New targets for an old drug

Methotrexate has been a clinical agent used in cancer, immunosuppression, rheumatoid arthritis, and other highly proliferative diseases for many years, yet its underlying molecular mechanism of action in these therapeutic areas is still unclear. We have previously reported using a chemical proteomics technique on several other potential pharmacodynamic targets of methotrexate. Here, using a frontal affinity chromatography with mass spectrometry detection, we confirm one of these targets, hypoxanthine-guanine amidophosphoribosyltransferase, as a true binder of methotrexate with a K_d of 4.2 μM. These results complement and confirm our recent study, but more importantly, shed light into the mechanism of action of methotrexate in oncology and other highly proliferative diseases and may help explain some unaccounted for effects of this drug. For example, despite the fact that DNA salvage pathway enzymes are highly active, methotrexate can be effective if it only targets enzymes of the de novo pathway.     

Proteomic strategies in the search for novel pancreatic cancer biomarkers and drug targets: recent advances and clinical impact

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers; despite a low incidence rate it is the fourth leading cause of cancer-related death in the world. Improvement of the diagnosis, prognosis and treatment remains the main focus of pancreatic cancer research. Rapid developments in proteomic technologies has improved our understanding of the pancreatic cancer proteome. Here, the authors summarise the recent proteomic strategies undertaken in the search for: novel biomarkers for early diagnosis, pancreatic cancer-specific proteins which may be used for novel targeted therapies and proteins which may be useful for monitoring disease progression post-therapy. Recent advances and findings discussed here provide great promise of having a significant clinical impact and improving the outcome of patients with this malignancy.     

New derivatives of eremomycin containing 15N or F atoms for NMR study

New semisynthetic derivatives of eremomycin containing ¹⁵N or F atoms were obtained for studying the antibiotic-target interaction in intact cells of Gram-positive bacteria by REDOR NMR method. Interaction of the terminal carboxyl group of amino acid 7 (AA7) of eremomycin with amines in the presence of PyBOP and TBTU reagents resulted in the corresponding [¹⁵N]-amide, p-fluorobenzylamide, p-fluorophenylpiperazide, and 6-N-(p-fluorobenzyl)aminohexylamide. A selective method of [¹⁵N]-amidation of carboxyl group of amino acid 3 (AA3) of carboxyeremomycin was developed, and the amide of eremomycin containing [¹⁵N] in AA3 amide group near the antibiotic binding pocket was obtained. Carboxyeremomycin bisamides substituted at AA3 and AA7 and containing two atoms of [¹⁵N] or F were obtained from carboxyeremomycin and [¹⁵N]NH₄Cl or the corresponding p-fluorobenzylamine hydrochloride in the presence of PyBOP at pH ～8. The Edman degradation of eremomycin p-fluorobenzylamide gave de-(D-MeLeu)-eremomycin p-fluorobenzylamide, a hexapeptide derivative incapable of the antibiotic binding with-D-Ala-D-Ala fragment of growing cell wall peptidoglycan. Among the compounds studied, carboxyeremomycin bis-p-fluorobenzylamide showed the best activity against both the glycopeptides-sensitive and glycopeptides-resistant strains of staphylococci and enterococci.     

Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway

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Use of proteomics for the identification of novel drug targets in brain diseases

In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases.     

New therapeutic targets for the treatment of high‐risk neuroblastoma

High‐risk neuroblastoma remains a major problem in pediatric oncology, accounting for 15% of childhood cancer deaths. Although incremental improvements in outcome have been achieved with the intensification of conventional chemotherapy agents and the addition of 13‐cis‐retinoic acid, only one‐third of children with high‐risk disease are expected to be long‐term survivors when treated with current regimens. In addition, the cost of cure can be quite high, as surviving children remain at risk for additional health problems related to long‐term toxicities of treatment. Further advances in therapy will require the targeting of tumor cells in a more selective and efficient way so that survival can be improved without substantially increasing toxicity. In this review we summarize ongoing clinical trials and highlight new developments in our understanding of the molecular biology of neuroblastoma, emphasizing potential targets or pathways that may be exploitable therapeutically. J. Cell. Biochem. 107: 46–57, 2009. © 2009 Wiley‐Liss, Inc.     

