In vitro anti-influenza assessment of anionic compounds ascorbate, acetate and citrate.

BACKGROUND: Influenza A virus (IAV) infection remains a serious public health threat. Due to drug resistance and side effects of the conventional antiviral drugs, repurposing the available natural compounds with high tolerability and fewer side effects has attracted researchers' attention. The aim of this study was to screen in vitro anti-influenza activity of three anionic compounds ascorbate, acetate, and citrate. METHODS: The non-cytotoxic concentration of the compounds was determined by MTT assay and examined for the activity against IAV in simultaneous, pre-, and post-penetration combination treatments over 1 h incubation on Madin-Darby Canine Kidney (MDCK) cell line. The virus titer and viral load were determined using hemagglutination assay (HA) and qPCR, respectively. Few pro-inflammatory and anti-inflammatory cytokines were evaluated at RNA and protein levels by qPCR and ELISA, respectively. RESULTS: The non-cytotoxic concentrations of the ascorbate (200 mg/ml), acetate and citrate (both 3 mg/ml) reduced the viral titer by 6.5, 4.5, and 1.5 logs in the simultaneous combination treatment. The M protein gene copy number decreased significantly in simultaneous treatment (P < 0.01). The expression of cytokines was also affected by the treatment of these compounds. CONCLUSIONS: These anionic compounds could affect the influenza virus load, thereby reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines levels.

Turn-folded magainin lipopeptide analog induces cytoplasmic vacuoles in MDA-MB-231 cells through G2-phase arrest.

Selective induced non-canonical programmed deaths in the lipid raft type 1-enriched MDA-MB-231 is a promising treatment approach. Cationic amphiphilic peptides conjugated to relatively long fatty acyl chains that tend to self-aggregate are prone to upregulate necroptotic and paraptotic signaling. We investigated the toxic effects of an N-terminally palmitoylated magainin derivate (P1MK5E) in the MDA-MB-231 cells in relation to its structure at molecular level. The modeling showed that the palmitoylation reinforces a turn-like structural motif in the lipopeptide which is likely required for its activity. P1MK5E triggered intracellular generation of reactive oxygen species (ROS), G2-phase arrest, mitochondrial membrane potential (ΔΨmt) disturbance and presumable flopping of phosphatidylserine (PtdSer) to the cancer cell membrane outer surface in a comparable manner to doxorubicin (DOX) that induces apoptotic signaling. Despite forming extensive congregates of different sizes at the cell surface, P1MK5E had little impacts on the MDA-MB-231 membrane integrity. The cell death upon exposure to the lipopeptide was, however, caspase 3 independent and characterized by cytoplasmic vacuolation and no distinct nuclear fragmentation that is to be privileged in the treatment of apoptotic resistance pathways in triple-negative breast cancers (TNBCs).

Molecular Insight into the Interaction between Camptothecin and Acyclic Cucurbit[4]urils as Efficient Nanocontainers in Comparison with Cucurbit[7]uril: Molecular Docking and Molecular Dynamics Simulation.

Cucurbit[n]urils (CB[n], n = 5, 6, 7, 8, 10, 14) and their derivatives due to the hydrophobic cavities and polar carbonyl portals have been considerably explored for their potential uses as drug delivery systems. It is important to understand how these macrocyclic compounds interact with guests. Camptothecin (CPT), as a natural alkaloid, is a topoisomerase inhibitor with antitumor activity against breast, pancreas, and lung cancers. The application of this drug in cancer therapy is restricted due to its low aqueous solubility and high toxicity. Recently, the complex formation between the cucurbit[7]uril (CB[7])/acyclic cucurbit[4]uril (aCB[4]) nanocontainers and CPT have been evaluated to overcome the potential drawbacks of the related drug. Herein, using computational methods, we identified the interaction mechanism of CPT with CB[7]/aCB[4]s, which consist of benzene and naphthalene sidewalls (aCB[4]benzene and aCB[4]naphthalene, respectively) since the experimental approaches have not completely provided information at the molecular level. Our molecular docking and molecular dynamics (MD) simulations show that CB[7] and its two acyclic derivatives form stable inclusion complexes with CPT especially through hydrophobic interactions. We also found that aCB[4]s with the aromatic sidewalls can attach to CPT through π-π interactions. This investigation highlights aCB[4]s due to the structural properties and flexible nature as better nanocontainers for controlled release delivery of pharmaceutical agents in comparison with the CB[7] nanocontainer.

