

Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1-ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds.

Integrated workflow for the identification of new GABAA R positive allosteric modulators based on the in†silico screening with further in†vitro validation. Case study using Enamine's stock chemical space.

Numerous studies reported an association between GABAA R subunit genes and epilepsy, eating disorders, autism spectrum disorders, neurodevelopmental disorders, and bipolar disorders. This study was aimed to find some potential positive allosteric modulators and was performed by combining the in silico approach with further in vitro evaluation of its real activity. We started from the GABAA R-diazepam complexes and assembled a lipid embedded protein ensemble to refine it via molecular dynamics (MD) simulation. Then we focused on the interaction of α1ß2γ2 with some Z-drugs (non-benzodiazepine compounds) using an Induced Fit Docking (IFD) into the relaxed binding site to generate a pharmacophore model. The pharmacophore model was validated with a reference set and applied to decrease the pre-filtered Enamine database before the main docking procedure. Finally, we succeeded in identifying a set of compounds, which met all features of the docking model. The aqueous solubility and stability of these compounds in mouse plasma were assessed. Then they were tested for the biological activity using the rat Purkinje neurons and CHO cells with heterologously expressed human α1ß2γ2 GABAA receptors. Whole-cell patch clamp recordings were used to reveal the GABA induced currents. Our study represents a convenient and tunable model for the discovery of novel positive allosteric modulators of GABAA receptors. A High-throughput virtual screening of the largest available database of chemical compounds resulted in the selection of 23 compounds. Further electrophysiological tests allowed us to determine a set of 3 the most outstanding active compounds. Considering the structural features of leader compounds, the study can develop into the MedChem project soon.

Single-Walled Carbon Nanotubes Inhibit TRPC4-Mediated Muscarinic Cation Current in Mouse Ileal Myocytes.

Single-walled carbon nanotubes (SWCNTs) are characterized by a combination of rather unique physical and chemical properties, which makes them interesting biocompatible nanostructured materials for various applications, including in the biomedical field. SWCNTs are not inert carriers of drug molecules, as they may interact with various biological macromolecules, including ion channels. To investigate the mechanisms of the inhibitory effects of SWCNTs on the muscarinic receptor cation current (mICAT), induced by intracellular GTPγs (200 µM), in isolated mouse ileal myocytes, we have used the patch-clamp method in the whole-cell configuration. Here, we use molecular docking/molecular dynamics simulations and direct patch-clamp recordings of whole-cell currents to show that SWCNTs, purified and functionalized by carboxylation in water suspension containing single SWCNTs with a diameter of 0.5-1.5 nm, can inhibit mICAT, which is mainly carried by TRPC4 cation channels in ileal smooth muscle cells, and is the main regulator of cholinergic excitation-contraction coupling in the small intestinal tract. This inhibition was voltage-independent and associated with a shortening of the mean open time of the channel. These results suggest that SWCNTs cause a direct blockage of the TRPC4 channel and may represent a novel class of TRPC4 modulators.

C60 fullerene against SARS-CoV-2 coronavirus: an in silico insight.

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.

Modelling of an autonomous Nav1.5 channel system as a part of in silico pharmacology study.

A homology model of Nav1.5, based mainly on the crystal structures of Nav1.2/1.5 was built, optimized and successfully inserted into the membrane bilayer. We applied steered and free MD simulation protocols for the visualization of the mechanism of Nav1.5 activation. We constrained dihedrals of S4 trigger to introduce a structural tension with further rearrangement and movement of secondary structure elements. From these, we observed an intracellular gate opening and movement of the Lys1419 residue caused by a gradual displacement of the distal S6 α-helix with the extended S4 3-10 helix of voltage-sensing domains (VSD). A construction containing the Lys1419 residue in P-loop also changed its position due to the extension of this helix and subsequent induction of the pore-forming helixes motion. From this point, a double membrane system was generated, implying a free of ligand Nav1.5 protein and on the opposite side its copy containing a docked bupivacaine molecule inside the pore channel. The system can be used for the design of selective inhibitors against the Nav1.7 channel, instead of mixed effect on both channels.

