Lysine ε-aminolysis and incorporation of sulfhydryl groups into human brain tau 4R/1N and 306VQIVYK311 enhances the formation of beta structures and toxicity.

In the present study, we investigated the effects of N-homocysteine thiolactone (tHcy) modification on expressed and purified tau protein and the synthesized VQIVYK target peptide. The modified constructs were subjected to comprehensive validation using various methodologies, including mass spectrometry. Subsequently, in vivo, in vitro, and in silico characterizations were performed under both reducing and non-reducing conditions, as well as in the presence and absence of heparin as a cofactor. Our results unequivocally confirmed that under reducing conditions and in the presence of heparin, the modified constructs exhibited a greater propensity for aggregation. This enhanced aggregative behavior can be attributed to the disruption of lysine positive charges and the subsequent influence of hydrophobic and p-stacking intermolecular forces. Notably, the modified oligomeric species induced apoptosis in the SH-SY5Y cell line, and this effect was further exacerbated with longer incubation times and higher concentrations of the modifier. These observations suggest a potential mechanism involving reactive oxygen species (ROS). To gain a deeper understanding of the molecular mechanisms underlying the neurotoxic effects, further investigations are warranted. Elucidating these mechanisms will contribute to the development of more effective strategies to counteract aggregation and mitigate neurodegeneration.

Dihydropyrimidine derivatives as MDM2 inhibitors.

One of the chief pathways to regulate p53 levels is MDM2 protein, which negatively controls p53 by direct inhibition. Many cancers overproduce MDM2 protein to interrupt p53 functions. Therefore, impeding MDM2's binding to p53 can reactivate p53 in tumor cells may suggest an effective approach for tumor therapy. Here, some Monastrol derivatives were designed in silico as MDM2 inhibitors, and their initial cytotoxicity was evaluated in vitro on MFC-7 and MDA-MB-231 cells. A small library of Monastrol derivatives was created, and virtual screening (VS) was performed on them. The first-ranked compound, which was extracted from VS, and the other six compounds 5a-5f were selected to carry out the single-docking and docking with explicit waters. The compound with the best average results was then subjected to molecular dynamic (MD) simulation. Compounds 5a-5f were chemically synthesized and evaluated in vitro for their initial cytotoxicity on MFC-7 and MDA-MB-231 cells by MTT assay. The best compound was compound 5d with ΔGave = -10.35 kcal/mol. MD simulation revealed a median potency in comparison with Nutlin-3a. The MTT assay confirmed the docking and MD experiments. 5d has an IC50 of 60.09 µM on MCF-7 cells. We attempted to use Monastrol scaffold as a potent inhibitor of MDM2 rather than an Eg5 inhibitor using in silico modification. The results obtained from the in silico and in vitro evaluations were noteworthy and warranted much more effort in the future.

The effect of mutation on neurotoxicity reduction of new chimeric reteplase, a computational study.

Background and purpose: Excitotoxicity in nerve cells is a type of neurotoxicity in which excessive stimulation of receptors (such as N-methyl-d-aspartate glutamate receptors (NMDAR)) leads to the influx of high-level calcium ions into cells and finally cell damage or death. This complication can occur after taking some of the plasminogen activators like tissue plasminogen activator and reteplase. The interaction of the kringle2 domain in such plasminogen activator with the amino-terminal domain (ATD) of the NR1 subunit of NMDAR finally leads to excitotoxicity. In this study, we assessed the interaction of two new chimeric reteplase, mutated in the kringle2 domain, with ATD and compared the interaction of wild-type reteplase with ATD, computationally. Experimental approach: Homology modeling, protein docking, molecular dynamic simulation, and molecular dynamics trajectory analysis were used for the assessment of this interaction. Findings/Results: The results of the free energy analysis between reteplase and ATD (wild reteplase: -2127.516 ± 0.0, M1-chr: -1761.510 ± 0.0, M2-chr: -521.908 ± 0.0) showed lower interaction of this chimeric reteplase with ATD compared to the wild type. Conclusion and implications: The decreased interaction between two chimeric reteplase and ATD of NR1 subunit in NMDAR which leads to lower neurotoxicity related to these drugs, can be the start of a way to conduct more tests and if the results confirm this feature, they can be considered potential drugs in acute ischemic stroke treatment.

Evolutionary trajectories of beta-lactamase NDM and DLST cluster in Pseudomonas aeruginosa: finding the putative ancestor.

