Stafia-1: a STAT5a-Selective Inhibitor Developed via Docking-Based Screening of in Silico O-Phosphorylated Fragments

We present a new approach for the identification of inhibitors of phosphorylation-dependent protein–protein interaction domains, in which phenolic fragments are adapted by in silico O-phosphorylation before docking-based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product-derived phenolic fragments, which were virtually O-phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia-1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia-1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small-molecule inhibitor.     

Cyclization of Nitrospirobenzopyrans to Bridged Benzoxazepino[3,2-a]indoles

Condensation of 1-substituted 1,2,3,9a-tetrahydro-9H-imidazo[1,2-a]indol-2-ones with 5-nitrosalicylaldehyde afforded 1′-[(N-monosubstituted carbamoyl)methyl]indoline nitrospirobenzopyrans. Treatment of the latter with strong base led to the formation of a mixture of cis/trans-5a,13-methano-1,3-benzoxazepino[3,2-a]indoles. Results of semiempirical calculations gave evidence that such a transformation of nitrospirobenzopyrans to bicyclic indole derivatives could proceed via a single transition state, where the negatively charged carbon atom attacks the vinylic double bond of the spiropyran system.     

Targeted Photodynamic Therapy of Human Head and Neck Squamous Cell Carcinoma with Anti-epidermal Growth Factor Receptor Antibody Cetuximab and Photosensitizer IR700DX in the Mouse Skin-fold Window Chamber Model

Targeted photodynamic therapy (PDT) in head/neck cancer patients with a conjugate of the anti-epidermal growth factor receptor (EGFR) antibody, Cetuximab and a phthalocyanine photosensitizer IR700DX is under way, but the exact mechanisms of action are still not fully understood. In this study, the EGFR-overexpressing human head/neck OSC-19-luc2-cGFP tumor with transfected GFP gene was used in a skin-fold window chamber model in BALB/c nude mice. The uptake and localization of the conjugate in the tumor and its surrounding normal tissues were studied by an intravital confocal laser scanning microscopy with image analyses. The tumor was also irradiated with 690 nm laser light 24 h after conjugate administration. The vascular and tumor responses were examined by morphological evaluation and immunohistochemistry (IHC). The amount of conjugate in the tumor peaked at 24–48 h after injection. Image analyses of colocalization correlation parameters demonstrated a high fraction of the conjugate IR700DX colocalized in the GFP-expressing tumor cells. PDT-treated tumors showed extensive necrotic/apoptotic destruction with little vascular damage, while IHC showed no HIF-1α expression and decreased EGFR and Ki67 expression with activated caspase-3 overexpression, indicating a direct killing of tumor cells through both necrotic and apoptotic cell death.     

The solvent venting injection technique in capillary supercritical fluid chromatography

A solvent venting technique for injection of volumes up to 1 μl on 50 μm i.d. SFC columns has been compared to direct injection methods. The peak broadening and peak splitting observed with direct injection have been examined and found to be related to the starting pressure, the column temperature, and the sample solvent, in addition to the sample volume. The solvent venting technique removed peak splitting and improved the column efficiency. With a proper selection of experimental conditions, the sample recovery was 100%. The major part of the solvent was eluted in 15–20 s. Several applications have been demonstrated.     

Raman spectroscopic study of tungsten(VI) oxosulfato complexes in WO3-K2S2O7-K2SO4 molten mixtures: stoichiometry, vibrational properties, and molecular structure