An efficient method for recombinant production of human alpha synuclein in Escherichia coli using thioredoxin as a fusion partner.

Herein, we describe a simple and efficient approach to produce recombinant human α-synuclein (hAS) with high purity from Escherichia coli (E. coli). The cDNA for hAS was inserted into plasmid pET32a and expressed in E. coli BL21 (DE3) with an N-terminal tag containing E. coli thioredoxin (trx), followed by a histidine hexapeptide, and a tobacco etch virus (TEV) protease cleavage site (trx-6His-TEV). The fusion protein, trx-hAS, was initially released by osmotic shock treatment from the host cells and subsequently purified using a nickel affinity chromatography. A TEV protease cleavage step was performed to liberate the target protein, hAS, from the fusion partner, trx. Finally, an additional nickel affinity chromatography was performed to further purify the digested product. The yield of this method is ∼25 mg of tag-less protein (with ∼99% purity) per liter of culture volume. Reverse phase HPLC (RP-HPLC) and electrospray ionization (ESI) mass spectrometry confirmed the purity and authenticity of the purified protein. Thioflavin T (ThT) fluorescence assay, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy demonstrated that the purified proteins form fibrils. Our protocol not only provides a convenient procedure for preparing highly pure hAS, but also requires very little specialized laboratory techniques.

Characterization of a new member of kunitz-type protein family from the venom of Persian false-horned viper, Pseudocerastes persicus.

A new member of kunitz-type protein family, PPTI (PseudocerastesPersicusTrypsin Inhibitor), was isolated from the venom of Persian false horned viper Pseudocerastes persicus and characterized. Mass spectrometry and amino acid sequencing revealed that PPTI is a 68 amino acid protein with molecular weight of about 7.6 kDa. The first amino acid residue of PPTI is N-terminally blocked via a post translational modification to pyroglutamyl. Sequence comparison against UniProtKB shows a high sequence similarity of PPTI with kunitz-type proteins, especially serine protease inhibitors and dendrotoxins (DTXs). The number of cysteines and disulfide bonding pattern of PPTI are the same as kunitz-type proteins. Based on sequence derive information, anti-protease activity of PPTI against trypsin was experimentally examined. The constructed homology models of PPTI confirmed the ability of PPTI to fold similarly to kunitz domain. The presence of characteristic basic-hydrophobic functional dyad of DTXs in PPTI supports its inhibitory potential against potassium channels. In summary, this study hypothesized the dual functionality of PPTI according to its inhibitory effect on trypsin and its potential ability in blocking potassium channel.

Arginine/Tryptophan-Rich Cyclic α/ß-Antimicrobial Peptides: The Roles of Hydrogen Bonding and Hydrophobic/Hydrophilic Solvent-Accessible Surface Areas upon Activity and Membrane Selectivity.