Anticoronavirus Activity of Water-Soluble Pristine C60 Fullerenes: In Vitro and In Silico Screenings.

INTRODUCTION: The emergence of a new member of the Coronaviridae family, which caused the 2020 pandemic, requires detailed research on the evolution of coronaviruses, their structure and properties, and interaction with cells. Modern nanobiotechnologies can address the many clinical challenges posed by the COVID-19 pandemic. In particular, they offer new therapeutic approaches using biocompatible nanostructures with "specific" antiviral activity. Therefore, the nanosized spherical-like molecule (0.72 nm in diameter) composed of 60 carbon atoms, C60 fullerene, is of interest in terms of fighting coronaviruses due to its high biological activity. In here, we aim to evaluate the effectiveness of anticoronavirus action of water-soluble pristine C60 fullerene in the model and in vitro systems. As a model, apathogenic for human coronavirus, we used transmissible gastroenteritis virus of swine (TGEV), which we adapted to the BHK-21 cell culture (kidney cells of a newborn Syrian hamster). METHODS: The shape and size of the particles present in C60 fullerene aqueous colloidal solution (C60FAS) of given concentration, as well as C60FAS stability (value of zeta potential) were studied using microscopic (STM, scanning tunneling microscopy, and AFM, atomic force microscopy) and spectroscopic (DLS, dynamic light scattering) methods. The cytopathic effect of TGEV was determined with the help of a Leica DM 750 microscope and the degree of monolayer changes in cells was assessed. The microscopy of the viral suspension was performed using a high resolution transmission electron microscope (HRTEM; JEM-1230, Japan). Finally, the search for and design of optimal possible complexes between C60 fullerene and target proteins in the structure of SARS-CoV-2 coronavirus, evaluation of their stability in the simulated cellular environment were performed using molecular dynamics and docking methods. RESULTS: It was found that the maximum allowable cytotoxic concentration of C60 fullerene is 37.5 ± 3.0 µg/ml. The investigated C60FAS reduces the titer of coronavirus infectious activity by the value of 2.00 ± 0.08 TCID50/ml. It was shown that C60 fullerene interacts directly with SARS-CoV-2 proteins, such as RdRp (RNA-dependent RNA polymerase) and 3CLpro (3-chymotrypsin-like protease), which is critical for the life cycle of the coronavirus and, thus, inhibits its functional activity. In both cases, C60 fullerene fills the binding pocket and gets stuck there through stacking and steric interactions. CONCLUSION: Pioneer in vitro study to identify the anticoronavirus activity of water-soluble pristine C60 fullerenes indicates that they are highly promising for further preclinical studies, since a significant inhibition of the infectious activity of swine coronavirus of transmissible gastroenteritis in BHK-21 cell culture was found. According to molecular modeling results, it was shown that C60 fullerene can create the stable complexes with 3CLpro and RdRp proteins of SARS-CoV-2 coronavirus and, thus, suppress its functional activity.

Peptidyl inhibition of Spt4-Spt5: Protein-protein inhibitors for targeting the transcriptional pathway related to C9orf72 expansion repeats.

Spt4/Spt5 is an useful target as it is likely a transcription factor that has implications for long non-coding RNA repeats related to frontotemporal dementia (FTD) found in the C9orf72 disease pathology. Inhibitors for Spt4/Spt5 using peptides as a starting point for assays as a means for developing small molecules, which could likely lead to therapeutic development for inhibition for Spt4/Spt5 with CNS characteristics. To elucidate the specific steps of identification and modification of key interacting residues from Spt4/Spt5 complex with further effect prediction, a set of different computational methods was applied. Newly characterized, theoretically derived peptides docked on Spt4/Spt5 models, based on X-ray crystallography sources, allowed us to complete molecular dynamics simulations and docking studies for peptide libraries that give us high confident set of peptides for use to screen for Spt4/Spt5 inhibition. Several peptides with increased specificity to the Spt4/Spt5 interface were found and can be screened in cell-based assays and enzymatic assays for peptide screens that lead to small molecule campaigns. Spt4/Spt5 comprises an attractive target for neurological diseases, and applying these peptides into a screening campaign will promote the goal of therapeutic searches for FTD drug discovery.

