Biological Evaluation of Anti-Cholinesterase Activity, in Silico Molecular Docking Studies, and DFT Calculations of Green Synthesized Thiadiazolo[3,2-a]pyrimidine Derivatives.

A series of [1,3,4] thiadiazolo[3,2-a]pyrimidine-6-carboxylate derivatives 4(a-n) have been designed and synthesized as inhibitors of acetylcholinesterase (AChE). Synthesizing of thiadiazolo[3,2-a] pyrimidines was carried out in a single step, one-pot reaction using aromatic aldehydes, ethyl acetoacetate and different derivatives of 1,3,4-thiadiazoles (with molar ratio of 1 : 2 : 1, respectively) in conjunction with the catalyst, anhydrous iron(III) chloride by a grinding method under solvent-free conditions at room temperature. The in-vitro studies exhibited good potency for inhibiting AChE comparable with donepezil as the reference drug. The best results were obtained by Ethyl 2-(4-nitroophenyl)-7-methyl-5-(pyridin-3-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxylate 4n with IC50 value of 0.082±0.001 µM which was comparable with AChE inhibitory effects of donepezil (IC50 =0.079 µM).

Correlation between total phenolic and flavonoid contents and biological activities of 12 ethanolic extracts of Iranian propolis.

Propolis is a resinous substance produced by honey bees that is very popular as a natural remedy in traditional medicine. The current research is the first study on the biological properties of ethanolic extracts of propolis (EEP) from several different regions (12) of Iran. Total phenolic and flavonoid contents (TPC and TFC) of Iranian EEPs were variable between 26.59-221.38 mg GAE/g EEP and 4.8-100.03 mg QE/g EEP. The DPPH scavenging assay showed all the studied EEP samples, except for the sample with the lowest TPC and TFC (P6), have suitable antioxidant activity. All the EEPs inhibited both cholinesterase enzymes (acetylcholinesterase: AChE, butyrylcholinesterase: BuChE) but most of them exhibited a distinct selectivity over BuChE. Evaluation of the antibacterial activity of the EEP samples using four pathogenic bacteria (B. cereus, S. aureus, A. baumannii, and P. aeruginosa) demonstrated that the antibacterial properties of propolis are more effective on the gram-positive bacterium. Spearman correlation analysis showed a strong positive correlation between TPC and TFC of the Iranian EEPs and their antioxidant, anticholinesterase, and antibacterial activities. Considering that there is ample evidence of anticholinesterase activity of flavonoids and a significant correlation between the anticholinesterase activity of the studied Iranian EEPs and their total flavonoid content was observed, the interaction of 17 well-known propolis flavonoids with AChE and BuChE was explored using molecular docking. The results indicated that all the flavonoids interact with the active site gorge of both enzymes with high affinity. Summing up, the obtained results suggest that Iranian propolis possesses great potential for further studies.

Anti-AChE and Anti-BuChE Screening of the Fermentation Broth Extracts from Twelve Aspergillus Isolates and GC-MS and Molecular Docking Studies of the Most Active Extracts.

Nowadays, the administration of cholinesterase enzyme (acetylcholinesterase: AChE and butyrylcholinesterase: BuChE) inhibitors is very common for the symptomatic treatment of Alzheimer's disease and the other forms of dementia and CNS disorders. In this paper, the anti-AChE and anti-BuChE activities of the fermentation broth ethyl acetate extracts from twelve Aspergillus isolates were evaluated by Ellman method. The results showed that A1 (Aspergillus flavus) and A5 (Aspergillus tubingensis, isolate 1) extracts with IC50 values of 46.77 µg/mL and 75.85 µg/mL possess the greatest ability to inhibit AChE and BuChE, respectively. GC-MS analysis of the extracts (A1 and A5) demonstrated that two alkaloids named 14-methyl-16-azabicyclo[10.3.1]hexadeca-1(15),12(16),13-triene (MAHT) and 6-chloro-2-methyl-7,8,9,10-tetrahydro-phenanthridine (CMTP) account for the highest percentage of A1 (26.95%) and A5 (25.5%) extracts, respectively. A 2-pyrazoline derivative, 5-hydroxy-3-(4-pyridinyl)-5-trifluoromethyl-1-(2,4,6-trimethylphenoxyacetyl)- (PHPTT), also constituted the high percentage (9.54%) of A5 extract. The anticholinesterase and neuroprotective effects of some 2-pyrazoline derivatives have been previously reported. The interaction study of MAHT with human AChE and CMTP and PHPTT with human BuChE using molecular docking indicated that these alkaloids bind to the active site gorge of the enzymes with high affinity. The best docking scores of MAHT, CMTP, and PHPTT were -7.1, -8.2, and -9.7 kcal/mol, respectively.

Effects of synonymous mutations on kinetic properties and structure of firefly luciferase: Molecular dynamics simulation, molecular docking, RNA folding, and experimental study.