The bacterial selectivity of an amphiphilic library of small cyclic α/ß-tetra-, α/ß-penta-, and α/ß-hexapeptides rich in arginine/tryptophan (Arg/Trp) residues, which contains asymmetric backbone configurations and differ in hydrophobicity and alternating d,l-amino acids, was investigated against Bacillus subtilis and Escherichia coli. The structural analyses showed that the peptides tend to form assemblies of different shapes. All-l-peptides, especially the most hydrophobic pentamers, were more strongly anti-B. subtilis. With the exception to cyclo(Phe-d-Trp-ß3 hArg-Arg-d-Trp) (Phe=phenylalanine), the peptides had no effects on inner membrane of E. coli, but lyzed the lipopolysaccharide layer according to their activity pattern. The activities adversely changed with a decrease in the number of amide intramolecular hydrogen bonds in assemblies of diastereomeric peptides and the ratio of hydrophobic/hydrophilic solvent-accessible surface areas. The remarkable enhanced entropic contribution for the partitioning of the least conformationally constrained cyclo(Trp-d-Phe-ß3 hTrp-Arg-d-Arg) sequence into the membranes supported the strong self-assembly behavior, therefore making the peptide less penetrable through the E. coli outer layer.

Turn plasticity distinguishes different modes of amyloid-ß aggregation.

Pathogenesis of Alzheimer's disease (AD) is associated with aggregation of the amyloid-ß (Aß) peptide into oligomeric and fibrillar assemblies; however, little is known about the molecular basis of aggregation of Aß into distinct assembly states. Here we demonstrate that phosphorylation at serine 26 (S26) impairs Aß fibrillization while stabilizing its monomers and nontoxic soluble assemblies of nonfibrillar morphology. NMR spectroscopy and replica-exchange molecular dynamics indicate that introduction of a phosphate group or phosphomimetic at position 26 diminishes Aß's propensity to form a ß-hairpin, rigidifies the region around the modification site, and interferes with formation of a fibril-specific salt bridge between aspartic acid 23 and lysine 28. The combined data demonstrate that phosphorylation of S26 prevents a distinct conformational rearrangement that is required for progression of Aß aggregation toward fibrils and provide a basis for a possible role of phosphorylation at serine 26 in AD.

A molecular dynamics simulation study of nanomechanical properties of asymmetric lipid bilayer.

A very important part of the living cells of biological systems is the lipid membrane. The mechanical properties of this membrane play an important role in biophysical studies. Investigation as to how the insertion of additional phospholipids in one leaflet of a bilayer affects the physical properties of the obtained asymmetric lipid membrane is of recent practical interest. In this work a coarse-grained molecular dynamics simulation was carried out in order to compute the pressure tensor, the lateral pressure, the surface tension and the first moment of lateral pressure in each leaflet of such a bilayer. Our simulations indicate that adding more phospholipids into one monolayer results in asymmetrical changes in the lateral pressure of the individual bilayer leaflets. Interestingly, it has been observed that a change in phospholipid density in one leaflet affects the physical properties of unperturbed leaflet as well. The asymmetric behavior of the physical properties of the two leaflets as a result of a change in the contribution of the various intermolecular forces in the presence of additional phospholipids may be expressed formally.

Nanomechanical properties of lipid bilayer: asymmetric modulation of lateral pressure and surface tension due to protein insertion in one leaflet of a bilayer.

The lipid membranes of living cells form an integral part of biological systems, and the mechanical properties of these membranes play an important role in biophysical investigations. One interesting problem to be evaluated is the effect of protein insertion in one leaflet of a bilayer on the physical properties of lipid membrane. In the present study, an all atom (fine-grained) molecular dynamics simulation is used to investigate the binding of cytotoxin A3 (CTX A3), a cytotoxin from snake venom, to a phosphatidylcholine lipid bilayer. Then, a 5-microsecond [corrected] coarse-grained molecular dynamics simulation is carried out to compute the pressure tensor, lateral pressure, surface tension, and first moment of lateral pressure in each monolayer. Our simulations reveal that the insertion of CTX A3 into one monolayer results in an asymmetrical change in the lateral pressure and corresponding spatial distribution of surface tension of the individual bilayer leaflets. The relative variation in the surface tension of the two monolayers as a result of a change in the contribution of the various intermolecular forces may potentially be expressed morphologically.

Familial Alzheimer's Disease-Related Mutations Differentially Alter Stability of Amyloid-Beta Aggregates.