Synthesis, biological evaluation, and modeling studies of 1,3-disubstituted cyclobutane-containing analogs of combretastatin A4.

With the aim of circumventing the adverse cis/trans-isomerization of combretastatin A4 (CA4), a naturally occurring tumor-vascular disrupting agent, we designed novel CA4 analogs bearing 1,3-cyclobutane moiety instead of the cis-stilbene unit of the parent compound. The corresponding cis and trans cyclobutane-containing derivatives were prepared as pure diastereomers. The structure of the target compounds was confirmed by X-ray diffraction study. The title compounds were evaluated for their cytotoxic properties in human cancer cell lines HepG2 (hepatocarcinoma) and SK-N-DZ (neuroblastoma), and the overall activity was found in micromolar range. Molecular docking studies and molecular dynamics simulation within the colchicine binding site of tubulin were in good agreement with the obtained cytotoxicity data.

C60 Fullerene Governs Doxorubicin Effect on Metabolic Profile of Rat Microglial Cells In Vitro.

Background: C60 fullerenes and their derivatives are actively investigated for the use in neuroscience. Applications of these nanoscale materials require the examination of their interaction with different neural cells, especially with microglia, because these cells, like other tissue resident phagocytes, are the earliest and most sensitive responders to nanoparticles. The aim of this study was to investigate the effect of C60 fullerene and its nanocomplex with doxorubicin (Dox) on the metabolic profile of brain-resident phagocytes-microglia-in vitro. Methods: Resting microglial cells from adult male Wistar rats were used in experiments. Potential C60 fullerene targets in microglial cells were studied by computer simulation. Microglia oxidative metabolism and phagocytic activity were examined by flow cytometry. Griess reaction and arginase activity colorimetric assay were used to explore arginine metabolism. Results: C60 fullerene when used alone did not influence microglia oxidative metabolism and phagocytic activity but shifted arginine metabolism toward the decrease of NO generation. Complexation of C60 fullerene with Dox (C60-Dox) potentiated the ability of the latter to stimulate NO generation. Conclusion: The capability of C60 fullerenes used alone to cause anti-inflammatory shift of microglia arginine metabolism makes them a promising agent for the correction of neuroinflammatory processes involved in neurodegeneration. The potentiating action of C60 fullerene on the immunomodulatory effect of Dox allows us to consider the C60 molecule as an attractive vehicle for this antitumor agent.

Diarylethene moiety as an enthalpy-entropy switch: photoisomerizable stapled peptides for modulating p53/MDM2 interaction.

Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein-protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the "open" configuration, but up to eight times larger for the corresponding "closed" isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the "closed" isomers is mostly enthalpy-driven, whereas the "open" photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.

Water-Soluble Pristine C60 Fullerenes Inhibit Liver Fibrotic Alteration and Prevent Liver Cirrhosis in Rats.

Liver cirrhosis is an outcome of a wide range of liver chronic diseases. It is attributed to oxidative stress; therefore, antioxidant usage could be a promising treatment of that. So, exploring the impact of effective free radical scavenger pristine C60 fullerenes on liver fibrosis and cirrhosis and their ability to interact with main growth factor receptors involved in liver fibrogenesis was aimed to be discovered. We used N-diethylnitrosamine/carbon tetrachloride-induced simulations of rat liver fibrosis (10 weeks) and cirrhosis (15 weeks). Pristine C60 fullerene aqueous colloid solution (C60FAS) was injected daily at a dose of 0.25 mg/kg throughout the experiment. Liver morphology and functional and redox states were assessed. C60 fullerenes' ability to interact with epidermal, vasoendothelial, platelet-derived, and fibroblast growth factor receptors (EGFR, VEGFR, PDGFR, and FGFR, respectively) was estimated by computational modeling. We observed that C60FAS reduced the severity of fibrosis in fibrotic rats (0.75 vs. 3.0 points according to Ishak score), attenuated the hepatocyte injury, normalized elevated blood serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH), and mitigated oxidative stress manifestation in liver tissue restoring its redox balance. When applied to cirrhotic animals, C60FAS reduced connective tissue deposition as well (2.4 vs. 5.4 points according to Ishak score), diminished ALP and LDH (by 16% and 61%), and normalized conjugated and nonconjugated bilirubin, restoring the liver function. Altered liver lipid and protein peroxides and glutathione peroxidase activity were also leveled. Within a computer simulation, it was shown that C60 fullerenes can block hinge prohibiting ATP binding for EGFR and FGFR and thus blocking associated signal pathways. This ability in addition to their antioxidant properties may contribute to C60 fullerene's antifibrotic action. Thus, C60FAS may have a substantial therapeutic potential as an inhibitor of liver fibrosis and cirrhosis.