Although synonymous mutations have long been thought to lack striking results, a growing body of research shows these mutations have highly variable effects. In this study, the impact of synonymous mutations in the development of thermostable luciferase was investigated using a combination of experimental and theoretical approaches. Using bioinformatics analysis, the codon usage features in the Lampyridae family's luciferases were studied and four synonymous mutations of Arg in luciferase were created. An exciting result was that the analysis of kinetic parameters showed a slight increase in the thermal stability of the mutant luciferase. AutoDock Vina, %MinMax algorithm, and UNAFold Server were used to perform molecular docking, folding rate, and RNA folding, respectively. Here, it was assumed that in the region (Arg337) with a moderate propensity for coil, synonymous mutation altered the rate of translation, which in turn may lead to a slight change in the structure of the enzyme. According to the molecular dynamics simulation data, local minor global flexibility is observed in the context of the protein conformation. A plausible explanation is that this flexibility may strengthen hydrophobic interactions due to its sensitivity to a molecular collision. Accordingly, thermostability originated mainly from hydrophobic interaction.

Determine genetic variations in heat shock factor gene family (HSFs) and study their effect on the functional and structural characterization of protein in Tali goat.

In this study, the effect of genetic variations of four heat shock transcription factor genes (HSF1, HSF2, HSF4, and HSF5) on the 3 D protein structure and function were studied. We defined the breed-specific genetic variations of pooled DNA of Tali goat that differed from the goat reference sequence (CHI2.0). Disordered regions of HSF proteins were predicted using PONDR. Post-translation changes were studied by several predicted online servers. Then, the structure of the order region of proteins was anticipated by using the Swiss model. Tali goat HSF genes contain a total number of 181, 679, 91, and 301 SNPs for HSF1, 2, 4, and 5, respectively. Also, 5 and 3 variants were identified as nsSNPs in the coding region of HSF4 and HSF5, respectively. (r.145A/S), (r.322P/Y), (r.379T/C) in HSF4 and (r.300Q/P), (r.573E/Q) in HSF5 obtained the tolerant and high confidence (SIFT score) for nsSNPs. More than half of these proteins are predicted to be disordered (56, 50, 52, and 50%, respectively for HSF1, 2, 4, and 5). Phosphorylation, acetylation, glycosylation, and Sumoylation sites of HSFs were compared between Tali goat and reference goat. Three residues S145, S263, and S322 of HSF4 in Tali goat were phosphorylation sites, and in HSF5, the reference goat has a phosphorylation site in S593.

Green and four-component cyclocondensation synthesis and in silico docking of new polyfunctionalized pyrrole derivatives as the potential anticholinesterase agents.

Synthesis of new substituted pyrrole scaffolds containing substituted thiadiazol-2-amine moiety was successfully developed through one-pot and multi-component tandem condensation reaction utilizing of triethyl ammonium hydrogen sulfate ([Et3NH][HSO4]) ionic liquid as a green media under solvent-free conditions. The chemical structures of all newly synthesized compounds were fully characterized by spectroscopic methods (IR, 1H NMR, 13C NMR) and elemental analyzes. The molecular docking studies were also performed to predict the possible binding sites of the derivatives on the active site gorge of cholinesterase enzymes (AChE and BuChE). The results showed that all the seventeen derivatives interact with the enzymes with high affinity and among them 7d and 7f possess the greatest ability to bind to AChE and BuChE, respectively.

Design, synthesis and biological assessment of acridine derivatives containing 1,3,4-thiadiazole moiety as novel selective acetylcholinesterase inhibitors.

A novel series of acridine derivatives containing substituted thiadiazol-2-amine moiety was synthesized via multi-component condensation reaction of dimedone, aromatic aldehyde and 5-aryl-1,3,4-thiadiazol-2-amines in the presence of LaCl3 as a catalyst under solvent-free conditions. Anticholinesterase (AChE and BuChE) activity evaluation of the derivatives showed that all the derivatives are capable of inhibiting both enzymes and are highly selective towards AChE. Among them, the ability of 4i and 4d with respective IC50 values of 0.002 and 0.006 µM to inhibit AChE was higher than the reference compound tacrine (IC50 = 0.016 µM). The kinetics studies demonstrated that 4i and 4d inhibit AChE through a competitive/non-competitive mixed mechanism. The HEPG2 cell viability assay evidenced that 4i and 4d significantly exhibit lower hepatotoxicity compared with tacrine. Blind docking experiments performed on TcAChE (PDB ID: 2ACE) indicated that an unknown site is preferred for binding by all the derivatives over classic binding site of the enzyme, site 1 (CAS/PAS). Identification of the residues by protein structure alignment confirmed that this site is site 2 which was recently recognized as a new allosteric site of hAChE. The binding modes of 4i and 4d were also investigated using local docking studies on site 1 and site 2.

