Привлечение банков к венчурному финансированию в Республике Беларусь

Материалы XIII Междунар. науч.-техн. конф. студентов, магистрантов и молодых ученых, Гомель, 25–26 апр. 2013 г

STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions

STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided

Small-molecule inhibitor of OGG1 suppresses pro-inflammatory gene expression and inflammation

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo

Microfluidic electrocapture technology in protein and peptide analysis

After sequencing the genomes of several organisms, science in the postgenomic era now aims at a thorough study of the proteins present in a given tissue or organism. Since this task requires an enormous analytical effort, integrated microfluidic systems are envisioned as the solution to automated high throughput analysis of biomolecules. This thesis is focused on a microfluidic methodology and device that present several advantages over present technologies. The microfluidic device utilizes an electric field to capture molecules traveling in a flow stream. After capture, another medium is injected into the system that is of a desirable chemical composition or carries reagents, which are brought into contact with the captured molecules. The microfluidic device was employed as a concentrator for capillary electrophoresis (CE). Samples containing a mixture of proteins were concentrated and injected into a CE instrument. Detection limits were thereby improved from µM to nM protein levels. The device was further applied to desalting and removal of contaminants before MALDI-MS analysis. Polypeptides were captured followed by the injection of a solvent suitable for NIS analysis. Significant desalting and removal of CHAPS detergent was obtained for efficient analysis of peptides and proteins by MALDI-MS. In further study, the utilization of the electrocapture device to carry out microreactions is described. After the capture of a target protein, another medium containing enzymes and/or reagents is injected. Reduction, alkylation, and trypsin digestion, as well as sample cleanup, were carried out for peptide mass mapping by MALDI-MS. The use of the electrocapture device as a separation tool is also described. The separation process involves the capture and subsequent sequential release of peptides according to their electrophoretic mobility. Tryptic peptides from digestion of a mixture of proteins were separated and analyzed by MALDI-MS. A final study concerns the capture mechanism. It was found that negatively charged molecules are in fact immobilized in the flow stream due to a steady-state phenomenon created by the generation of areas with different electric field strengths along the fluidic channel. Herein we describe a flexible microfluidic device capable of processing polypeptides to resolve key analytical problems in protein and peptide analysis

Multistep microreactions with proteins using electrocapture technology

A method to perform multistep reactions by means of electroimmobilization of a target molecule in a microflow stream is presented. A target protein is captured by the opposing effects between the hydrodynamic and electric forces, after which another medium is injected into the system. The second medium carries enzymes or other reagents, which are brought into contact with the target protein and react. The immobilization is reversed by disconnecting the electric field, upon which products are collected at the outlet of the device for analysis. On-line reduction, alkylation, and trypsin digestion of proteins is demonstrated and was monitored by MALDI mass spectrometry

Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related gamma-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb) 2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth