Synthesis,pharmacological screening and in silico studies of new class of Diclofenac analogues as a promising anti-inflammatory agents

A novel series of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-(substituted)-1,3,4-oxadiazol-2(3H)-thione (4a–k) derivatives have been synthesized by the Mannich reaction of 5-[2-(2,6-dichlorophenylamino)benzyl]-1,3,4-oxadiazol-2(3H)-thione (3) with an appropriately substituted primary/secondary amines, in the presence of formaldehyde and absolute ethanol. Structures of these novel compounds were characterized on the basis of physicochemical, spectral and elemental analysis. The title compounds (4a–k) were screened for in vivo acute anti-inflammatory and analgesic activities at a dose of 10 mg/kg b.w. Compound 4k exhibited the most promising and significant anti-inflammatory profile while compounds 4a, 4d, 4e, 4i, and 4j showed moderate to good inhibitory activity at 2nd and 4th h, respectively. These compounds were also found to have considerable analgesic activity (acetic acid induced writhing model) and antipyretic activity (yeast induced pyrexia model). In addition, the tested compounds were also found to possess less degree of ulcerogenic potential as compared to the standard NSAIDs. Compounds that displayed promising anti-inflammatory profile were further evaluated for their inhibitory activity against cyclooxygenase enzyme (COX-1/COX-2), by colorimetric COX (ovine) inhibitor screening assay method. The results revealed that the compounds 4a, 4e, 4g and 4k exhibited effective inhibition against COX-2. In an attempt to understand the ligand–protein interactions in terms of the binding affinity, docking studies were performed using Molegro Virtual Docker (MVD-2013, 6.0) for those compounds, which showed good anti-inflammatory activity. It was observed that the binding affinities calculated were in agreement with the IC₅₀ values.     

Biophysical analysis of the EPEC translocated intimin receptor-binding domain

Enteropathogenic Escherichia coli (EPEC) are Gram (-) bacteria responsible for widespread illness in the form of diarrhea. EPEC cells attach to the intestinal epithelium using a Type III secretion system common to many Gram (-) bacteria. The translocated intimin receptor (TIR) is the first protein secreted through the EPEC secretion complex, and is absolutely required for pathogenesis. It inserts into the intestinal epithelium, serving as an anchor responsible for the attachment of EPEC to the host epithelial cell. Intimin is a transmembrane protein displayed on the EPEC cell surface with an extracellular domain that binds TIR. Observation of a TIR-TIR dimer in the X-ray co-crystal structure of the extracellular domains of intimin and TIR raised the question of how these protein domains interact and function in solution. Herein we report that the extracellular domain of TIR exists in a folded and active monomeric state in solution, as confirmed by analytical ultracentrifugation, analytical size-exclusion HPLC, isothermal titration calorimetry, and surface plasmon resonance.     

Comparative thermodynamics for monomer and dimer sequence-dependent binding of a heterocyclic dication in the DNA minor groove

Phenylamidine cationic groups linked by a furan ring (furamidine) and related symmetric diamidine compounds bind as monomers in the minor groove of AT sequences of DNA. DB293, an unsymmetric derivative with one of the phenyl rings of furamidine replaced with a benzimidazole, can bind to AT sequences as a monomer but binds more strongly to GC-containing minor-groove DNA sites as a stacked dimer. The dimer-binding mode has high affinity, is highly cooperative and sequence selective. In order to develop a better understanding of the correlation between structural and thermodynamic aspects of DNA molecular recognition, DB293 was used as a model to compare the binding of minor-groove agents with AT and mixed sequence DNA sites. Isothermal titration calorimetry and surface plasmon resonance results clearly show that the binding of DB293 and other related compounds into the minor groove of AT sequences is largely entropy-driven while the binding of DB293 as a dimer into the minor groove of GC-containing sequences is largely enthalpy-driven. At 25 degrees C, for example, the AT binding has DeltaG degrees, DeltaH degrees and TDeltaS degrees values of -9.6, -3.6 and 6.0 kcal/mol while the values for dimer binding to a GC-containing site are -9.0, -10.9 and -1.9 kcal/mol (per mol of bound compound), respectively. These results show that the thermodynamic components for binding of compounds of this type to DNA are very dependent on the structure, solvation and sequence of the DNA binding site.     

The ionic strength effect on the DNA complexation by DOPC - gemini surfactants liposomes

Liposome dispersions obtained from the mixture of gemini surfactants of the type alkane-α,ω-diyl-bis(alkyldimethylammonium bromide) and helper lipid DOPC create complexes with DNA showing a regular inner microstructure, identified by small angle X-ray diffraction as condensed lamellar phase (L_α^c). In addition to the L_α^c phase, a coexisting lamellar phase L_B was also identified in the complexes formed, with periodicities in the range ~ 8.8-5.7 nm, at ionic strengths corresponding to 50-200 mM NaCl. The periodicities of L_B phase did not correspond to those identified in liposome dispersion without DNA using small angle neutron scattering. The observed phase separation is shown to depend on the interplay between the surface charge density of cationic liposomes, ionic strength and method of complex preparation. The effect of ionic strength on complex formation was studied by isothermal titration calorimetry and zeta potential measurements. High ionic strength reduces the fraction of bound DNA in the complexes, and the isoelectric point is attained at a ratio of DNA/gemini surfactant which is lower than the one that can be estimated by calculation based on nominal charges of CLs and DNA.     

Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.     

STEME系统:一种助力体内定向进化的新工具

通过定向进化(directed evolution)可以快速进行蛋白工程改良及重要基因功能研究,以获得新型农艺性状突变体。近期,中国科学院遗传与发育生物学研究所高彩霞团队和李家洋团队合作构建了新型的饱和靶向内源诱变编辑器(saturated targeted endogenous mutagenesis editors, STEMEs),并在植物中实现了基因的定向进化和功能筛选。该系统融合了现有的2种单碱基编辑技术,成功实现在植物体内同时诱导C:G>T:A、A:T>G:C双碱基编辑,通过靶向OsACC羧基转移酶结构域编码序列定向进化出水稻除草剂抗性植株。这种在体内进行基因定向进化的新方法,对于今后农作物重要农艺性状的筛选和功能基因研究具有重要作用。本文对STEME系统的组成、编辑效率和应用原理进行介绍,并与已有的定向进化方法进行比较,为加速作物种质资源创新研究提供参考。     

Expression of base excision repair key factors and miR17 in familial and sporadic breast cancer

Understanding of BRCA1/2 interaction with the base excision repair (BER) pathway could improve therapy based on ‘synthetic lethality'', whose effectiveness is based on homologous recombination deficiency in cells lacking functional BRCA genes. However, poly (ADP-ribose) polymerase (PARP) inhibitors failed in some patients and for this reason we explored BER key enzyme expression. In this study, the expression of BER enzymes (redox factor 1/apurinic-apyrimidinic endonuclease 1 (REF1/APEX1), NTH endonuclease III-like 1 (NTHL1), 8-oxoguanine DNA glycosylase (OGG1), PARP1) and of the scaffold protein XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1) were investigated in familial (BRCA-related and not) and sporadic breast cancer cases. Furthermore, miR17 expression was measured because of its role in the epigenetic regulation of BRCA1. Gene expression was evaluated in BRCA1-mutated cell lines, SUM149PT and SUM1315MO2, and in a BRCA1-proficient triple-negative MDA-MB-231 cell line. A cohort of 27 familial and 16 sporadic breast cancer patients was then examined to confirm results obtained from the cell line model. APEX1/REF1 was found to be upregulated in familial BRCA-wild-type and sporadic cases, indicating this enzyme as a potential therapeutic target. Furthermore, XRCC1 was overexpressed in BRCAX patients; consequently, we suggest to test the effectiveness of inhibitors targeting two different BER components in preclinical studies. XRCC1, which is also involved in the non-homologous end-joining pathway, was found to be downregulated in BRCA2-related patients concurrently with no change in PARP1 expression. Interestingly, no difference in PARP1 and miR17 expression was found in BRCA-related and sporadic breast cancer cases. PARP1 and miR17 could therefore be further investigated as molecular biomarkers of ‘BRCAness'' phenotype, indicating patients which could really benefit from PARP inhibitor therapies.     

Drosophila oogenesis as a bio-marker responding to EMF sources

The model biological organisms Drosophila melanogaster and Drosophila virilis have been utilized to assess effects on apoptotic cell death of follicles during oogenesis and reproductive capacity (fecundity) decline. A total of 280 different experiments were performed using newly emerged flies exposed for short time daily for 3–7?d to various EMF sources including: GSM 900/1800?MHz mobile phone, 1880–1900?MHz DECT wireless base, DECT wireless handset, mobile phone-DECT handset combination, 2.44?GHz wireless network (Wi-Fi), 2.44?GHz blue tooth, 92.8?MHz FM generator, 27.15?MHz baby monitor, 900?MHz CW RF generator and microwave oven’s 2.44?GHz RF and magnetic field components. Mobile phone was used as a reference exposure system for evaluating factors considered very important in dosimetry extending our published work with D. melanogaster to the insect D. virilis. Distance from the emitting source, the exposure duration and the repeatability were examined. All EMF sources used created statistically significant effects regarding fecundity and cell death-apoptosis induction, even at very low intensity levels (0.3?V/m blue tooth radiation), well below ICNIRP’s guidelines, suggesting that Drosophila oogenesis system is suitable to be used as a biomarker for exploring potential EMF bioactivity. Also, there is no linear cumulative effect when increasing the duration of exposure or using one EMF source after the other (i.e. mobile phone and DECT handset) at the specific conditions used. The role of the average versus the peak E-field values as measured by spectrum analyzers on the final effects is discussed.