Improvement of kinetic properties and thermostability of recombinant Lepidium draba peroxidase (LDP) upon exposed to osmolytes.

In this study, effects of different concentrations of glycine and D-sorbitol were analyzed on the activity and thermostability of recombinant Lepidium draba peroxidase (LDP). Based on the results, activity of the enzyme increased in the presence of various concentrations of these osmolytes. Maximum activity was detected for the enzyme in the presence of 300 mM glycine and 600 mM sorbitol. In presence of the aforementioned doses of osmolytes, enzyme affinity for substrate (3,3',5,5'-tetramethylbenzidine and H2O2) and Vmax increased. According to the results, enzyme stability improved against temperature and H2O2. Furthermore, structural changes of the enzyme upon exposure to the osmolytes were revealed by the use of far-UV circular dichroism and fluorescence methods. The results showed, whereas the secondary structure of the enzyme was not significantly changed upon exposed to the osmolytes, the fluorescence studies revealed microenvironment of the aromatic residues dramatically affected by them. Overall, it may be speculated, structural changes of the enzyme upon exposed to the osmolytes, lead to the improvement of its kinetic properties and stability that can be benefit for using of it in in vitro applications.

Evaluation of Apoptosis in Multipotent Hematopoietic Cells of Bone Marrow by Anthracycline Antibiotics.

Anthracycline antibiotics are potent anticancer drugs widely used in the treatment of solid tumors and hematological malignancies. Because of their extensive clinical use and their toxic effect on normal cells, in the present study the effect of these drugs on multipotent hematopoietic bone marrow cells was investigated employing, viability tests, PARP cleavage, Hoechst 33258 staining, DNA fragmentation and superoxide anion production techniques. The results revealed that daunorubicin and doxorubicin exhibited time and dose dependent cytotoxicity against the cells and upon increasing the drugs concentrations, apoptosis was occurred after 4 h of incubation and at low concentration of the drugs. The cleavage of poly ADP-ribose polymerase (PARP) demonstrated by daunorubicin and doxorubicin treatment of the cells, suggest that the apoptotic process is PARP dependent. The drugs induced DNA fragmentation and also anion superoxide production was increased upon rising drugs concentrations. From the results it is concluded that anthracycline antibiotics represent cytotoxic effect on hematopoietic progenitor/stem cells of bone marrow, inducing apoptosis and in this process toxicity of daunorubicin is more pronounced compared to doxorubicin.

Bioinformatic Analysis of Codon Usage and Phylogenetic Relationships in Different Genotypes of the Hepatitis C Virus.

BACKGROUND: The hepatitis C virus (HCV) has six major genotypes. The purpose of this study was to phylogenetically investigate the differences between the genotypes of HCV, and to determine the types of amino acid codon usage in the structure of the virus in order to discover new methods for treatment regimes. METHODS: The codon usage of the six genotypes of the HCV nucleotide sequence was investigated through the online application available on the website Gene Infinity. Also, phylogenetic analysis and the evolutionary relationship of HCV genotypes were analyzed with MEGA 7 software. RESULTS: The six genotypes of HCV were divided into two groups based on their codon usage properties. In the first group, genotypes 1 and 5 (74.02%), and in the second group, genotypes 2 and 6 (72.43%) were shown to have the most similarity in terms of codon usage. Unlike the results with respect to determining the similarity of codon usage, the phylogenetic analysis showed the closest resemblance and correlation between genotypes 1 and 4. The results also showed that HCV has a GC (guanine-cytosine) abundant genome structure and prefers codons with GC for translation. CONCLUSIONS: Genotypes 1 and 4 demonstrated remarkable similarity in terms of genome sequences and proteins, but surprisingly, in terms of the preferred codons for gene expression, they showed the greatest difference. More studies are therefore needed to confirm the results and select the best approach for treatment of these genotypes based on their codon usage properties.

Evidence for the binding affinity of daunomycin to HMGB1 protein in chromatin and in solution.

In this study the interaction of daunomycin with HMGB1 nonhistone chromatin protein in the chromatin context using hydroxyapatite (HAP) column chromatography and free in solution was investigated employing fluorescence, circular dichroism spectroscopy and thermal denaturation techniques. The results demonstrate that HMGB1 fraction eluted from HAP column contained the most amount of daunomycin. Upon addition of daunomycin to HMGB1 solution, fluorescence emission intensity was dependent on the drug concentration used whereas the ellipticity in CD spectra was decreased at both 205 and 220 nm extremes implying that quenching of the drug with the HMGB1 chromospheres alters secondary structure of the protein. Although daunomycin slightly increased the melting point of HMGB1, but exhibited a significant hyperchromicity at low concentrations and hypochromicity at higher concentrations of daunomycin. The results suggest that daunomycin binds to HMGB1 protein which may influence its interaction with DNA in nucleosomes and other cellular processes.