Pyrrole derivatives as potential anti-cancer therapeutics: synthesis, mechanisms of action, safety.

Pyrrole derivatives (PDs) chloro-1-(4-chlorobenzyl)-4-((3-(trifluoromethyl)phenyl)amino)-1H-pyrrole-2,5-dione (MI-1) and 5-amino-4-(1,3-benzothyazol-2-yn)-1-(3-methoxyphenyl)-1,2-dihydro-3H-pyrrole-3-one (D1) were synthesised as inhibitors of several protein kinases including EGFR and VEGFR. The aim of the study was to reveal the exact mechanisms of PDs' action EGFR and VEGFR are involved in. We observed, that both PDs could bind with EGFR and VEGFR and form stable complexes. PDs entered into electrostatic interactions with polar groups of phospholipid heads in cell membrane, and the power of interaction depended on the nature of PD radical substituents (greater for MI-1 and smaller for D1). Partial intercalation of MI-1 into the membrane hydrophobic zone also occurred. PDs concentrations induced apoptosis in malignant cells but normal ones had different sensitivity to those. MI-1 and D1 acted like antioxidants in inflamed colonic tissue, as evidenced by reduce of lipid and protein peroxidation products (by 43-67%) and increase of superoxide dismutase activity (by 40 and 58%) with restoring these values to control ones. MI-1 restored reduced haemoglobin and normalised elevated platelets and monocytes in settings of colorectal cancer, whereas D1 normalised only platelets. Thus, MI-1 and D1 could be used as competitive inhibitors of EGFR and VEGFR and antioxidants, which might contribute to realisation of their anti-inflammatory, proapoptotic and antitumor activity.

C60 fullerenes selectively inhibit BKCa but not Kv channels in pulmonary artery smooth muscle cells.

Possessing unique physical and chemical properties, C60 fullerenes are arising as a potential nanotechnological tool that can strongly affect various biological processes. Recent molecular modeling studies have shown that C60 fullerenes can interact with ion channels, but there is lack of data about possible effects of C60 molecule on ion channels expressed in smooth muscle cells (SMC). Here we show both computationally and experimentally that water-soluble pristine C60 fullerene strongly inhibits the large conductance Ca2+-dependent K+ (BKCa), but not voltage-gated K+ (Kv) channels in pulmonary artery SMC. Both molecular docking simulations and analysis of single channel activity indicate that C60 fullerene blocks BKCa channel pore in its open state. In functional tests, C60 fullerene enhanced phenylephrine-induced contraction of pulmonary artery rings by about 25% and reduced endothelium-dependent acetylcholine-induced relaxation by up to 40%. These findings suggest a novel strategy for biomedical application of water-soluble pristine C60 fullerene in vascular dysfunction.

Synthesis and biological activity of 4-amino-3-chloro-1H-pyrrole-2,5-diones.

