Sensing the insulin signaling pathway with an antibody array

The development of insulin resistance and type 2 diabetes is determined by various factors, including defects within the insulin signaling pathway. Mediators of insulin resistance operate through activation of various protein kinase C isoforms, IκB kinase β (IKKβ), and/or c‐Jun N‐terminal kinase, and subsequent inhibition of the proximal insulin signaling pathway via the insulin receptor substrate 1 and Akt. These mechanisms are still largely unresolved because of the complexity of the molecular events. In this study, an expression and activation state profiling of multiple known key signaling biomolecules involved in insulin metabolic and mitogenic signaling pathways was evaluated using a phosphospecific antibody array platform. The results of the arrayed antibodies were verified by the multiplexed bead array assay and conventional Western blot analysis, and confirmed the well‐known inhibitory effects of phorbol esters on insulin signaling pathway activation. Of interest, the increase in protein kinase C signaling responses with phorbol esters was associated with activation of the lipid phosphatase PTEN and a 27 kDa HSP. Thus, this insulin signaling antibody array provides a powerful and effective way to investigate the mechanism of insulin resistance and likely assist the development of innovative therapeutic drugs for type 2 diabetes.     

Role of phosphoproteomics in the development of personalized cancer therapies

Cell signalling pathways driven by protein and lipid kinases contribute to the onset and progression of virtually all cancer types. Consequently, several inhibitors against these enzymes have clinical utility for the treatment of different forms of cancer. A problem that hampers further development is that not all patients respond equally well to kinase inhibitors and a significant proportion of those that initially respond eventually develop resistance. This review considers how an integrative analysis of kinase signalling may be used to address this issue. Advances in the biophysics of mass spectrometry, in biochemical procedures for phosphopeptide enrichment, and in computational approaches for label-free quantification have contributed to the development of phosphoproteomics workflows compatible with the analysis of clinical material. These developments, together with new bioinformatics tools to derive information on signalling circuitry from phosphoproteomics data, allow investigating kinase networks with unprecedented depth. Phosphoproteomics technology is starting to be used in translational research and, with further developments, such methods may also be able to measure the circuitry of cancer signalling networks in routine clinical assays. This review reflects on how this information could be used to accurately predict the best kinase inhibitor for each individual cancer patient.     

Selective Ion Extraction for High Throughput Screening with a Microfluidic Enzyme Assay

This proof-of-concept paper describes the application of selective ion extraction to an assay of protein kinase A on a microfluidic chip platform. Selective ion extraction is a flux balance technique, where a combination of independent pressure control and voltage are used to selectively extract one ion from a mixture. The assay product is completely separated and diverted into a separate channel from the waste stream containing the unconverted substrate and enzyme. By detecting only product, background noise generated by the substrate is removed which increases the signal to noise ratio and assay sensitivity. This technique is intended for adapting kinase or protease assays with low conversion rates to an on-chip reaction format for HTS screening.     

Cellular Dielectric Spectroscopy: A Label-Free Technology for Drug Discovery

CDS is a technology that enables comprehensive pharmacological evaluation of cell surface receptors in real time. It is a universal assay that allows measurement of multiple types of receptors, including G protein-coupled receptors (GPCRs), tyrosine kinase receptors (TKRs), and steroid receptors using the same platform, without the need for transfections or special reagents. The CDS-based platform is sensitive enough to consistently monitor ligand-mediated activation of endogenously expressed receptors, and for this reason, has the ability to generate more physiologically relevant data than obtained with genetically and chemically manipulated cells.The label-free CDS measurement is a measure of cell function as opposed to binding events that are typical of many current label-free approaches: classical (BIAcore's SPR technology) and more recent (Corning's EPIC, SRU Biosystems' BIND, CSEM's WIOS, and Akubio's RAP technology). A detailed comparison of these and other impedance-based measurement systems is available in a recent review of label-free technologies.⁵⁸To illustrate the power of the CDS technology, data has been presented demonstrating the use of this label-free approach in pharmacological evaluation of ligands for a number of receptors in a variety of cell types, including primary cells. Specifically, the CDS technology has been applied to hit confirmation, receptor selectivity analysis, ligand potency, and Schild analysis of receptor-selective antagonists. CDS measurements quantitatively align with results from other cell-based assays in determining the potency and ranking of agonists and antagonists. The robust and reproducible nature of CDS assays is reflected in low coefficients of variation, high signal-to-background, and excellent Z'-scores of the data. The data generated using CDS technology are unique in that the response profiles reflect receptor-mediated signaling pathways and are characteristic of main subsets of GPCRs within a cell line. Clustering analysis has been performed using response profiles from receptors that lead to potential novel applications including identification of the G-protein coupling of unknown GPCRs and the cataloging of active endogenous receptors in cell lines. Because the CDS assay requires no stable or transiently transfected cells or special reagents, assay development and data acquisition are simple and fast. These studies demonstrate that the CDS platform provides an easy-to-use, label-free, universal assay with applications in secondary screening, hit identification and lead optimization areas of drug discovery. The CellKey™ system, based on CDS technology, will be commercially available in late 2005.²⁹     

