A real-time,fluorescence-based assay for Rho-associated protein kinase activity

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z’-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC₅₀ values of 3.6 μM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.     

Analysis of volatile organic compounds in indoor environments using thermal desorption with comprehensive two‐dimensional gas chromatography and high‐resolution time‐of‐flight mass spectrometry

Building‐related health effects are frequently observed. Several factors have been listed as possible causes including temperature, humidity, light conditions, presence of particulate matter, and microorganisms or volatile organic compounds. To be able to link exposure to specific volatile organic compounds to building‐related health effects, powerful and comprehensive analytical methods are required. For this purpose, we developed an active air sampling method that utilizes dual‐bed tubes loaded with TENAX‐TA and Carboxen‐1000 adsorbents to sample two parallel air samples of 4 L each. For the comprehensive volatile organic compounds analysis, an automated thermal desorption comprehensive two‐dimensional gas chromatography high‐resolution time‐of‐flight mass spectrometry method was developed and used. It allowed targeted analysis of approximately 90 known volatile organic compounds with relative standard deviations below 25% for the vast majority of target volatile organic compounds. It also allowed semiquantification (no matching standards) of numerous nontarget air contaminants using the same data set. The nontarget analysis workflow included peak finding, background elimination, feature alignment, detection frequency filtering, and tentative identification. Application of the workflow to air samples from 68 indoor environments at a large hospital complex resulted in a comprehensive volatile organic compound characterization, including 178 single compounds and 13 hydrocarbon groups.     

Potential Screening at Electrode/Ionic Liquid Interfaces from In Situ X-ray Photoelectron Spectroscopy

A new approach to investigate potential screening at the interface of ionic liquids (ILs) and charged electrodes in a two-electrode electrochemical cell by in situ X-ray photoelectron spectroscopy has been introduced. Using identical electrodes, we deduce the potential screening at the working and the counter electrodes as a function of applied voltage from the potential change of the bulk IL, as derived from corresponding core level binding energy shifts for different IL/electrode combinations. For imidazolium-based ILs and Pt electrodes, we find a significantly larger potential screening at the anode than at the cathode, which we attribute to strong attractive interactions between the imidazolium cation and Pt. In the absence of specific ion/electrode interactions, asymmetric potential screening only occurs for ILs with different cation and anion sizes as demonstrated for an imidazolium chloride IL and Au electrodes, which we assign to the different thicknesses of the electrical double layers. Our results imply that potential screening in ILs is mainly established by a single layer of counterions at the electrode.     

Possible observation of Kondo screening cloud in Yb14MnSb11 *

ABSTRACT

Single crystals of the underscreened Kondo ferromagnet Yb₁₄MnSb₁₁ were investigated using polarised neutron diffraction and magnetisation measurements. The magnitude and direction of the magnetisation at each of the 9 distinct crystallographic sites was measured using polarised neutron diffraction. A large moment of close to 4.2?μ_B was found at the unique Mn site. A compensating antiferromagnetic moment of ≈?0.5?μ_B was found to be distributed uniformly among the remaining Yb and Sb sites, which was not expected from a previous theoretical calculation. The compensating magnetisation, however, is consistent both in size and magnitude with the expected Kondo screening cloud.     

Chemical composition and anti-complement activity of Glechomae Herba collected in different months

Glechomae Herba (GH) is derived from the dried aerial part of Glechoma longituba (Nakai) Kupr., which is harvested from spring to autumn. It has the effects of clearing heat and detoxification. The aim of this paper was to study the chemical composition and the anti-complement activity of GH collected in different months. Ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry based on predicted compounds screening and diagnostic ion filter strategy was developed for identifying the chemical composition of GH collected in different months. A total of 102 compounds—40 chlorogenic acids (CGAs), 32 phenolic acids, and 30 flavonoids—were reasonably identified in GH. Thirty-four CGAs were discovered in GH for the first time. The correlations between chemical compositions and anti-complement activities of GH collected in different months were analyzed. Phenolic acids and flavonoids were found to be negatively correlated with anti-complement activity, and CGAs were positively correlated with anti-complement activity. At the same time, six CGA standards had obvious anti-complement activity. It was demonstrated that different harvest months had a significant impact on the difference in chemical composition and anti-complement activity of GH. And CGAs might play an important role in the anti-complement activity of GH.     

Synthesis,characterization, and biological screening of metal complexes of novel sulfonamide derivatives: Experimental and theoretical analysis of sulfonamide crystal

