Non-electrostatic interactions associated with aggregate formation between polyallylamine and Escherichia coli.

Bacterial aggregation by mixing with polymers is applied as pretreatment to identify pathogens in patients with infectious diseases. However, the detailed interaction between polymers and bacteria has yet to be fully understood. Here, we investigate the interaction between polyallylamine and Escherichia coli by isothermal titration calorimetry. Aggregation was observed at pH 10 and the binding was driven by favorable enthalpic gain such as the electrostatic interaction. Neither aggregation nor the apparent heat of binding was observed at pH 4.0, despite the strong positive charge of polyallylamine. These results suggest that intermolecular repulsive forces of the abundant positive charge of polyallylamine cause an increased loss of conformational entropy by binding. Non-electrostatic interaction plays a critical role for aggregation.

Crystal structure of ß-L-arabinobiosidase belonging to glycoside hydrolase family 121.

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of ß-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of ß-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular ß-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing ß-1,2-linked arabinofuranose disaccharide (ß-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) ß-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the ß-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 ß-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. ß-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the ß-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

Solubility-Improved 10-O-Substituted SN-38 Derivatives with Antitumor Activity.

With the objective of improving the poor water solubility of the potent antitumor compound SN-38, 10-O-substituted SN-38 derivatives were developed by the introduction of fluoroalkyl, fluorobenzoyl, or bromobenzoyl groups. The 10-O-fluoropropyl-substituted compound 2 {(S)-4,11-diethyl-9-(3-fluoropropoxy)-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione} was found to be 17-fold more soluble than SN-38 in phosphate-buffered saline, and it exhibited a level of biological activity ≈50 % that of SN-38 in a cytotoxicity assay using the prostate cancer cell line PC-3. Five other derivatives did not show solubility improvements to the same extent, but their activities in cytotoxicity assays were nearly the same as that of SN-38. In vivo studies of 2 with PC-3 tumor-bearing mice revealed that it has higher antitumor activity than SN-38, even at lower dosage. These results will promote the medicinal chemistry application of 10-O-modifications of SN-38 and help reestablish the potential this drug. Furthermore, the inclusion of fluoro and bromo substituents means that the synthetic strategy developed here may be used to obtain 18 F- or 76 Br-labeled SN-38 derivatives for in vivo positron emission tomography studies.

MEK and PI3K catalytic activity as predictor of the response to molecularly targeted agents in triple-negative breast cancer.

Hyper-activation of the MAPK and PI3K-AKT pathways is linked to tumour progression in triple-negative breast cancer (TNBC). However, clinically effective predictive markers for drugs targeted against protein kinases involved in these pathways have not been identified. We investigated the ability of MEK and PI3K catalytic activity to predict sensitivity to trametinib and wortmannin in TNBC. MEK and PI3K activities correlated strongly with each other only in cell lines showing wortmannin-specific sensitivity, as shown by a linear regression curve (R = 0.951). Accordingly, we created a new parameter that distinguishes trametinib and wortmannin sensitivity in vitro and in vivo. Our findings suggest that the catalytic activities of MEK and PI3K might predict the response of TNBC to trametinib and wortmannin.

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family.

Extensin is a glycoprotein that is rich in hydroxyprolines linked to ß-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel ß-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-ß1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara(4)-Hyp) or Araf-ß1,2-Araf-ß-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked ß-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.