4-Amino-3-chloro-1H-pyrrole-2,5-dione derivatives were designed and synthesized as potential tyrosine kinase inhibitors. One of them has been shown to inhibit growth of cancer cell lines and in vivo tumors. To determine the impact of side groups on biological activity the ability of different 4-amino-3-chloro-1H-pyrrole-2,5-diones to interact with ATP-binding domains of growth factor receptors and with model cell membranes were aimed to be discovered. The methods of molecular docking, short-molecular dynamics (in silico) and non-steady cyclic current-voltage characteristics (in vitro) were used. Five 4-amino-3-chloro-1H-pyrrole-2,5-diones were synthesized from 3,4-dichloro-1H-pyrrole-2,5-diones. All of them demonstrated the potential ability to form complexes with ATP-binding domains of EGFR and VEGFR2. These complexes were more stable compared to those with ANP. 4-Amino-3-chloro-1H-pyrrole-2,5-diones while interact with different bilayer lipid membranes caused an increase of their specific conductance and electric capacity, demonstrating the certain disturbance in lipid packing. Obtained data allowed us to suggest that proposed chemicals can interact with the surface of lipid bilayer, do likely intercalate into the membrane and form stable complexes with EGFR and VEGFR2. So, the prospect of developed chemicals to be effective EGFR and VEGFR2 inhibitors and therefore realize antitumor activity was concluded.

C60 fullerene and its nanocomplexes with anticancer drugs modulate circulating phagocyte functions and dramatically increase ROS generation in transformed monocytes.

BACKGROUND: C60 fullerene-based nanoformulations are proposed to have a direct toxic effect on tumor cells. Previous investigations demonstrated that C60 fullerene used alone or being conjugated with chemotherapeutic agents possesses a potent anticancer activity. The main aim of this study was to investigate the effect of C60 fullerene and its nanocomplexes with anticancer drugs on human phagocyte metabolic profile in vitro. METHODS: Analysis of the metabolic profile of phagocytes exposed to C60 fullerene in vitro revealed augmented phagocytic activity and down-regulated reactive nitrogen species generation in these cells. Additionally, cytofluorimetric analysis showed that C60 fullerene can exert direct cytotoxic effect on normal and transformed phagocytes through the vigorous induction of intracellular reactive oxygen species generation. RESULTS: Cytotoxic action as well as the pro-oxidant effect of C60 fullerene was more pronounced toward malignant phagocytes. At the same time, C60 fullerenes have the ability to down-regulate the pro-oxidant effect of cisplatin on normal cells. These results indicate that C60 fullerenes may influence phagocyte metabolism and have both pro-oxidant and antioxidant properties. CONCLUSIONS: The antineoplastic effect of C60 fullerene has been observed by direct toxic effect on tumor cells, as well as through the modulation of the functions of effector cells of antitumor immunity.

Straightforward hit identification approach in fragment-based discovery of bromodomain-containing protein 4 (BRD4) inhibitors.

A combination approach of a fragment screening and "SAR by catalog" was used for the discovery of bromodomain-containing protein 4 (BRD4) inhibitors. Initial screening of 3695-fragment library against bromodomain 1 of BRD4 using thermal shift assay (TSA), followed by initial hit validation, resulted in 73 fragment hits, which were used to construct a follow-up library selected from available screening collection. Additionally, analogs of inactive fragments, as well as a set of randomly selected compounds were also prepared (3 × 3200 compounds in total). Screening of the resulting sets using TSA, followed by re-testing at several concentrations, counter-screen, and TR-FRET assay resulted in 18 confirmed hits. Compounds derived from the initial fragment set showed better hit rate as compared to the other two sets. Finally, building dose-response curves revealed three compounds with IC50 = 1.9-7.4 µM. For these compounds, binding sites and conformations in the BRD4 (4UYD) have been determined by docking.

An Old Story in the Parallel Synthesis World: An Approach to Hydantoin Libraries.

An approach to the parallel synthesis of hydantoin libraries by reaction of in situ generated 2,2,2-trifluoroethylcarbamates and α-amino esters was developed. To demonstrate utility of the method, a library of 1158 hydantoins designed according to the lead-likeness criteria (MW 200-350, cLogP 1-3) was prepared. The success rate of the method was analyzed as a function of physicochemical parameters of the products, and it was found that the method can be considered as a tool for lead-oriented synthesis. A hydantoin-bearing submicromolar primary hit acting as an Aurora kinase A inhibitor was discovered with a combination of rational design, parallel synthesis using the procedures developed, in silico and in vitro screenings.