Synthesis,biological evaluation,and molecular docking studies of novel 2-styryl-5-nitroimidazole derivatives containing 1,4-benzodioxan moiety as FAK inhibitors with anticancer activity

A series of 2-styryl-5-nitroimidazole derivatives containing 1,4-benzodioxan moiety (3a–3r) has been designed, synthesized and their biological activities were also evaluated as potential antiproliferation and focal adhesion kinase (FAK) inhibitors. Among all the compounds, 3p showed the most potent activity in vitro which inhibited the growth of A549 with IC₅₀ value of 3.11 μM and Hela with IC₅₀ value of 2.54 μM respectively. Compound 3p also exhibited significant FAK inhibitory activity (IC₅₀ = 0.45 μM). Docking simulation was performed for compound 3p into the FAK structure active site to determine the probable binding model.     

Multiple Modes of Protein–Protein Interactions Promote RNP Granule Assembly

Eukaryotic cells are known to contain a wide variety of RNA–protein assemblies, collectively referred to as RNP granules. RNP granules form from a combination of RNA–RNA, protein–RNA, and protein–protein interactions. In addition, RNP granules are enriched in proteins with intrinsically disordered regions (IDRs), which are frequently appended to a well-folded domain of the same protein. This structural organization of RNP granule components allows for a diverse set of protein–protein interactions including traditional structured interactions between well-folded domains, interactions of short linear motifs in IDRs with the surface of well-folded domains, interactions of short motifs within IDRs that weakly interact with related motifs, and weak interactions involving at most transient ordering of IDRs and folded domains with other components. In addition, both well-folded domains and IDRs in granule components frequently interact with RNA and thereby can contribute to RNP granule assembly. We discuss the contribution of these interactions to liquid–liquid phase separation and the possible role of phase separation in the assembly of RNP granules. We expect that these principles also apply to other non-membrane bound organelles and large assemblies in the cell.     

Development of M2BPGi: a novel fibrosis serum glyco-biomarker for chronic hepatitis/cirrhosis diagnostics

Many proteins in the living body are glycoproteins, which present glycans linked on their surface. Glycan structures reflect the degree of cell differentiation or canceration and are cell specific. These characteristics are advantageous in the development of various disease biomarkers. Glycoprotein-based biomarkers (glyco-biomarkers) are developed by utilizing the specific changes in the glycan structure on a glycoprotein secreted from the diseased cells of interest. Therefore, quantification of the altered glycan structures is the key to developing a new glyco-biomarker. Glycoscience is a relatively new area of molecular science, and recent advancement of glycotechnologies is remarkable. In the author’s institute, new glycoscience technologies have been designed to be efficiently utilized for the development of new diagnostic agents. This paper introduces a strategy for glyco-biomarker development, which was successfully applied in the development of Wisteria floribunda agglutinin-positive Mac-2 binding protein M2BPGi, a liver fibrosis marker now commercially available for clinical use.     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

Collective sampling of intact anionic polysaccharide components and application in quantitative determination by LC-MS

Anionic polysaccharides, such as glycosaminoglycans (GAGs) and alginate, readily undergo source-induced fragmentation when analyzed by electrospray mass spectrometry with the use of high source cone voltage. The dissociation chemistry converts all components of a polysaccharide into a small set of structurally characteristic small saccharides. This chemistry enables the collective detection of a polysaccharide through the detection of one or more small saccharides. This ability, combined with the elution of polysaccharides as relatively compact bands using ion-pairing reverse phase liquid chromatography, created a unique opportunity for the development of LC–MS methods suitable for the quantitative analysis of intact anionic polysaccharides. Feasibility of this approach is demonstrated with a mixture of heparin, chondroitin sulfate A, and alginate.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.