Amyloid-beta (Aß) deposition as senile plaques is a pathological hallmark of Alzheimer's disease (AD). AD is characterized by a large level of heterogeneity in amyloid pathology, whose molecular origin is poorly understood. Here, we employ NMR spectroscopy and MD simulation at ambient and high pressures and investigate how AD-related mutations in Aß peptide influence the stability of Aß aggregates. The pressure-induced monomer dissociation from Aß aggregates monitored by NMR demonstrated that the Iowa (D23N), Arctic (E22G), and Osaka (ΔE22) mutations altered the pressure stability of Aß40 aggregates in distinct manners. While the NMR data of monomeric Aß40 showed only small localized effects of mutations, the MD simulation of mutated Aß fibrils revealed their distinct susceptibility to elevated pressure. Our data propose a structural basis for the distinct stability of various Aß fibrils and highlights "stability" as a molecular property potentially contributing to the large heterogeneity of amyloid pathology in AD.

Investigation of Antioxidant and Anti-inflammatory Properties of Berberine Nanomicelles: In vitro and In vivo Studies.

INTRODUCTION: In the present study, neuroprotective effects of berberine (BBR) and berberine nanomicelle (BBR-NM) against lipopolysaccharides (LPS)-induced stress oxidative were investigated, and compared by evaluating their antioxidant and anti-inflammatory activities in PC12 cells, and rat brains. A fast, green, and simple synthesis method was used to prepare BBR-NMs. METHOD: The prepared BBR-NMs were then characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). In vitro experiments were carried out on the LPS-treated PC12 cell lines to investigate the anti-cytotoxic and antioxidant properties of BBR-NM and BBR. The results showed that BBR-NMs with a diameter of ~100 nm had higher protective effects against ROS production and cytotoxicity induced by LPS in PC12 cells in comparison with free BBR. RESULTS: Moreover, in vivo experiments indicated that the activity levels of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), increased in the brain of LPS-treated rats administrated with BBR-NM at the optimum dose of 100 mg.kg-1 . BBR-NM administration also resulted in decreased concentration of lipid peroxidation (MDA) and pro-inflammatory cytokines, such as Serum interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α). CONCLUSION: Overall, BBR-NM demonstrated higher neuroprotective effects than free BBR, making it a promising treatment for improving many diseases caused by oxidative stress and inflammation.

Crocin Inhibits the Fibrillation of Human α-synuclein and Disassembles Mature Fibrils: Experimental Findings and Mechanistic Insights from Molecular Dynamics Simulation.

The aggregation of human alpha-synuclein (hαS) is pivotally implicated in the development of most types of synucleinopathies. Molecules that can inhibit or reverse the aggregation process of amyloidogenic proteins have potential therapeutic value. The anti-aggregating activity of multiple carotenoid compounds has been reported over the past decades against a growing list of amyloidogenic polypeptides. Here, we aimed to determine whether crocin, the main carotenoid glycoside component of saffron, would inhibit hαS aggregation or could disassemble its preformed fibrils. By employing a series of biochemical and biophysical techniques, crocin was exhibited to inhibit hαS fibrillation in a dose-dependent fashion by stabilizing very early aggregation intermediates in off-pathway non-toxic conformations with little ß-sheet content. We also observed that crocin at high concentrations could efficiently destabilize mature fibrils and disassemble them into seeding-incompetent intermediates by altering their ß-sheet conformation and reshaping their structure. Our atomistic molecular dynamics (MD) simulations demonstrated that crocin molecules bind to both the non amyloid-ß component (NAC) region and C-terminal domain of hαS. These interactions could thereby stabilize the autoinhibitory conformation of the protein and prevent it from adopting aggregation-prone structures. MD simulations further suggested that ligand molecules prefer to reside longitudinally along the fibril axis onto the edges of the inter-protofilament interface where they establish hydrogen and hydrophobic bonds with steric zipper stabilizing residues. These interactions turned out to destabilize hαS fibrils by altering the interstrand twist angles, increasing the rigidity of the fibril core, and elevating its radius of gyration. Our findings suggest the potential pharmaceutical implication of crocin in synucleinopathies.