Label-free quantitative proteomic analysis of the inhibitory activities of juglone against translation and energy metabolism in Escherichia coli

Plant-derived antimicrobial agents have received increasing attention owing to their potential to control pathogens and excellent efficacy despite the growing prevalence of antibiotic resistance. However, the antibacterial mechanism of juglone, a traditional medicine used to cure skin infections, is still unclear. Therefore, in this study, in order to elucidate the mechanisms underlying the antibacterial activity of juglone, label-free quantitative proteomic technology was applied for analysis of the 417 proteins that were differentially expressed in Escherichia coli after treatment with juglone at one-half of the minimum inhibitory concentration. Gene ontology enrichment analysis of differentially expressed proteins suggested that juglone effectively repressed the expression of dehydrogenase and cytochrome oxidase, indicating that energy generation was blocked. Additionally, juglone induced RNA formation and ribosome assembly, resulting in inhibition of translation. This is the first study to adopt a proteomic approach to investigate the antibacterial mechanism of action of juglone against E. coli.     

Multi-omic Characterization of the Mode of Action of a Potent New Antimalarial Compound,JPC-3210, Against Plasmodium falciparum

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Highlights

• •Multi-omics analysis on mode of action of novel antimalarial, JPC-3210
• •JPC-3210 has rapid parasite killing kinetics.
• •Metabolomics and peptidomics demonstrated JPC-3210 inhibits hemoglobin digestion.
• •Proteomics demonstrated JPC-3210 enriches for translation regulation proteins.

     

基于细胞热漂移测定(CETSA)技术鉴定活细胞内药物靶标的标准操作流程

【目的】细胞热漂移测定(cell thermal shift assay, CETSA)技术是一种检测细胞内药物(配体)和蛋白质(靶标)相互作用的技术,原理是当蛋白质结合药物后,其热稳定性会发生变化,通过测定这种变化去鉴定药物和蛋白之间的相互作用。本研究以治疗多发性骨髓瘤的靶向药帕比司他(panobinostat)为例,建立基于蛋白印迹杂交(Western blotting)和CETSA技术的药物靶蛋白鉴定的标准操作流程。【方法】首先用药物panobinostat处理培养的K562细胞,然后加热处理细胞、裂解细胞及提取可溶性蛋白,以及用抗靶蛋白的抗体经Western blotting定量可溶性蛋白。【结果】经Western blotting定量及曲线拟合,成功得到3个蛋白——组蛋白去乙酰化酶(histone deacetylase,HDAC1)、人突触蛋白(humansyntaxin-4,STX4)以及四三肽重复结构域(tetratricopeptiderepeat domain38,TTC38)随温度变化的热熔解曲线和恒定温度条件下的药物剂量反应曲线。【结论】HDAC1、STX4及T...     

疾病干预的新靶点:嘌呤与嘧啶受体

内源性核苷、核苷酸通过嘌呤与嘧啶受体(P受体),参与机体组织器官多种功能的调节。在肿瘤、细胞凋亡、局部缺血、伤口愈合、骨质疏松、药物毒性、炎症及痛觉等病理状态下,P受体的内源性配体核苷与核苷酸发挥保护作用,P1、P2受体及其受体亚型的选择性激动剂和桔抗剂具有宽广的临床应用前景。     

Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets

Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major setback. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA). We have evaluated this system for usefulness in deducing the MOA for three test compounds. To achieve this, we constructed a database with 21 known antimicrobials, which served as a comparison for grouping our unknown MOA compounds. Pearson correlation and Ward linkage calculations were used to generate a dendrogram that produced clustering largely by known MOA, although there were exceptions. Of the three unknown compounds, one was definitively placed as an antifolate. The second and third compounds' MOA were not clearly identified, likely because the unique MOA was not represented within the database. The availability of the database generated in this report for Staphylococcus aureus ATCC 29213 will increase the accessibility of this technique to other investigators. From our analysis, the Phenotype Microarray system can group compounds with clear MOA, but the distinction of unique or broadly acting MOA at this time is less clear.     

Functional elucidation of hypothetical proteins for their indispensable roles toward drug designing targets from Helicobacter pylori strain HPAG1

Helicobacter pylori is a flagellated and slow growing gram-negative bacterium that persistently infects about half of the entire world population. In present study, we examined the proteome of H. pylori strain HPAG1 for identification of key uncharacterized proteins toward their novel regulatory functions. The complete proteome of this strain consists of 1539 proteins, out of which 520 proteins are annotated as hypothetical. Based on the functional motifs in their primary sequences, we were able to classify 254 of these hypothetical proteins into 6 functional categories. Further, KEGG database was used to find the roles of these hypothetical proteins in several pathways and structural prediction was done by homology modeling methods. Thirty-three of these hypothetical proteins were found to have strong association in various pathways including signaling and defense mechanisms. We noted that 27 of these proteins are specific to H. pylori and can be selected for drug designing targets, based on their virulence and regulatory role. We were able to successfully model the 3D structures of three of these proteins: YP_626977.1, YP_626786.1, and YP_628146.1. The stability of these proteins was also validated using molecular dynamics simulations, and their possible role in the regulation of different pathways was explained. These novel annotations may contribute to the understanding of disease mechanism at molecular level and provide novel potential targets for designing new drugs against H. pylori strain HPAG1.     