Pseudomonas aeruginosa has different antibiotic resistance pathways, such as broad-spectrum lactamases and metallo-ß-lactamases (MBL), penicillin-binding protein (PBP) alteration, and active efflux pumps. Polymerase chain reaction (PCR) and sequencing methods were applied for double-locus sequence typing (DLST) and New Delhi metallo-ß-lactamase (NDM) typing. We deduced the evolutionary pathways for DLST and NDM genes of P. aeruginosa using phylogenetic network. Among the analyzed isolates, 62.50% of the P. aeruginosa isolates were phenotypically carbapenem resistance (CARBR) isolates. Characterization of isolates revealed that the prevalence of blaNDM, blaVIM, blaIMP, undetermined carbapenemase, and MexAB-OprM were 27.5%, 2%, 2.5%, 12.5%, and 15%, respectively. The three largest clusters found were DLST t20-105, DLST t32-39, and DLST t32-52. The network phylogenic tree revealed that DLST t26-46 was a hypothetical ancestor for other DLSTs, and NDM-1 was as a hypothetical ancestor for NDMs. The combination of the NDM and DLST phylogenic trees revealed that DLST t32-39 and DLST tN2-N3 with NDM-4 potentially derived from DLST t26-46 along with NDM-1. Similarly, DLST t5-91 with NDM-5 diversified from DLST tN2-N3 with NDM-4. This is the first study in which DLST and NDM evolutionary routes were performed to investigate the origin of P. aeruginosa isolates. Our study showed that the utilization of medical equipment common to two centers, staff members common to two centers, limitations in treatment options, and prescription of unnecessary high levels of meropenem are the main agents that generate new types of resistant bacteria and spread resistance among hospitals.

One-pot synthesis of polyhydroquinoline-1,2,3-triazole hybrids in deep eutectic solvent as anti-leishmanial agents and molecular modeling studies.

The novel hybrids with 1,2,3-triazole and polyhydroquinoline scaffolds were successfully synthesized by multicomponent reaction of propargyloxybenzaldehyde, 1,3-cyclohexadione, ethylacetoacetate and ammonium acetate followed through click reaction in the presence of deep eutectic solvent ChCl/ZnCl2 as an efficient catalyst. Their anti-leishmanial activity was evaluated against amastigote and promastigote forms of L. tropica, L. major, and two different species of L. infantum. Furthermore, to determine the cytotoxicity of the hybrids, they were evaluated against the murine macrophage cell line J774.A1. Based on the results, three hybrids showed the highest antileishmanial activity. However, they revealed low cytotoxicity. Hybrid 6j was the most potent compound against both the forms of all leishmanial types, with IC50 = 13.5 and 11.9 µg/mL for L. major, 37.5 and 25 µg/mL for L. tropica, 17.5 and 20 µg/mL for L. infantum (MCAN/IR//96/LON49) and 35.5 and 30 µg/mL for L. infantum (MCAN/ES/98/LIM-877), respectively. Finally, molecular docking and molecular dynamics simulations were also performed to identify possible mechanism antileishmanial activity.Communicated by Ramaswamy H. Sarma.

A theoretical survey to find potential natural compound for inhibition of binding the RBD domain to ACE2 receptor based on plant antivirals.

The spike protein of coronavirus is crucial in binding and arrival of the virus to the human cell via binding to the human ACE2 receptor. In this study, at first 25 antiviral phytochemicals were docked into the RBD domain of spike protein, and then all complexes and free RBD domains were separately subjected to molecular dynamics simulation for 100 ns and MM/PBSA binding free energy calculation. In this phase, four ligands were chosen as hit compounds and a natural compound database (NPASS) was screened based on high similarity with these ligands, and 367 ligands were found. Then the same previous procedure was repeated for these ligands and ADME properties were investigated. Finally, virtual screening and 4400 ns MD simulation and MM/PBSA calculation revealed that new ligands including NPC67959, NPC157855, NPC248793, and NPC216361 can inhibit the RBD domain of spike protein and we propose them as potential drugs for experimental studies.Communicated by Ramaswamy H. Sarma.

Preparing a database of corrected protein structures important in cell signaling pathways.