S494 O-glycosylation site on the SARS-CoV-2 RBD affects the virus affinity to ACE2 and its infectivity; a molecular dynamics study.

SARS-CoV-2 is a strain of Coronavirus family that caused the ongoing pandemic of COVID-19. Several studies showed that the glycosylation of virus spike (S) protein and the Angiotensin-Converting Enzyme 2 (ACE2) receptor on the host cell is critical for the virus infectivity. Molecular Dynamics (MD) simulations were used to explore the role of a novel mutated O-glycosylation site (D494S) on the Receptor Binding Domain (RBD) of S protein. This site was suggested as a key mediator of virus-host interaction. By exploring the dynamics of three O-glycosylated models and the control systems of unglcosylated S4944 and S494D complexes, it was shown that the decoration of S494 with elongated O-glycans results in stabilized interactions on the direct RBD-ACE2. Calculation of the distances between RBD and two major H1, H2 helices of ACE2 and the interacting pairs of amino acids in the interface showed that the elongated O-glycan maintains these interactions by forming several polar contacts with the neighbouring residues while it would not interfere in the direct binding interface. Relative binding free energy of RBD-ACE2 is also more favorable in the O-glycosylated models with longer glycans. The increase of RBD binding affinity to ACE2 depends on the size of attached O-glycan. By increasing the size of O-glycan, the RBD-ACE2 binding affinity will increase. Hence, this crucial factor must be taken into account for any further inhibitory approaches towards RBD-ACE2 interaction.

Structural characterization of PPTI, a kunitz-type protein from the venom of Pseudocerastes persicus.

The main purpose of this report is to investigate the structural property and new potential function of PPTI (Pseudocerastes Persicus Trypsin Inhibitor), a kunitz-type protein with inhibitory effect against trypsin proteolytic activity. Besides kunitz-type serine protease inhibitors, PPTI shows clear-cut similarities with dendrotoxins (DTXs), the other kunitz-type protein subfamily. The most important reason is the presence of functionally important residues of DTXs at correspondingly the same positions in PPTI. As such, we proposed the new ability of PPTI for inhibiting voltage-gated potassium channels and consequently its dual functionality. At first, we determined the solution structure of PPTI via Nuclear Magnetic Resonance (NMR) spectroscopy. Then by homology modeling, we constructed the model structure of trypsin-PPTI complex to confirm the same interaction pattern as trypsin-BPTI at complex interface. Finally, by Brownian dynamics (BD) simulations of PPTI NMR derived ensemble structure as ligand against homology model of human Kv1.1 potassium channel as receptor, we evaluated the potential DTX-like activity of PPTI. The results of our study support the proposed dual functionality of PPTI.

Paclitaxel interaction with cucurbit [7]uril and acyclic Cucurbit[4]uril nanocontainers: A computational approach.

Paclitaxel (PTX) is a natural terpenoid compound that has been broadly studied for its antitumor activities and widely used as a chemotherapy medication. The treatment efficacy of PTX is affected by its low aqueous solubility, thus causing a subject of extensive research. In recent years, synthetic molecular containers such as cucurbit[n]urils (CB[n]s) and their derivatives have been significantly developing because of their remarkable ability to bind hydrophobic and cationic drugs. Recent experimental studies have shown that acyclic CB[n]-type containers (aCB[n]s), as new derivatives of the family of CB[n]s, increase the solubility of insoluble pharmaceuticals. However, the nature by which the drug interacts with carriers remains largely unknown. In this study, molecular docking and molecular dynamics (MD) simulation were performed to understand how CB[7] and aCB[4] nanocontainers interact with PTX which affect its aqueous solubility. The results clarify how the flexibility of containers is influenced by their structure and how this affects their interactions with PTX. Our results reveal that although both CB[7] and aCB[4] are capable of binding to PTX, the affinity to aCB[4] is higher than that of CB[7]. It has also been shown that the binding to both CB[7] and aCB[4] is probably an entropy-driven process. This research supports the potential use of the cucurbit[n]urils and their acyclic derivatives as drug delivery systems.