This study reports the synthesis of sulfonamide-derived Schiff bases as ligands L ¹ and L ² as well as their transition metal complexes [VO(IV), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II)]. The Schiff bases (4-{E-[(2-hydroxy-3-methoxyphenyl)methylidene]amino}benzene-1-sulfonamide ( L ¹ ) and 4-{[(2-hydroxy-3-methoxyphenyl)methylidene]amino}-N-(5-methyl-1,2-oxazol-3-yl)benzene-1-sulfonamide ( L ² ) were synthesized by the condensation reaction of 4-aminobenzene-1-sulfonamide and 4-amino-N-(3-methyl-2,3-dihydro-1,2-oxazol-5-yl)benzene-1-sulfonamide with 2-hydroxy-3-methoxybenzaldehyde in an equimolar ratio. Sulfonamide core ligands behaved as bidentate ligands and coordinated with transition metals via nitrogen of azomethine and the oxygen of the hydroxyl group. Ligand L ¹ was recovered in its crystalline form and was analyzed by single-crystal X-ray diffraction technique which held monoclinic crystal system with space group (P2₁/c). The structures of the ligands L ¹ and L ² and their transition metal complexes were established by their physical (melting point, color, yields, solubility, magnetic susceptibility, and conductance measurements), spectral (UV–visible [UV–Vis], Fourier transform infrared spectroscopy, ¹H NMR, ¹³C NMR, and mass analysis), and analytical (CHN analysis) techniques. Furthermore, computational analysis (vibrational bands, frontier molecular orbitals (FMOs), and natural bonding orbitals [NBOs]) were performed for ligands through density functional theory utilizing B3LYP/6-311+G(d,p) level and UV–Vis analysis was carried out by time-dependent density functional theory. Theoretical spectroscopic data were in line with the experimental spectroscopic data. NBO analysis confirmed the extraordinary stability of the ligands in their conjugative interactions. Global reactivity parameters computed from the FMO energies indicated the ligands were bioactive by nature. These procedures ensured the charge transfer phenomenon for the ligands and reasonable relevance was established with experimental results. The synthesized compounds were screened for antimicrobial activities against bacterial (Streptococcus aureus, Bacillus subtilis, Eshcheria coli, and Klebsiella pneomoniae) species and fungal (Aspergillus niger and Aspergillus flavous) strains. A further assay was designed for screening of their antioxidant activities (2,2-diphenyl-1-picrylhydrazine radical scavenging activity, total phenolic contents, and total iron reducing power) and enzyme inhibition properties (amylase, protease, acetylcholinesterase, and butyrylcholinesterase). The substantial results of these activities proved the ligands and their transition metal complexes to be bioactive in their nature.     

Similar,or dissimilar,that is the question. How different are methods for comparison of compounds similarity?

Comparison of compounds similarity is one of the main strategies of virtual screening protocols. Both similarity and dissimilarity concepts are of great importance during the search for new active compounds. Similarity is important due to the assumption that underlies the process of searching for new drug candidates: structurally similar compounds should induce similar biological response. On the other hand, we are also interested in dissimilarity, as we usually aim to find structurally novel ligands. In the study, we compared several approaches of evaluating compound similarity. Various representations and metrics were applied and we indicated the rate of variation of the results that can occur when shifting from one strategy to another. We compared both general similarity of datasets using different approaches, as well as examined the changes in the set of nearest neighbors when changing one compound representation into another, and the influence of representation/metric settings on the clustering outcome. We hope that the study will be of great help during the preparation of virtual screening experiments, stressing the need for careful selection of the way, the compound similarity is assessed. The differences in the results that can be obtained via the application of particular strategy can significantly influence the outcome of comparison studies; therefore, its settings should be carefully selected beforerunning the comparison.     

A column‐switching HPLC‐MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disorders

Lysosomal storage disorders comprise a group of rare genetic diseases in which a deficit of specific hydrolases leads to the storage of undegraded substrates in lysosomes. Impaired enzyme activities can be assessed by MS/MS quantification of the reaction products obtained after incubation with specific substrates. In this study, a column‐switching HPLC‐MS/MS method for multiplex screening in dried blood spot of the lysosomal enzymes activities was developed. Mucopolysaccharidosis type I, Fabry, Gaucher, Krabbe, Niemann–Pick A/B and Pompe diseases were simultaneously assayed. Dried blood spots were incubated with substrates and internal standards; thereafter, supernatants were collected with minor manipulations. Samples were injected, trapped into an online perfusion column and, by a six‐port valve, switched online through the C₁₈ analytical column to perform separation of metabolites followed by MS/MS analysis. A total of 1136 de‐identified newborn screening samples were analyzed to determine references for enzymes activity values. As positive controls, we analyzed dried blood spots from three patients with Pompe, one with Fabry, one with Krabbe disease and two with MPS I, and in all cases the enzyme activities were below the cutoff values measured for newborns, except for an MPS I patient after successful hematopoietic stem cell transplantation. Copyright © 2014 John Wiley & Sons, Ltd.     

Chiral Amine Synthesis Using ω‐Transaminases: An Amine Donor that Displaces Equilibria and Enables High‐Throughput Screening

The widespread application of ω‐transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by‐product removal using only one equivalent of a diamine donor (ortho‐xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)‐ and (S)‐selective ω‐TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1‐indanone). Significantly, spontaneous polymerization of the isoindole by‐product generates colored derivatives, providing a high‐throughput screening platform to identify desired ω‐TA activity.     

Optimizing Antimicrobial Peptide Dendrimers in Chemical Space

We used nearest‐neighbor searches in chemical space to improve the activity of the antimicrobial peptide dendrimer (AMPD) G3KL and identified dendrimer T7 , which has an expanded activity range against Gram‐negative pathogenic bacteria including Klebsiellae pneumoniae, increased serum stability, and promising activity in an in vivo infection model against a multidrug‐resistant strain of Acinetobacter baumannii. Imaging, spectroscopic studies, and a structural model from molecular dynamics simulations suggest that T7 acts through membrane disruption. These experiments provide the first example of using virtual screening in the field of dendrimers and show that dendrimer size does not limit the activity of AMPDs.