Background and purpose: Precise structures of macromolecules are important for structure-based drug design. Due to the limited resolution of some structures obtained from X-ray diffraction crystallography, differentiation between the NH and O atoms can be difficult. Sometimes a number of amino acids are missing from the protein structure. In this research, we intend to introduce a small database that we have prepared for providing the corrected 3D structure files of proteins frequently used in structure-based drug design protocols. Experimental approach: 3454 soluble proteins belonging to the cancer signaling pathways were collected from the PDB database from which a dataset of 1001 was obtained. All were subjected to corrections in the protein preparation step. 896 protein structures out of 1001 were corrected successfully and the decision on the remained 105 proposed twelve for homology modeling to correct the missing residues. Three of them were subjected to molecular dynamics simulation for 30 ns. Findings / Results: 896 corrected proteins were perfect and homology modeling on 12 proteins with missing residues in the backbone resulted in acceptable models according to Ramachandran, z-score, and DOPE energy plots. RMSD, RMSF, and Rg values verified the stability of the models after 30 ns molecular dynamics simulation. Conclusion and implication: A collection of 1001 proteins were modified for some defects such as adjustment of the bond orders and formal charges, and addition of missing side chains of residues. Homology modeling corrected the amino missing backbone residues. This database will be completed for quite a lot of water-soluble proteins to be uploaded to the internet.

In silico screening and molecular dynamics simulations toward new human papillomavirus 16 type inhibitors.

Background and purpose: Human papillomavirus (HPV) is known as the main reason for cervical cancer. According to carcinogenic risk, HPV can be located into two classes, counting the low-risk virus, which is the main cause of genital warts and low-grade cervical epithelial lesions. HPV-16 is one of the high-risk HPV subtypes in the spectrum of cervical diseases. Experimental approach: The PubChem database was screened in order to identify potential anti-HPV hits followed by ADMET predictions. Then, molecular docking was performed to improve the accuracy of screening and also to find the details of the interactions of the hit compounds with the active site. Finally, molecular dynamic (MD) simulations and free binding energy on top-ranked structures CID_73212812, CID_91059286, CID_69838075, cidofovir, and jaceosidin were carried out with protein to compute the interaction energies and stability of the top-ranked compounds at the active site. Findings/Results: Based on molecular docking studies, three compounds including CID_73212812, CID_91059286, and CID_69838075 exhibited the best results among compounds against the E6 protein of HPV-16. Furthermore, RMSD, RMSF, hydrogen binds, Rg, and energy analysis during MD simulation certainly indicated the stable binding of selected compounds with E6 protein of HPV-16 active site. Conclusion and implications: Docking and MD results revealed that hydrophobic contacts and optimum hydrogen bonds were determinant factors in the interactions of hits and the E6 protein of HPV-16. In addition, the binding energy portions exposed that Van der Waals and non-polar interactions were fundamental factors in the molecule binding.

In silico and in vitro studies of thiosemicarbazone-indole hybrid compounds as potent α-glycosidase inhibitors.

It is essential to study α-glucosidase enzyme (EC 3.2.1.20) inhibitors because of their physiological role as well as their clinical relevance. In previous research, a novel series of thiosemicarbazone-indole hybrid compounds were synthesized and reported. In the current research, α-glucosidase inhibitory activity of the derivatives was evaluated and then in silico studies were carried out on screened compounds. All derivatives exhibited a magnificent α-glucosidase inhibitory activity (IC50 = 27.0 ± 1.0-97.4 ± 1.5 µM) toward the acarbose as reference drug (IC50 = 750.0 ± 1.5 µM). Compound 1i having phenyl ring at the thiosemicarbazone moiety and the trimethoxymethyl substituent at phenyl moiety of C2 position of indole ring was the most potent compound (IC50 = 27.0 ± 1.0 µM) among other compounds. A kinetic study of 1i revealed that is a competitive inhibitor against α-glucosidase. Moreover, the molecular docking studies established that screened derivatives interacted with the essential amino acids in the active site. Finally, based on the molecular dynamics simulations and free binding energy calculations, complexes 1d, 1i and 1k with α-glucosidase showed a good stability in the active site. Van der Waals and electrostatic interactions also exhibited the most contributions to the stability of these complexes. Moreover, all the screened compounds showed agreeable ADME properties for oral bio-availability, and good drug-likeness.

Absorption of daunorubicin and etoposide drugs by hydroxylated and carboxylated carbon nanotube for drug delivery: theoretical and experimental studies.

