Profiling the structural determinants for the selectivity of representative factor-Xa and thrombin inhibitors using combined ligand-based and structure-based approaches

The current study deciphers the combined ligand- and structure-based computational insights to profile structural determinants for the selectivity of representative diverse classes of FXa-selective and thrombin-selective as well as dual FXa-thrombin high affinity inhibitors. The thrombin-exclusive insertion 60-loop (D-pocket) was observed to be one of the most notable recognition sites for the known thrombin-selective inhibitors. Based on the topological comparison of four common active-site pockets (S1-S4) of FXa and thrombin, the greater structural disparity was observed in the S4-pocket, which was more symmetrical (U-shaped) in FXa as compared to thrombin mainly due to the presence of L99 and I174 residues in latter in place of Y99 and F174 respectively in former protease. The S2 pocket forming partial roof at the entry of 12 ? deep S1-pocket, with two extended β-sheets running antiparallel to each other by undergoing U-turn (～180?), has two conserved glycine residues forming H-bonds with the bound ligand for governing ligand binding affinity. The docking, scoring, and binding pose comparison of the representative high-affinity and selective inhibitors into the active sites of FXa and thrombin revealed critical residues (S214, Y99, W60D) mediating selectivity through direct- and long-range electrostatic interactions. Interestingly, most of the thrombin-selective inhibitors attained S-shaped conformation in thrombin, while FXa-selective inhibitors attained L-shaped conformations in FXa. The role of residue at 99th position of FXa and thrombin toward governing protease selectivity was further substantiated using molecular dynamics simulations on the wild-type and mutated Y99L FXa bound to thrombin-selective inhibitor 2. Furthermore, predictive CoMFA (FXa q2 = 0.814; thrombin q2 = 0.667) and CoMSIA (FXa q2 = 0.807; thrombin q2 = 0.624) models were developed and validated (FXa r2(test) = 0.823; thrombin r(2)(test) = 0.816) to feature molecular determinants of ligand binding affinity using the docking-based conformational alignments (DBCA) of 141 (88(train)+53(test)) and 39 (27(train)+11(test)) nonamidine class of potent FXa (0.004 ≤ K(i) (nM) ≤ 4700) and thrombin (0.001 ≤ K(i) (nM) ≤ 940) inhibitors, respectively. Interestingly, the ligand-based insights well corroborated with the structure-based insights in terms of the role of steric, electrostatic, and hydrophobic parameters for governing the selectivity for the two proteases. The new computational insights presented in this study are expected to be valuable for understanding and designing potent and selective antithrombotic agents.     

Structural, microstructural and magnetic properties of NiFe2O4, CoFe2O4 and MnFe2O4 nanoferrite thin films

The structural, microstructural and magnetic properties of nanoferrite NiFe₂O₄ (NF), CoFe₂O₄ (CF) and MnFe₂O₄ (MF) thin films have been studied. The coating solution of these ferrite films was prepared by a chemical synthesis route called sol-gel combined metallo-organic decomposition method. The solution was coated on Si substrate by spin coating and annealed at 700 °C for 3 h. X-ray diffraction pattern has been used to analyze the phase structure and lattice parameters. The scanning electron microscopy (SEM) and atomic force microscopy (AFM) have been used to show the nanostructural behavior of these ferrites. The values of average grain's size from SEM are 44, 60 and 74 nm, and from AFM are 46, 61 and 75 nm, respectively, measured for NF, CF and MF ferrites. At room temperature, the values of saturation magnetization, M_s∼50.60, 33.52 and 5.40 emu/cc, and remanent magnetization, M_r∼14.33, 15.50 and 1.10 emu/cc, respectively, are observed for NF, CF and MF. At low temperature measurements of 10 K, the anisotropy of ferromagnetism is observed in these ferrite films. The superparamagnetic/paramagnetic behavior is also confirmed by χ′(T) curves of AC susceptibility by applying DC magnetizing field of 3 Oe. The temperature dependent magnetization measurements show the magnetic phase transition temperature.     

Synthesis and characterization of glycol ether and oxime modified precursors of niobium(V) for soft transformation to niobia

A glycol ether modified precursor, [Nb{O(CH₂CH₂O)₂}(OPrⁱ)₃] (A) was prepared by the reaction of Nb(OPrⁱ)₅ with O(CH₂CH₂OH)₂ in 1:1 molar ratio in anhydrous benzene. Further reactions of A with a variety of internally functionalized oximes in different molar ratios, yielded heteroleptic complexes of the type, [Nb{O(CH₂CH₂O)₂}(OPrⁱ)_3?n{ON = C(CH₃)(Ar)}_n] (1–9) {where Ar = C₄H₃O-2, n = 1 [1], n = 2 [2], n = 3 [3]; C₄H₃S-2, n = 1 [4], n = 2 [5], n = 3 [6]; C₅H₄N-2, n = 1 [7], n = 2 [8], n = 3 [9]}. All the above derivatives have been characterized by elemental analyses, FT-IR, NMR (¹H, ¹³C {¹H}) and FAB mass studies. Spectral studies of 1–9 suggest the presence of mono- and bi-dentate mode of oxime moieties, in the solution and in the solid states, respectively. FAB mass studies indicate monomeric nature for 3 and dimeric nature for A. TG curves of A and 6 show their low thermal stability. Soft transformation of A and 3 to pure niobia, a and b, respectively have been carried out by sol–gel technique. The XRD patterns of niobia a and b suggest the formation of nano-size crystallites of average size of 10.8 and 19.5 nm, respectively. The XRD patterns also indicate the formation of monoclinic phase of the niobia in both the cases. Absorption spectra of a and b suggest energy band gaps of 4.95 and 4.39 eV, respectively.     

Radical‐Cation Dimerization Overwhelms Inclusion in [n]Pseudorotaxanes

Suppression of the dimerization of the viologen radical cation by cucurbit[7]uril ( CB7 ) in water is a well‐known phenomenon. Herein, two counter‐examples are presented. Two viologen‐containing thread molecules were designed, synthesized, and thoroughly characterized by ¹H DOSY NMR spectrometry, UV/Vis absorption spectrophotometry, square‐wave voltammetry, and chronocoulometry: BV ⁴⁺, which contains two viologen subunits, and HV ¹²⁺, which contains six. In both threads, the viologen subunits are covalently bonded to a hexavalent phosphazene core. The corresponding [3]‐ and [7]pseudorotaxanes that form on complexation with CB7 , that is, BV ⁴⁺?( CB 7)₂ and HV ¹²⁺?( CB 7)₆, were also analyzed. The properties of two monomeric control threads, namely, methyl viologen ( MV ²⁺) and benzyl methyl viologen ( BMV ²⁺), as well as their [2]pseudorotaxane complexes with CB7 ( MV ²⁺? CB7 and BMV ²⁺? CB7 ) were also investigated. As expected, the control pseudorotaxanes remained intact after one‐electron reduction of their viologen‐recognition stations. In contrast, analogous reduction of BV ⁴⁺?( CB 7)₂ and HV ¹²⁺?( CB 7)₆ led to host–guest decomplexation and release of the free threads BV ^{2( . +)} and HV ^{6( . +)}, respectively. ¹H DOSY NMR spectrometric and chronocoulometric measurements showed that BV ^{2( . +)} and HV ^{6( . +)} have larger diffusion coefficients than the corresponding [3]‐ and [7]pseudorotaxanes, and UV/Vis absorption studies provided evidence for intramolecular radical‐cation dimerization. These results demonstrate that radical‐cation dimerization, a relatively weak interaction, can be used as a driving force in novel molecular switches.     