NanoArrays, The Next Generation Molecular Array Format for High Throughput Proteomics, Diagnostics and Drug Discover.

Biomolecular array technology is an invaluable tool for rapid screening of nucleic acid mixtures. This approach has been tremendously successful both in its breadth of application and its commercial value. Entire genomes, including the human genome, have been screened by molecular array techniques. Arrays are a rapid and now routine method for analysis of expression patterns and their association with physiological states. Such a rapid, high throughput analysis of cellular expression is key to the expansion of our basic knowledge of the relationship between gene expression and organismal function, as well as to the understanding of the genetic component of disease states and the predisposition to disease.Despite the success of array technology for nucleic acid applications, a similar trend for proteins has not occurred. Due, in part, to the difficulties involved in production and labeling of proteins for solid state analysis, solid state arrays of proteins are not widely utilized. Protein function and interaction have been traditionally addressed by the combination of 2D gel electrophoretic separation and mass spectrometry to examine individual protein spots, a slow, tedious and expensive process. Another approach uses in vivo methods for examining protein-protein interactions by the two-hybrid system in yeast and mammalian cells [1]. Although the two-hybrid system has shown some success in finding new interaction between proteins in important cellular pathways, it is far more difficult, costly and time consuming than the solid state methods used for nucleic acids.BioForce Laboratory, Inc., has developed a solid state method for examining the interaction between a wide range of molecules in an array format. This technology involves several key technological innovations.     

GPCR assay automation for leveraging lead optimization

The drug discovery process is dependent on the ability of screening efforts to identify and optimize lead compounds with therapeutic potential. Because G-protein-coupled receptors (GPCRs) account for the most "druggable" targets, the development of high-throughput, low-cost, and high-density GPCR assays to accommodate the increasing size of compound collections is significant. In this study, we report the application of an advanced LEADseeker robotic platform equipped with customized IT solutions for rapid data transfer to reduce assay cycles times for support of GPCR panel screening. The advantages of assay throughput, format, automation design, data management flow, and data reproducibility are discussed in terms of gains in productivity for lead optimization. The GPCR robotic platform demonstrates how automation technology can leverage traditional drug discovery processes by providing consistent and reliable data packages to expedite lead optimization efforts.     

Automation and miniaturization of the bioluminescent UGT-Glo assay for screening of UDP-glucuronosyltransferase inhibition by various compounds

The uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes is involved in the metabolism of various compounds. These enzymes transfer a hydrophilic glucuronic acid moiety to their substrates, rendering them more water soluble and amenable to excretion. The UGTs act on various endogenous substrates, such as bilirubin, 17β-estradiol, and testosterone, and drugs and other xenobiotics. The function of these enzymes is essential for the clearance of drugs and toxicants, and alteration of UGT activity is a potential cause of adverse drug-drug interactions in vivo. This has stimulated an increased interest in the study of UGT function and inhibition, and the desire to profile new drug entities against UGT enzymes, similar to CYP450 profiling. However, certain factors have hindered the development of a robust method for UGT profiling. Current methods for assessing UGT enzyme activity are laborious and involve protein precipitation and/or chromatographic separation steps, which are not amenable to rapid screening applications for UGT inhibitors or substrates. The approach presented here is a bioluminescent assay for measuring UGT enzyme activity and inhibition in vitro. Using flexible, robust instrumentation in a 384-well microplate format, this study highlights the quick and easy assay implementation for estimation of inhibition kinetics with a variety of known and suspected UGT substrates and inhibitors.     