Anti-cancer daunorubicin and etoposide drugs are mostly used in chemotherapy medicine to treat a wide variety of cancers. Many of the side effects and specific delivery to a target tissue are the main challenges of using chemotherapeutic agents. To avoid serious toxic side effects and improve treatment outcomes, functionalized carbon nanotubes (f-CNTs) are considered promising nano-carriers for the delivery of chemotherapeutic drugs to cancerous cells. We examined the effects of -OH and -COO- groups on CNTs surface for absorption of two anticancer drugs including daunorubicin and etoposide using molecular dynamics simulation and experimental assays. To evaluate the absorption of each drug in each CNT, the complexes of drugs/CNTs in water were simulated separately. Theoretical investigation demonstrated that CNT-OH and CNT-COO- are more suitable for absorption of daunorubicin and etoposide, respectively. Experimental findings also confirmed molecular dynamics simulation results. Communicated by Ramaswamy H. Sarma.

Finding a prospective dual-target drug for the treatment of coronavirus disease by theoretical study.

Spike protein of coronavirus is a key protein in binding and entrance of virus to the human cell via binding to the receptor-binding domain (RBD) domain of S1 subunit to peptidase domain region of ACE2 receptor. In this study, the possible effect of 24 antiviral drugs on the RBD domain of spike protein was investigated via docking and molecular dynamics simulation for finding a dual-target drug. At first, all drugs were docked to the RBD domain of spike protein, and then all complexes and free RBD domains were separately used for molecular dynamics simulation for 50 ns via amber18 software. The simulation results showed that 10 ligands from 28 ligands were separated from the RBD domain, and among 18 remained ligands, baloxavir marboxil, and danoprevir drugs, besides endonuclease activity and protease inhibitory, can bind to key residues of the RBD domain. Then these drugs have a dual target and should be more effective than current drugs, and experimental studies should be done on baloxavir marboxil and danoprevir as more potential drugs for coronavirus disease Communicated by Ramaswamy H. Sarma.

In-silico design of peptides for inhibition of HLA-A*03-KLIETYFSK complex as a new drug design for treatment of multiples sclerosis disease.

Multiple sclerosis is recognized as a chronic inflammatory disease. Human leukocyte antigen (HLA) plays an important role in initiating adaptive immune responses. HLA class I is present in almost all nucleated cells and presents the cleaved endogenous peptide antigens to cytotoxic T cells. HLA-A*03 is one of the HLA class I alleles, which is reported as substantially related HLA to MS disease. In 2011, the structure of the HLA-A*03 in complex was identified with an immunodominant proteolipid protein (PLP) epitope (KLIETYFSK). This complex has been reported as an important autoantigen-presenting complex in MS pathogenesis. In this study, new peptides were designed to bind to this complex that may prevent specific pathogenic cytotoxic T cell binding to this autoantigen-presenting complex and CNS demyelination. Herein, 14 new helical peptides containing 19 amino acids were designed and their structures were predicted using the PEP-FOLD server. The binding of each designed peptide to the mentioned complex was then performed. A mutation approach was used by the BeAtMuSiC server to improve the binding affinity of the designed peptide. In each position, amino acid substitutions leading to an increase in the binding affinity of the peptide to the mentioned complex were determined. Finally, the resulting complexes were simulated for 40 ns using AMBER18 software. The results revealed that out of 14 designed peptides, "WRYWWKDWAKQFRQFYRWF" peptide exhibited the highest affinity for binding to the mentioned complex. This peptide can be considered as a potential drug to control multiple sclerosis disease in patients carrying the HLA-A*03 allele.

In silico evaluation of the downstream effect of mutated glucagon is consistent with higher blood glucose homeostasis in Galliformes and Strigiformes.

In contrast to mammals, glucagon is reported as a much more potent blood glucose modulator in birds. Interestingly, we have found p.Thr16Ser mutation, a variation in the highly conserved glucagon hormone, in Galliformes as well as Strigiformes. To check the effect of this mutation on the receptor binding of glucagon, we predicted the ancestral glucagon receptor sequence of all available Galliformes and Strigiformes species. Subsequently, we analysed their binding to the mutated and wild type glucagon (ancestral) by molecular dynamics simulation. At first, we made a model of ancestral glucagon receptor and ancestral mutated, and wild type glucagon in the order Galliformes and Strigiformes. Then we performed molecular dynamics for each Galliformes and Strigiformes receptor as well as each glucagon peptide, respectively. The final structures were used for docking simulation of glucagon to their receptors. The results of the docking simulations showed a stronger binding affinity of mutated glucagon to glucagon receptors. Afterward, we obtained blood glucose concentrations of all available Galliformes members, as well as all available members of its only taxonomic neighbour (order Anseriformes) in superorder Galloanserae. Interestingly the p.Thr16Ser mutation could finely cluster these two orders into two groups: higher blood glucose concentration (order Galliformes, 17.64 ± 1.66 mMol/L) and lower blood glucose concentration (order Anseriformes, 11.34 ± 1.11 mMol/L). Strigiformes which carry the mutated glucagon peptide show also high blood glucose concentrations (17.40 ± 1.51 mMol/L). Therefore, the results suggest this mutation, which leads to stronger binding affinity of mutated glucagon to its receptor, may be a driving force for higher blood glucose homeostasis in the related birds.

