Discovery of novel Akt1 inhibitors by an ensemble-based virtual screening method, molecular dynamics simulation, and in vitro biological activity testing.

Akt1, as an important member of the Akt family, plays a controlled role in cancer cell growth and survival. Inhibition of Akt1 activity can promote cancer cell apoptosis and inhibit tumor growth. Therefore, in this investigation, a multilayer virtual screening approach, including receptor-ligand interaction-based pharmacophore, 3D-QSAR, molecular docking, and deep learning methods, was utilized to construct a virtual screening platform for Akt1 inhibitors. 17 representative compounds with different scaffolds were identified as potential Akt1 inhibitors from three databases. Among these 17 compounds, the Hit9 exhibited the best inhibitory activity against Akt1 with inhibition rate of 33.08% at concentration of 1 µM. The molecular dynamics simulations revealed that Hit9 and Akt1 could form a compact and stable complex. Moreover, Hit9 interacted with some key residues by hydrophobic, electrostatic, and hydrogen bonding interactions and induced substantial conformation changes in the hinge region of the Akt1 active site. The average binding free energies for the Akt1-CQU, Akt1-Ipatasertib, and Akt1-Hit9 systems were - 34.44, - 63.37, and - 39.14 kJ mol-1, respectively. In summary, the results obtained in this investigation suggested that Hit9 with novel scaffold may be a promising lead compound for developing new Akt1 inhibitor for treatment of various cancers with Akt1 overexpressed.

Chitosan oligosaccharide ameliorates acute lung injury induced by blast injury through the DDAH1/ADMA pathway.

OBJECTIVE: To investigate the protective effect of chitosan oligosaccharide (COS) on acute lung injury (ALI) caused by blast injury, and explore possible molecular mechanisms. METHODS: A mouse model of blast injury-induced ALI was established using a self-made explosive device. Thirty mice were randomly assigned to control, ALI and ALI + COS groups. An eight-channel physiological monitor was used to determine the mouse physiological index. Enzyme linked immunosorbent assay was used to measure serum inflammatory factors. Hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluorescence staining, real time-polymerase chain reaction and western blot assay were used to detect inflammatory reactions, oxidative stress and apoptosis. RESULTS: Mice were sacrificed 24 hours after successful model induction. Compared with the ALI group, the heart rate, respiration and PCO2 were significantly lower, but the PO2, TCO2 and HCO3- were significantly higher in the ALI + COS group. Compared to ALI alone, COS treatment of ALI caused a significant decrease in the wet/dry lung weight ratio, indicating a reduction in lung edema, inflammatory cell infiltration, levels of tumor necrosis factor-α, interleukin (IL)-1ß, IL-4, IL-6 and nuclear factor kappa B mRNA and protein expression were reduced and IL-10 mRNA and protein expression was increased (P < 0.05). COS significantly inhibited reactive oxygen species, MDA5 and IREα mRNA and protein expressions, cell apoptosis and Bax and Caspase-3 mRNA and protein expressions, and significantly increased superoxide dismutase-1 mRNA expression, and Bcl-2 and Caspase-8 mRNA and protein expression (all P<0.05). COS significantly increased dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein expression, and reduced ADMA and p38 protein expression (P< 0.05). CONCLUSION: Blast injury causes inflammation, oxidative stress and apoptosis in the lung tissues of mice. COS has protective effects on blast injury-induced ALI, possibly by promoting DDAH1 expression and inhibiting ADMA and mitogen-activated protein kinase pathways.

[Purification and characterization of a novel alpha-galactosidase from penicillium sp. F63 CGMCC1669].

An a-galactosidase-producing fungus was screened out of 26 filamentous fungi isolated from soil by us. Phylogenetic analysis based on the alignment of 18S rDNA sequences, combined with the morphological identification, indicated that the strain F63 was a member of the genus Penicillium. The a-galactosidase from Penicillium sp. F63 was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. The molecular size of the purified enzyme is approximately 82kDa estimated by SDS-PAGE. The a-galactosidase has an optimum pH of 5.0 and an optimum temperature of 45 degrees C. The enzyme is stable between pH5.0 and 6.0 below 40 degrees C. The a-galactosidase activity is slightly inhibited by Ag+ , which is dissimilar to other a-galactosidases. Kinetic studies of the a-galactosidase showed that the Km and the Vmax for pNPG are 1.4mmol/L and 1.556mmol/L. min(-1) x mg- 1, respectively. The enzyme is able to degrade natural substrates such as melibiose, raffinose and stachyose but not galactose-containing polysaccharides. The alpha-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The results show that the a-galactosidase is a novel one.

[Overexpression of Escherchia coli phytase with high specific activity].

High-level expression of phytase with high specific activity is an effective way to improve phytase fermentation potency and reduce its production cost. The gene appA encoding Escherchia coli phytase AppA with high specific activity was modified and artificially synthesized according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence of the AppA. The modified gene, appA-m, was inserted in the Pichia pastoris expression vector pPIC9, then introduced into the host Pichia pastoris by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The result of Southern blotting analysis of the recombinant yeast indicated that only one copy of the appA-m gene was integrated into the genome of Pichia pastoris. The result of Northern analysis of the recombinant yeast showed that the modified gene was effectively transcribed. SDS-PAGE analysis of the phytase expressed in Pichia pastoris revealed that the phytase was overexpressed and secreted into the medium supernatant. There are three phytase proteins with apparent molecular weight in approximately 50kD, 52kD and 54kD respectively in the media, which are larger in the size than the native phytase from E. coli. The results of N-terminal sequecing and deglycosylation of the expressed phytase in Pichia pastoris proved that the expressed phytase were glycosylated protein with different glycosylation degree. The expressed phytase Pichia pastoris shared similar pH and temperature optima to those of the natural phytase from E. coli and had highly resistant to pepsin digestion. In 5-L fermentor, after induced by 0.5% methanol for 120 h, the expression level of phytase protein was 2.5 mg/mL, and the phytase activity (fermentation potency) exceeded 7.5 x 10(6) IU/mL, which was the highest among those of all kinds of recombinant strains reported now.