Aurora 蛋白激酶抑制剂研究综述

Aurora蛋白激酶是一类在细胞增殖过程起重要作用的丝/苏氨酸蛋白激酶,在人体中有Aurora A、Aurora B和Aurora C三个家族成员.Aurora蛋白激酶参与细胞有丝分裂过程,其异常表达会损害有丝分裂进程,并引起遗传不稳定性,最终导致细胞癌变.目前研究发现Aurora蛋白激酶在多种肿瘤细胞中高表达,并参与了这些肿瘤的发生发展.鉴于以上研究,以Aurora激酶为靶点的抗肿瘤药物研发为癌症治疗提供了新的策略.目前,已经有相当多的Aurora激酶抑制剂被开发出来,其中部分抑制剂展现出了显著的抑瘤效果,且已进入临床试验阶段.     

Identification of p38α MAP kinase inhibitors by pharmacophore based virtual screening

The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co-crystallised inhibitors were used in the development and validation of ligand and structure based pharmacophore modeling approached. The validated pharmacophore was utilized in database screening to identify potential hits. After Lipinski's rule of five filter and molecular docking analysis, nineteen hits were purchased and selected for in vitro analysis. The virtual hits exhibited promising activity against tumor necrosis factor-α (TNF-α) with 23–98% inhibition at 10 μM concentration. Out of these seven compounds has shown potent inhibitory activity against p38 MAP kinase with IC₅₀ values ranging from 12.97 to 223.5 nM. In addition, the toxicity study against HepG2 cells was also carried out to confirm the safety profile of identified virtual hits.     

Multi-step virtual screening to develop selective DYRK1A inhibitors

Developing selective inhibitors for a particular kinase remains a major challenge in kinase-targeted drug discovery. Here we performed a multi-step virtual screening for dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) inhibitors by focusing on the selectivity for DYRK1A over cyclin-dependent kinase 5 (CDK5). To examine the key factors contributing to the selectivity, we constructed logistic regression models to discriminate between actives and inactives for DYRK1A and CDK5, respectively, using residue-based binding free energies. The residue-based parameters were calculated by molecular mechanics-generalized Born surface area (MM-GBSA) decomposition methods for kinase–ligand complexes modeled by computer ligand docking. Based on the findings from the logistic regression models, we built a three-dimensional (3D) pharmacophore model and chose filter criteria for the multi-step virtual screening. The virtual hit compounds obtained from the screening were assessed for their inhibitory activities against DYRK1A and CDK5 by in vitro assay. Our screening identified two novel selective DYRK1A inhibitors with IC₅₀ values of several μM for DYRK1A and >100 μM for CDK5, which can be further optimized to develop more potent selective DYRK1A inhibitors.     

Altered signaling associated with chronic arsenic exposure in human skin keratinocytes

Modulation of signaling pathways upon chronic arsenic exposure remains poorly studied. Here, we carried out SILAC‐based quantitative phosphoproteomics analysis to dissect the signaling induced upon chronic arsenic exposure in human skin keratinocyte cell line, HaCaT. We identified 4171 unique phosphosites derived from 2000 proteins. We observed differential phosphorylation of 406 phosphosites (twofold) corresponding to 305 proteins. Several pathways involved in cytoskeleton maintenance and organization were found to be significantly enriched (p<0.05). Our data revealed altered phosphorylation of proteins associated with adherens junction remodeling and actin polymerization. Kinases such as protein kinase C iota type (PRKCI), mitogen‐activated protein kinase kinase kinase 1 (MAP3K1), tyrosine‐protein kinase BAZ1B (BAZ1B) and STE20 like kinase (SLK) were found to be hyperphosphorylated. Our study provides novel insights into signaling perturbations associated with chronic arsenic exposure in human skin keratinocytes. All MS/MS data have been deposited to the ProteomeXchange with identifier PXD004868.     

计算机软件数据接口的应用分析

伴随计算机技术的不断发展进步,而各种不同的计算机软件由于开发商不同,其数据结构也各种各样,假如用户需要操作两套或者两套以上的软件,需要根据计算机提供对应的格式基础,按照计算机软件保留各种格式的数据结果,这样无形中就增加了用户的工作量,并且软件处理流程链也容易中断。而计算机软件的数据接口可以很好的解决这类问题。本文就针对计算机软件数据接口的应用进行分析研究。     

PowerBuilder的数据窗口打印技术

PowerBuilder作为比较流行的数据库前端开发工具,其显著的特点就是强大的数据窗口（Data Window）技术．利用此技术可生成各种复杂的报表,但在打印出各种特殊格式要求的报表时,往往需要编程实现,打印效果需要反复调整并打印输出,效率低下,因此利用数据窗口设计通用的报表打印控件很有必要     

流式文档排版效果自动化测试方法

为提高流式文档格式的标准符合性和兼容性测试效率,面向基于XML的流式文档,提出了一种排版效果自动化测试方法。结合流式文档特点和图像分析技术,通过逆向关联分析和排版格式分析实现对流式文档的排版效果分析,通过统计和层次分析法构建评价模型,对分析结果进行评价,给出量化的测试结果。相比人工测试可以大幅提高测试的自动化程度和准确率,对于文档格式标准的研制以及提高文字处理软件的质量均有重要的作用。     

Enhanced kinase sensing by radio-phosphorylation and oriented immobilization of a GBP-linked kinase substrate on a gold surface

A new strategy for enhanced sensing of kinases was developed using radio-phosphorylation and a polypeptide with an affinity for gold that was linked directly to kinase substrates. Gold-binding polypeptide (GBP)-linked GST-fusion substrates were efficiently immobilized on gold-coated slides, which is highly desirable for protein chips. The novel fusion substrates permitted oriented substrate immobilization on gold-coated slides, thereby enhancing the sensitivity of kinase assays. Thus, the proposed strategy is applicable to the development of protein microarrays and microfluidic lab-on-a-chip (LOC) sensing the activities of protein kinases.     

New design of nucleotide excision repair (NER) inhibitors for combination cancer therapy

Many cancer chemotherapy agents act by targeting the DNA of cancer cells, causing substantial damage within their genome and causing them to undergo apoptosis. An effective DNA repair pathway in cancer cells can act in a reverse way by removing these drug-induced DNA lesions, allowing cancer cells to survive, grow and proliferate. In this context, DNA repair inhibitors opened a new avenue in cancer treatment, by blocking the DNA repair mechanisms from removing the chemotherapy-mediated DNA damage. In particular, the nucleotide excision repair (NER) involves more than thirty protein–protein interactions and removes DNA adducts caused by platinum-based chemotherapy. The excision repair cross-complementation group 1 (ERCC1)-xeroderma pigmentosum, complementation group A (XPA) protein (XPA–ERCC1) complex seems to be one of the most promising targets in this pathway. ERCC1 is over expressed in cancer cells and the only known cellular function so far for XPA is to recruit ERCC1 to the damaged point. Here, we build upon our recent advances in identifying inhibitors for this interaction and continue our efforts to rationally design more effective and potent regulators for the NER pathway. We employed in silico drug design techniques to: (1) identify compounds similar to the recently discovered inhibitors, but more effective at inhibiting the XPA–ERCC1 interactions, and (2) identify different scaffolds to develop novel lead compounds. Two known inhibitor structures have been used as starting points for two ligand/structure-hybrid virtual screening approaches. The findings described here form a milestone in discovering novel inhibitors for the NER pathway aiming at improving the efficacy of current platinum-based therapy, by modulating the XPA–ERCC1 interaction.     

Efficient mapping algorithms for a class of hierarchical systems

Proposes techniques for mapping application algorithms onto a class of hierarchically structured parallel computing systems. Multiprocessors of this type are capable of efficiently solving a variety of scientific problems because they can efficiently implement both local and global operations for data in a two-dimensional array format. Among the set of candidate application domains, low-level and intermediate-level image processing and computer vision (IPCV) are characterized by high-performance requirements. Emphasis is given to IPCV algorithms. The importance of the mapping techniques stems from the fact that the current technology cannot be used to build cost-effective and efficient systems composed of very large numbers of processors, so the performance of various systems of lower cost should be investigated. Both analytical and simulation results prove the effectiveness and efficiency of the proposed mapping techniques     

数字水印技术在视频压缩格式中的研究与实现

随着信息技术的发展,数字媒体得到了空前广泛的应用,版权维护也成了数字出版商所关心的最大问题。基于当前数字版权维护中水印技术的发展现状和数字水印技术在视频压缩格式中的应用研究,提出一种以MPEG-4视频水印嵌入的方案,以期对相关数字版权保护工作提供一定的参考价值。     

Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors

Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC₅₀ values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN inhibitors. In conclusion, we report an exhaustively validated receptor model if IN that is useful for the efficient screening of large chemical compounds databases in the search of potent HIV-1 IN inhibitors.