Examining the interactions scorpion venom peptides (HP1090, Meucin-13, and Meucin-18) with the receptor binding domain of the coronavirus spike protein to design a mutated therapeutic peptide.

The spike protein of SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2) interacts with the ACE2 receptor in human cells and starts the infection of COVID-19 disease. Given the importance of spike protein's interaction with ACE2 receptor, we selected some antiviral peptides of venom scorpion such as HP1090, meucin-13, and meucin-18 and performed docking and molecular docking analysis of them with the RBD domain of spike protein. The results showed that meucin-18 (FFGHLFKLATKIIPSLFQ) had better interaction with the RBD domain of spike protein than other peptides. We also designed some mutations in meucin-18 and investigated their interactions with the RBD domain. The results revealed that the A9T mutation had more effective interaction with the RBD domain than the meucin-18 and was able to inhibit spike protein's interaction with ACE2 receptor. Hence, peptide "FFGHLFKLTTKIIPSLFQ" can be considered as the potential drug for the treatment of COVID-19 disease.

Discovery of potential inhibitors against New Delhi metallo-ß-lactamase-1 from natural compounds: in silico-based methods.

New Delhi metallo-ß-lactamase variants and different types of metallo-ß-lactamases have attracted enormous consideration for hydrolyzing almost all ß-lactam antibiotics, which leads to multi drug resistance bacteria. Metallo-ß-lactamases genes have disseminated in hospitals and all parts of the world and became a public health concern. There is no inhibitor for New Delhi metallo-ß-lactamase-1 and other metallo-ß-lactamases classes, so metallo-ß-lactamases inhibitor drugs became an urgent need. In this study, multi-steps virtual screening was done over the NPASS database with 35,032 natural compounds. At first Captopril was extracted from 4EXS PDB code and use as a template for the first structural screening and 500 compounds obtained as hit compounds by molecular docking. Then the best ligand, i.e. NPC120633 was used as templet and 800 similar compounds were obtained. As a final point, ten compounds i.e. NPC171932, NPC100251, NPC18185, NPC98583, NPC112380, NPC471403, NPC471404, NPC472454, NPC473010 and NPC300657 had proper docking scores, and a 50 ns molecular dynamics simulation was performed for calculation binding free energy of each compound with New Delhi metallo-ß-lactamase. Protein sequence alignment, 3D conformational alignment, pharmacophore modeling on all New Delhi metallo-ß-lactamase variants and all types of metallo-ß-lactamases were done. Quantum chemical perspective based on the fragment molecular orbital (FMO) method was performed to discover conserved and crucial residues in the catalytic activity of metallo-ß-lactamases. These residues had similar 3D coordinates of spatial location in the 3D conformational alignment. So it is posibble that all types of metallo-ß-lactamases can inhibit by these ten compounds. Therefore, these compounds were proper to mostly inhibit all metallo-ß-lactamases in experimental studies.

Design and production of new chimeric reteplase with enhanced fibrin affinity: a theoretical and experimental study.

Plasminogen activators (PAs) are widely used for treatment of disorders caused by clot formation. Fibrin specific PAs are safe drugs from this group because of reducing the incidence of hemorrhage. The newer generation of PAs like tenecteplase, reteplase and desmoteplase were designed with the aim of achieving desirable properties such as improving specificity and affinity to fibrin and increasing half-life. Protein engineering and using of theoretical methods can help to rational and reliable design of new PAs with a set of favorable properties. In the present study, two new chimeric reteplase named M1-chr and M2-chr were designed with the aim of enhancing fibrin affinity also some potential properties include of increasing resistance to plasminogen activator inhibitor-1 and decreasing neurotoxicity. So, finger domain of desmoteplase was added to reteplase as a high fibrin specific domain. Some other point mutations were considering to achieve other mentioned properties. Three dimensional structure of wild-type reteplase and mutants were created by homology modeling and were evaluated by molecular dynamic simulation. Then, mutants docked to fibrin by HADDOCK web tools. Result of theoretical section verified the stability of mutants' structures. Also showed better interaction between M1-chr with fibrin than M2-chr. Wild-type and mutants were produced in bacterial expression system. Experimental assessment showed both mutants have appropriate enzymatic activity also 1.9-fold fibrin binding ability compared to wild-type. Therefore, this study offers new thrombolytic drugs with desirable properties specially enhanced fibrin affinity so they can represent a promising future in cost-effective production of favorable thrombolytic drugs.Communicated by Ramaswamy H. Sarma.

In silico study of the association of the HLA-A*31:01 allele (human leucocyte antigen allele 31:01) with neuroantigenic epitopes of PLP (proteolipid protein), MBP (myelin basic protein) and MOG proteins (myelin oligodendrocyte glycoprotein) for studying the multiple sclerosis disease pathogenesis.

The main pathologic hallmark of multiple sclerosis is a demyelinating plaque that contains a prominent immunologic response dominated by T cells of the immune system. PLP (proteolipid protein), MPB (myelin basic protein), and Myelin oligodendrocyte glycoprotein (MOG) proteins are important autoantigens for the demyelinating of CNS in multiple sclerosis. There is good evidence indicating that T CD8+ cells and MHC class I molecules play an important role in this disease. The HLA-A*31:01 allele of MHC class I is a member of HLA-A3 superfamily and there is no clear report concerning the relationship of this allele with MS. Feeling this gap, we studied the possible association of the HLA-A*31:01 with MS by prediction of neuroantigenic epitopes of human MBP, PLP, and MOG proteins of myelin sheath using in silico methods. PLP did not show any neuroantigenic epitope, but the two epitopes of MBP and seven epitopes of MOG for HLA-A*31:01 were determined via bioinformatics servers. In silico study of the nine epitope showed that MOG195-204 (LIICYNWLHR) peptide of the membrane-associated/cytoplasmic part of human MOG has suitable binding affinity to the HLA-A*31:01 allele as a potential neuroantigenic epitope. Further investigations of this peptide revealed that the binding of C-terminal residue of this peptide has a more significant effect on binding to this allele than the N-terminal part of the peptide. Altogether, this combination of "LIICYNWLHR/A*31:01 allele "may play an important role in MS pathogenesis and this complex is suggested for further studies such as T cell receptor.Communicated by Ramaswamy H. Sarma.

Cycloarta-23-ene-3beta,25-diol a pentacyclic steroid from Euphorbia spinidens, as COX inhibitor with molecular docking, and in vivo study of its analgesic and anti-inflammatory activities in male swiss mice and wistar rats.

BACKGROUND AND AIMS: Euphorbia is a large genus of flowering plants. In Iran, some plants of this family have been used in the treatment of inflammatory disorders and also to relieve back pain. Euphorbia spinidens is a rich source of Cycloarta-23-ene-3beta,25-diol. Cycloartane structures are the starting material for the synthesis of plant steroids, and the aim of this study is to demonstrate COX inhibitory activity, molecular docking and in vivo approach of anti-inflammatory activity of cycloartane compound isolated from Euphorbia spinidens. MATERIAL AND METHODS: Plant material was extracted with acetone-chloroform and submitted to column chromatography for fractionation. Based on preliminary 1H-NMR spectra, cycloartane fraction was selected and purified by repeated recycle HPLC system. The structure and purity of compound were determined by 1H and 13C-NMR, HPTLC, and mass spectra. Inhibitory activities of the tested compounds on COX-1 and COX-2 were evaluated by a colorimetric COX (ovine) inhibitor screening method. Vero cells were used to assess the toxicity against the normal cells, and calculate the selectivity index. COX inhibitory activity results were evaluated and confirmed by molecular docking experiments. In the in vivo approach, analgesic activity was assessed by acetic acid-induced abdominal writhing and formalin tests. Croton oil-induced ear edema in mice and carrageenan-induced rat paw edema in rats were used to evaluate anti-inflammatory activity. Pain tests were carried out on male Swiss mice (25-35 g). Male Wistar rats (160-200 g) were used for the carrageenan test. RESULTS: Cycloart-23-ene-3ß,25-diol showedin vitro cyclooxygenase 1 and 2 inhibitory activities with more selectivity for COX-2. Molecular docking by predicting binding energies in COX protein receptors confirmed in vitro COX inhibitory results, and determined the best position for ligand in COX receptors along with its residue interactions in receptor pockets, which must be considered for designing of their inhibitors. In the in vivo studies, cycloartane inhibited significantly acetic acid-induced abdominal contractions and formalin-induced licking behavior at a dose of 200 mg/kg. The same dose reduced croton oil ear edema in mice and carrageenan-induced paw edema in rats. CONCLUSION: Therefore, according to these findings, cycloart-23-ene-3beta,25-diol showed promising analgesic and anti-inflammatory effects with low toxicity against normal cells and can be suggested as a template lead for designing anti-inflammatory compounds with good selectivity index, and potency for COX-2 inhibitory activity.