Differential electrochemical behaviour of phytofabricated and chemically synthesized silver nanoparticles towards hydrogen peroxide sensing**

Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials for biomedical applications. However, the impact of its synthesis by chemical and plant-mediated routes on its differential electrochemical behaviour has not been examined till date. Here, we report for the first time the differential study of the electrochemical behaviour of the AgNPs synthesized by different routes. First, the AgNPs were obtained by different routes (chemical and phytofabrication) and extensively characterized to compare their physical properties. Thereafter, a comparison of electron transfer kinetics between chemically synthesized (Ag−C) and phyto-fabricated (Ag-Phy) nanoparticles (NPs) has been studied by electrochemical techniques such as potentiodynamic cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). To further investigate the electrocatalytic properties of both types of AgNPs, we have used the peroxide moieties (H₂O₂), and the Ag−C NPs-based sensor probe has been reported to have four times better sensitivity than the Ag−Phy NPs-based sensor. The AgNPs modified sensor probes have also been tested in real-world environments to explore the consistency of their performance in complex matrices by using clinical urine samples, where we found comparable sensitivity to the standard conditions.     

Highly sensitive method for the determination of adenosine by LC-MS/MS-ESI: method validation and scope of application to a pharmacokinetic/pharmacodynamic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of adenosine in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electro‐spray ionization in the positive‐ion mode. The assay procedure involves extraction of adenosine and phenacetin (internal standard, IS) from rat plasma with a simple protein precipitation extraction process. The method was validated using rat plasma with extinguished adenosine endogenous levels. Chromatographic separation was achieved using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.50 mL/min on an Atlantis dC₁₈ column with a total run time of 4.0 min. The MS/MS ion transitions monitored were 268 → 136 for adenosine and 180 → 110 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.48 ng/mL and the linearity range extended from 0.48 to 1210 ng/mL. The intra‐ and inter‐day precisions were in the ranges 2.32–12.7 and 4.01–9.40%, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Targeting Akt/NF-κB/p53 Pathway and Apoptosis Inducing Potential of 1,2-Benzenedicarboxylic Acid,Bis (2-Methyl Propyl) Ester Isolated from Onosma bracteata Wall. against Human Osteosarcoma (MG-63) Cells

Onosma bracteata Wall. is an important medicinal and immunity-enhancing herbs. This plant is commonly used in the preparation of traditional Ayurvedic drugs to treat numerous diseases. Inspired by the medicinal properties of this plant, the present study aimed to investigate the antiproliferative potential and the primary molecular mechanisms of the apoptotic induction against human osteosarcoma (MG-63) cells. Among all the fractions isolated from O. bracteata, ethyl acetate fraction (Obea) showed good antioxidant activity in superoxide radical scavenging assay and lipid peroxidation assay with an EC₅₀ value of 95.12 and 80.67 µg/mL, respectively. Silica gel column chromatography of ethyl acetate (Obea) fraction of O. bracteata yielded a pure compound, which was characterized by NMR, FTIR, and HR-MS analysis and was identified as 1,2-benzene dicarboxylic acid, bis (2-methyl propyl) ester (BDCe fraction). BDCe fraction was evaluated for the antiproliferative potential against human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung carcinoma A549 cell lines by MTT assay and exhibited GI₅₀ values of 37.53 μM, 56.05 μM, and 47.12 μM, respectively. In MG-63 cells, the BDCe fraction increased the level of ROS and simultaneously decreased the mitochondria membrane potential (MMP) potential by arresting cells at the G₀/G₁ phase, suggesting the initiation of apoptosis. Western blotting analysis revealed the upregulation of p53, caspase3, and caspase9 while the expressions of p-NF-κB, p-Akt and Bcl-xl were decreased. RT-qPCR studies also showed upregulation in the expression of p53 and caspase3 and downregulation in the expression of CDK2, Bcl-2 and Cyclin E genes. Molecular docking analysis displayed the interaction between BDCe fraction with p53 (−151.13 kcal/mol) and CDK1 (−133.96 kcal/mol). The results of the present work suggest that the BDCe fraction has chemopreventive properties against osteosarcoma (MG-63) cells through the induction of cell cycle arrest and apoptosis via Akt/NF-κB/p53 pathways. This study contributes to the understanding of the utilization of BDCe fraction in osteosarcoma treatment.