Efficient construction of a diverse conformational library for amyloid-ß as an intrinsically disordered protein.

Structural characterization of intrinsically disordered proteins (IDPs) is paramount and challenging in structural biology. In this regard, a de novo computational protocol is introduced to build heterogeneous structural libraries for amyloid-ß (Aß) as a critical IDP. This method combines the strength of the simulated annealing - in jumping over energy barriers and escaping from traps - with short conventional molecular dynamics simulations to quickly explore local regions of the conformational space. The protocol efficiency and reliability in building Aß conformational library is compared with two widely used simulation methods, replica exchange molecular dynamics and multiple trajectory sampling. The probability distribution functions of various structural and energetic features are constructed for each library, and also the diversity and convergence rates in these protocols were compared. Our results show that the suggested protocol is a successful computational method in the generation of a diverse conformational library of the Aß monomer in agreement with experimental data. This method focuses on visiting more conformations in less computational time without paying attention to the statistical weight of each state in the library. We believe that the suggested computational technique can be used for generating a reasonable starting pool for subsequent reweighting with experimental data to obtain a statistical ensemble.

Conformational changes of alpha-chymotrypsin in a fibrillation-promoting condition: a molecular dynamics study.

Amyloid nanofibril formation appears to be a generic property of polypeptide chains. alpha-Chymotrypsin (aCT) was recently driven toward amyloid-like aggregation by the addition of trifluoroethanol (TFE) at intermediate concentrations. In this study we employed a molecular dynamics simulation to investigate the early events in TFE-induced conformational changes of aCT that precede amyloid formation, and compared the results of the simulation with previous experiments. TFE molecules were found to rapidly replace the water molecules closely associated with the protein surface. The gyration radius, together with total and hydrophobic solvent-accessible surface areas of aCT, was significantly increased. In accord with the experimental observations, the extended beta-conformation of backbone was increased. The secondary structural elements of aCT in water and TFE/water mixture showed a reasonable fit, whereas significant deviations were observed for several loops. These alterations originated largely from main-chain rotations at glycine residues. The catalytic active site and S1 binding pocket of the enzyme were also distorted in the TFE/water mixture. The obtained results are suggested to provide more insights into the conformational properties of the amyloid aggregation-prone protein species. Possible mechanisms of TFE-induced alterations in the conformation and dynamics of the protein structure are also discussed.

Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase.

Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase.

Structural and functional characterization of a mutant of Pseudocerastes persicus natriuretic peptide.

We hereby report on a mutational analysis of a novel natriuretic peptide (PNP), recently isolated by us from the Iranian snake venom. The PNP variant (mutPNP) with four substitutions (G16T, K18S, R21S, G23R) and a disulfide bonded ring shortened by 3 residues. mutPNP peptide was expressed in pET32 and purified by affinity separation on nickel resin followed by RP-HPLC chromatography. The conformation of mutPNP was characterized in solution by 1H nuclear magnetic resonance spectroscopy, where it was found that the 14-residue disulfide bonded ring, like the 17-residue ring in PNP, retains a high degree of conformational flexibility. The conformation of mutPNP bound to NPR-C receptor was predicted by homology protein structure modeling. When injected intravenously into rats, mutPNP, in contrast to PNP had no physiological effect on blood pressure or on diuresis. The loss of physiological activity is explained in terms of the modeled bound conformation and the ensemble of solution conformations obtained using the NMR constraints.