Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity‐Based Probes of Matriptase‐2

Matriptase‐2, a type II transmembrane serine protease, plays a key role in human iron homeostasis. Inhibition of matriptase‐2 is considered as an attractive strategy for the treatment of iron‐overload diseases, such as hemochromatosis and β‐thalassemia. In the present study, synthetic routes to nine dipeptidomimetic inactivators were developed. Five active compounds ( 41 – 45 ) were identified and characterized kinetically as irreversible inhibitors of matriptase‐2. In addition to a phosphonate warhead, these dipeptides possess two benzguanidine moieties as arginine mimetics to provide affinity for matriptase‐2 by binding to the S1 and S3/S4 subpockets, respectively. This binding mode was strongly supported by covalent docking analysis. Compounds 41 – 45 were obtained as mixtures of two diastereomers and were therefore separated into the single epimers. Compound 45 A , with S configuration at the N‐terminal amino acid and R configuration at the phosphonate carbon atom, was the most potent matriptase‐2 inactivator with a rate constant of inactivation of 2790 m ^?1 s^?1 and abolished the activity of membrane‐bound matriptase‐2 on the surface of intact cells. Based on the chemotyp of phosphono bisbenzguanidines, the design and synthesis of a fluorescent probe ( 51 A ) by insertion of a coumarin label is described. The in‐gel fluorescence detection of matriptase‐2 was demonstrated by applying 51 A as the first activity‐based probe for this enzyme.     

A rapid UPLC‐MS/MS method for the determination of oleanolic acid in rat plasma and liver tissue: application to plasma and liver pharmacokinetics

A reliable high‐throughput ultra‐high performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for oleanolic acid (OA) determination in rat plasma and liver tissue using glycyrrhetic acid as the internal standard (IS). Plasma and liver homogenate samples were prepared using solid‐phase extraction. Chromatographic separation was achieved on a C₁₈ column using an isocratic mobile phase system. The detection was performed by multiple reaction monitoring mode via positive electrospray ionization interface. The calibration curves showed good linearity (R² > 0.9997) within the tested concentration ranges. The lower limit of quantification for plasma and liver tissue was ≤0.75 ng/mL. The intra‐ and inter‐day precision and accuracy deviations were within ±15% in plasma and liver tissue. The mean extraction recoveries ranged from 80.8 to 87.0%. In addition, the carryover, matrix effect, stability and robustness involved in the method were also validated. The method was successfully applied to the plasma and hepatic pharmacokinetics of OA after oral administration to rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolism studies on prim‐O‐glucosylcimifugin and cimifugin in human liver microsomes by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Prim‐O‐glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti‐inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O‐glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP‐glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

Freezing the Dynamic Gap for Selectivity: Motion‐Based Design of Inhibitors of the Shikimate Kinase Enzyme

Shikimate kinase (SK), the fifth enzyme of the aromatic amino acid biosynthesis, is a recognized target for antibiotic drug discovery. The potential of the distinct dynamic apolar gap, which isolates the natural substrate from the solvent environment for catalysis, and the motion of Mycobacterium tuberculosis and Helicobacter pylori SK enzymes, which was observed by molecular dynamics simulations, was explored for inhibition selectivity. The results of the biochemical and computational studies reveal that the incorporation of bulky groups at position C5 of 5‐aminoshikimic acid and the natural substrate enhances the selectivity for the H. pylori enzyme due to key motion differences in the shikimic acid binding domain (mainly helix α5). These studies show that the less‐exploited motion‐based design approach not only is an alternative strategy for the development of competitive inhibitors, but could also be a way to achieve selectivity against a particular enzyme among its homologues.     

Protective effects of vitamin E against CCl<Subscript>4</Subscript>-induced hepatotoxicity in rabbits

The influence of CCl₄ on the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), the value of the total antioxidant status (TAS), and the concentration of malonic dialdehyde (MDA) and glutathione (GSH) was monitored in plasma or whole blood of rabbits. The administration of CCl₄ caused the increase of the SOD activity to approximately 150 % and the decrease in the activity of GPx and GR by about 50 %. These changes were accompanied with the increase in TAS value and MDA concentration and the decrease of GSH concentration. The effect of CCl₄ was suppressed by the previous 7 days lasting or simultaneous administration of vitamin E. Oxidative stress caused by CCl₄ was accompanied by the development of reactive oxygen forms, especially superoxide radical anion.     

