Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide–valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC₅₀ of 7 nM and EC_2×PT of 1.7 μM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide–valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.     

Substrate profiling of IGF-1R and InsR: identification of a potent pentamer substrate

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time. In contrast to already reported profiling methodologies, we were able to assess the k(cat)/K(M) ratio of peptides as short as tetramers. Notably, we were able to identify an efficient pentamer substrate that exhibited kinetic properties close to those of a 250-amino acid protein derived from IRS-1, a natural substrate of IGF-1R and InsR.     

Silk fibroin has a protective effect against high glucose induced apoptosis in HIT-T15 cells

High glucose levels induce cell death in many cell types, including pancreatic β-cells. Although protective agents against glucotoxicity have been searched for extensively, so far none have been found. In this report, we tested silk fibroin (SF) as a candidate material for antiglucotoxicity in the pancreatic β-cell (HIT-T15 cell) line. Approximately 50% of cells were killed after treatment with 80 mg/mL glucose. This reduction of cell number was recovered by the addition of SF at 50 mg/mL. SF treatment also decreased cellular reactive oxygen species (ROS) and increased proliferating cellular nuclear antigen (PCNA) immunoreactivity. In addition, TUNEL assays demonstrated that SF protects against glucose-induced apoptosis of HIT-T15 cells, suggesting that SF might protect cells from cell death by lowering cellular ROS levels. SF also induced expression of the insulin-like growth factor-1 (IGF-1) gene, and IGF-1 expression may be the cause of SF-induced protection against glucose toxicity. Taken together, these results suggest that SF could serve as a potential therapeutic agent to treat the hyperglycemia-induced death of pancreatic β-cells.     

Constitutive activation of nuclear factor-E2-related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte-specific deletion of Kelch-like ECH-associated protein 1

Chemicals that activate nuclear factor-E2-related factor 2 (Nrf2) often increase multidrug-resistance-associated protein (Mrp) expression in liver. Hepatocyte-specific deletion of Kelch-like ECH-associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte-specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild-type and hepatocyte-specific Keap1-null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate-cysteine ligase catalytic subunit expression were increased in livers of Keap1-null mice. Organic anion-transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild-type mice. By contrast, Mrp 1-5 mRNA and protein levels were increased in Keap1-null mouse livers, with Mrp4 expression being more than 15-fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions.     

PAX3基因及其蛋白的生物信息学分析

目的：对PAX3基因和PAX3蛋白进行生物信息学分析,更多的了解该基因的相关信息,为进一步研究PAX3与神经管畸形的相关性研究提供基础。方法：运用生物信息学方法对PAX3基因的基因结构、单核苷酸多态性位点（SNP）、PAX3基因与其他基因的相互作用网络、PAX3蛋白结构域、蛋白二级结构、蛋白间相互作用网络、以及PAX3蛋白所调控和影响的靶基因进行分析。结果：PAX3基因有9中可变剪切形式,编码区存在14个SNP位点,其中错意突变13个,移码突变1个。PAX3蛋白由479个氨基酸组成,分子量52968Da,PAX3蛋白可能调控和影响151个靶基因的转录和表达,与PAX3基因存在相互作用的基因和与PAX3蛋白存在相互作用的蛋白多数与发育相关。结论：通过对PAX3基因和PAX3蛋白的生物信息学分析获得了其相应的分子生物学特征,为进一步研究提供基础。     

伴海马硬化的颞叶癫痫患者海马组织内BDNF 表达变化

目的:研究伴海马硬化的难治性颞叶癫痫(TLE)患者海马组织内脑源性神经营养因子(brain derived neurotrophic factor,BDNF)的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法:采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应(RT-PCR)法检测BDNF mRNA表达,并与3例非海马硬化TLE患者对照。结果:与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNF mRNA表达明显增加(P<0.01)。结论:伴海马硬化的难治性TLE患者海马组织中BDNF mRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.     

Novel interactions of large P3 moiety and small P4 moiety in the binding of the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.