Rational design of a new mutant of tobacco etch virus protease in order to increase the in vitro solubility.

BACKGROUND AND PURPOSE: Tobacco etch virus (TEV) protease is a protease with high sequence specificity which is useful for the cleavage of fusion proteins. A major limitation of this enzyme is its relatively poor solubility. This study aimed to investigate the effects of some suggested mutations by online tools and molecular dynamics simulation to improve the solubility of TEV protease in vitro. EXPERIMENTAL APPROACH: We designed a rational multi-stage process to determine the solubilizing mutations of TEV protease. At the first stage, all the possible mutations were predicted using online tools such as PoPMuSiC and Eris servers, in which five mutations include N23F, N23L, Q74L, Q74V, and Q74I were suggested for further studies. In the next step, the three dimensional structure of the wild type (WT) and the best mutations were subjected to molecular dynamic simulations to evaluate the dynamic behaviour of the obtained structures. The selected mutation was introduced into the structure using site-directed mutagenesis and expressed in Escherichia coli BL21DE3. After purification, solubility and activity of the purified mutant and WT-TEV proteases were assayed. FINDINGS /RESULTS: By considering the analysis of various factors such as structural and solubility properties, one mutant, N23F, was selected for in vitro studies which led to a 1.5 times increase in the solubility compared to the WT while its activity was decreased somewhat. CONCLUSION AND IMPLICATIONS: We propose N23F mutation, according to computational and experimental analyses for TEV proteases which resulted in a 150% increase in solubility compared to the WT.

Exploring the binding mechanism of saccharin and sodium saccharin to promoter of human p53 gene by theoretical and experimental methods.

In the past few decades, extensive discussions have been on the impact of artificial sweeteners on the risk of cancer. The present study aimed to evaluate the interaction of saccharin (SA) and sodium saccharin (SSA) with the promoter of the human p53 gene. The binding ability was assessed using the spectroscopic technique, molecular docking and molecular dynamics (MD) simulation methods. Free energy of binding has been calculated using Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method. Fluorescence spectra of mentioned gene with concentration profiles of SA and SSA were obtained in a physiological condition. A gradual increase without any significant spectral shift in the fluorescence intensity of around 350 nm was evident, indicating the presence of an interaction between both compounds and gene. The docking results showed that both compounds were susceptible to bind to 5'-DG56DG57-3' nucleotide sequence of gene. Furthermore, the MD simulation demonstrated that the binding positions for SA and SSA were 5'-A1T3T4-3' and 5'-G44T45-3' sequences of gene, respectively. The binding of these sweeteners to gene made significant conformational changes to the DNA structure. Hydrogen and hydrophobic interactions are the major forces in complexes stability. Through the groove binding mode, the non-interactive DNA-binding nature of SSA and SA has been demonstrated by the results of spectrofluorometric and molecular modeling. This study could provide valuable insight into the binding mechanism of SA and its salt with p53 gene promoter as macromolecule at the molecular level in atomistic details. This work can contribute to the possibility of the potential hazard of carcinogenicity of this sweetener and to design and apply new and safer artificial sweeteners. AbbreviationsSASaccharinSSASodium SaccharinPp53gpromoter of human p53 geneMDMolecular dynamicsRMSDRoot-mean-square deviationRMSFRoot-mean-square fluctuationRgRadius of GyrationSASASolvent-Accessible Surface AreaADIAcceptable daily intakeMM/PBSAMolecular Mechanics/Poisson-Boltzmann Surface AreaCommunicated by Ramaswamy H. Sarma.