Effect of paclobutrazol on water stress amelioration through antioxidants and free radical scavenging enzymes in Arachis hypogaea L

An investigation was carried out to find out the extent of changes occurred in groundnut (Arachis hypogaea L.) cultivars in response to paclobutrazol (PBZ) treatment under water deficit stress. Two groundnut cultivars namely ICG 221 and ICG 476 were used for the study. Individual treatment with PBZ and drought stress showed an increase in ascorbic acid, -tocopherol and reduced glutathione, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities. PBZ with drought stressed plants maintained higher levels of antioxidant and scavenging enzymes. Significant differences were observed between cultivars and treatments. These results suggests that the adverse effects of water stress can be minimized by the application of PBZ by increasing the antioxidant levels and activities of scavenging enzymes such as SOD, APX and CAT. The Cv. ICG 221 appears to be more tolerant to water stress than the ICG 476.     

Changes of antioxidative enzymes and cell membrane osmosis in tomato colonized by arbuscular Mycorrhizae under NaCl stress

Salinity toxicity is a worldwide agricultural and eco-environmental problem. Many literatures show that arbuscular mycorrhizal fungi (AMF) can enhance salt tolerance of many plants and some physiological changes occurred in AM symbiosis under salt stress. However, the role of ROS-scavenging enzymes in AM tomato is still unknown in continuous salt stress. This study investigated the effect of Glomus mosseae on tomato growth, cell membrane osmosis and examined the antioxidants (superoxide-dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; peroxidase, POD) responses in roots of mycorrhizal tomato and control under different NaCl stress for 40 days in potted culture. NaCl solution (0, 0.5 and 1%) was added to organic soil in the irrigation water after 45 days inoculated by AMF (Glomus mosseae). (1) AMF inoculation improved tomato growth under salt or saltless condition and reduced cell membrane osmosis, MDA (malonaldehyde) content in salinity. So the salt tolerance of tomato was enhanced by AMF; (2) SOD, APX and POD activity in roots of AM symbiosis were significantly higher than corresponding non-AM plants in salinity or saltless condition. However, CAT activity was transiently induced by AMF and then suppressed to a level similar with non-AM seedlings; (3) higher salinity (1% level) and long stress time suppressed the effect of AMF on SOD, APX, POD and CAT activity; (4) this research suggested that the enhanced salt tolerance in AM symbiosis was mainly related with the elevated SOD, POD and APX activity by AMF which degraded more reactive oxygen species and so alleviated the cell membrane damages under salt stress. Whereas, the elevated SOD, POD and APX activity due to AMF depended on salinity environment.     

Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format

In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)₃₀ segment at the 5′ end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.     

微波辐射-酶耦合催化(MIECC)反应

将微波辐射用于非水相酶催化可以获得很多有别于常规加热下的反应结果.本文讨论了微波的非热效应在酶促反应中的表现,探讨了微波辐射对酶的结构、构象、活性及酶催化反应动力学的影响,以及微波辐射-酶耦合催化对反应的对映选择性、底物专一性、前手性选择性和区域选择性的影响.在大多数场合,适当的微波辐射不会损伤酶活而且可以提高反应速率,而对酶特异性的影响则不一而论.     

猪肝样品中莱克多巴胺测定的前处理方法

研究猪肝样品中莱克多巴胺测定的样品前处理方法。样品直接用HCl酸解并超声提取,同时添加70%~72%的高浓度HClO_4对酸解样品进行蛋白沉淀,然后经MCX固相萃取柱净化后,用液相色谱-串联质谱法检测。用正交试验考察了酸的种类、浓度、提取时间、提取温度和HClO_4体积对提取效率的影响,最佳条件为:HCl浓度为0.2mol/L,提取时间为40min,提取温度为25℃,HClO_4用量为200μL。从0.5、5.0和10.0μg/kg添加水平检测结果可以看出,方法平均回收率为82.0%~102%,日内和日间精密度4%,符合国家标准对检验方法的要求。该方法灵敏度高,精密度好,成本低,能简便、快速对样品中的莱克多巴胺进行提取,实现对动物源性食品的快速监测。