The dissolution reaction of WO3 in pure molten K2S2O7 and in molten K2S2O7-K2SO4 mixtures is studied under static equilibrium conditions in the XWO3(0) = 0-0.33 mol fraction range at temperatures up to 860 °C. High temperature Raman spectroscopy shows that the dissolution leads to formation of W(VI) oxosulfato complexes, and the spectral features are adequate for inferring the structural and vibrational properties of the complexes formed. The band characteristics observed in the W=O stretching region (band wavenumbers, intensities, and polarization characteristics) are consistent with a dioxo W(=O)2 configuration as a core unit within the oxosulfato complexes formed. A quantitative exploitation of the relative Raman intensities in the binary WO3-K2S2O7 system allows the determination of the stoichiometric coefficient, n, of the complex formation reaction WO3 + nS2O7(2-) --> C(2n-). It is found that n = 1; therefore, the reaction WO3 + S2O7(2-) > WO2(SO4)2(2-) with six-fold W coordination is proposed as fully consistent with the observed Raman features. The effects of the incremental dissolution and presence of K2SO4 in WO3-K2S2O7 melts point to a WO3 · K2S2O7 · K2SO4 stoichiometry and a corresponding complex formation reaction in the ternary molten WO3-K2S2O7-K2SO4 system according to WO3 + S2O7(2-) + SO4(2-) --> WO2(SO4)3(4-). The coordination sphere of W in WO2(SO4)2(2-) (binary system) is completed with two oxide ligands and two chelating sulfate groups. A dimeric [{WO2(SO4)2}2(μ-SO4)2](8-) configuration is proposed for the W oxosulfato complex in the ternary system, generated from inversion symmetry of aWO2(SO4)3(4-) moiety resulting in two bridging sulfates. The most characteristic Raman bands for the W(VI) oxosulfato complexes pertain to W(=O)2 stretching modes (i) at 972 (polarized) and 937 (depolarized) cm(-1) for the ν(s) and ν(as) W(=O)2 modes of WO2(SO4)2(2-), and (ii) at 933 (polarized) and 909 (depolarized) cm(-1) for the respective modes of [{WO2(SO4)2}2(μ-SO4)2](8-).     

Assessing transformation processes of organic contaminants by compound-specific stable isotope analysis

The analysis of variations in stable isotope composition is becoming an essential approach for evaluating enzymatic and abiotic reactions of organic contaminants in soils and aquatic systems. Different, sometimes complementary analytical techniques are currently used and developed to determine stable isotope ratios in individual organic compounds. Anticipating an increasing demand for compound-specific isotope analysis, this survey compiles information for choosing the most promising analytical approach to an isotope-related problem. To this end, we review the principles of instrumentation for compound-specific isotope analysis and show how they can be exploited to assess contaminant transformation processes. Using chlorinated solvents and triazine herbicides as illustrative examples, we discuss how the isotope-sensitive techniques impact the investigation of stable isotope fractionation in environmental chemistry and microbiology.     

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 μs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.     

Determination of low levels of polymer additives migrating from polypropylene to food simulated liquids by capillary SFC and solvent venting injection

Summary Methods for the determination of specific migration of polymer additives from polypropylene to aqueous acetic acid and isooctane have been studied. The migrants were extracted from aqueous acetic acid and characterized by supercritical fluid chromatography (SFC) with flame ionization detection (FID) and mass spectrometry (MS). The isooctane simulant was concentrated and injected directly into the SFC. Injection of extracts after incubation, using the solvent venting injection technique in the SFC, enabled additive concentrations as low as 10 ppb in 150 ml simulant to be determined, corresponding to a specific migration level of approximately 1 g additive/dm² polymer.     

On-line enzymatic reaction,extraction, and chromatography of fatty acids and triglycerides with supercritical carbon dioxide

Supercritical carbon dioxide can be utilized both as an introduction solvent in capillary SFC and as a reaction medium; both possibilities are illustrated in this study. An off-line SFE unit was modified for on-line SFE-SFC. To facilitate rapid depressurization of the extraction cell and to prevent memory effects, a 6-port valve was installed at the outlet line of the extraction unit. An increased background signal was obtained when PEEK polymer was used in the construction of the extraction vessel; when stainless steel was used, the blanks improved. The synthesis of methyl esters and butyl esters of fatty acids from triglycerides in edible fat was examined using an immobilized lipase as a catalyst in on-line SFE-SFC. As a result of 30 minutes reaction – extraction time, high yields of fatty acid esters were obtained at a pressure of 150 bar and a temperature of 50°C.