抑郁症易感基因和抗抑郁药物靶点相关[]神经细胞类型及相互作用网络分析

基于抑郁症的全基因组关联分析研究(GWAS),对于获得的单核苷酸多态性位点(SNP)使用Haploreg软件进行基因注释,得到SNP注释的102个易感基因.。使用MAGMA软件对GWAS的汇总统计数据做基因水平的分析,获得了270个校正之后显著的基因,两者合并共得到320个抑郁症易感基因。通过药物数据库Drugbank获取133个抗抑郁药物靶点基因。使用EWCE包对抑郁症易感基因和抗抑郁药物靶点在三套脑组织单细胞测序数据中,分别进行神经细胞类型富集分析。结果发现大脑皮质的GABA神经元(抑制性神经元)和谷氨酸能神经元(兴奋性神经元)是抑郁症易感基因和抗抑郁药物靶点共同的神经元。这两种类型的神经细胞可能是抗抑郁药物与抑郁症易感基因相互作用的神经细胞,另外少突胶质前体细胞可能是抑郁症特有的易感神经细胞。使用Network Calculator软件构建网络并进行进行网络拓扑学参数分析。结果表明抑郁症易感基因与抗抑郁药物靶点组成了一个具有显著的相互连接的网络。本研究从单细胞层面揭示抑郁症的遗传机制,在网络层面为寻找新的抗抑郁药物靶点提供了一定的启示。     

《昆明-蒙特利尔全球生物多样性框架》核心目标与我国的保护行动建议

在中国作为主席国的引领下,联合国《生物多样性公约》(以下简称《公约》)第十五次缔约方大会(以下简称COP15)第二阶段会议通过了62项决定,特别是达成了以变革理论为基础的《昆明-蒙特利尔全球生物多样性框架》(以下简称《昆蒙框架》),为全球生物多样性治理擘画了新的蓝图。该文就《昆蒙框架》的三个核心目标——保护地“3030目标”、资源调动、遗传资源数字序列信息进行解读,对保障《昆蒙框架》落地的相关决议进行简要介绍,并就我国未来的保护行动提出了相关建议:(1)加强生物多样性保护的主流化;(2)进一步制定详细的保护计划,明确保护区域的范围、目的和管理措施,并落实实施计划的责任部门和具体措施;(3)根据框架目标的监测要求,制定可操作的指标体系和监测计划;(4)继续加强生物多样性保护的意识和教育,提高公众对生物多样性保护的认识和重视程度,促进全社会的可持续生产和可持续消费;(5)大力推进国际合作,在更大尺度上探索和促进基于自然的解决方案,寻找对自然产生正面、积极效果的经济和社会发展路径。     

基于网络药理学探讨丹参-丹皮配伍抗脑缺血损伤的作用机制

本文旨在通过网络药理学和分子对接方法探讨丹参-丹皮活性成分治疗脑卒中的潜在分子机制.首先基于中药系统药理学分析平台筛选丹参、丹皮的活性成分及其作用靶点,利用CTD、TTD和GeneCards数据库收集脑卒中相关靶点.然后将药物和疾病靶点取交集,借助STRING数据库获取靶点间相互作用关系,利用R语言的Cluster-P...     

Triclosan - an update

The discovery in 1998 that triclosan has a site-specific action in the bacterial cell as an inhibitor of NADH- or NADPH-dependent enoyl-acyl carrier protein reductase led to a lively debate in the scientific press. The thesis of this debate was that such a mode of action may allow triclosan to induce resistance and cross-resistance in bacterial cells. The debate last saw review in 2004, and this paper aims at updating our knowledge in this area, given recent research on the topic.     

Genome-wide identification and comprehensive analysis of Excretory/Secretory proteins in nematodes provide potential drug targets for parasite control

Nematodes are responsible for causing severe diseases in plants, humans and other animals. Infection is associated with the release of Excretory/Secretory (ES) proteins into host cytoplasm and interference with the host immune system which make them attractive targets for therapeutic use. The identification of ES proteins through bioinformatics approaches is cost- and time-effective and could be used for screening of potential targets for parasitic diseases for further experimental studies. Here, we identified and functionally annotated 93,949 ES proteins, in the genome of 73 nematodes using integration of various bioinformatics tools. 30.6% of ES proteins were found to be supported at RNA level. The predicted ES proteins, annotated by Gene Ontology terms, domains, metabolic pathways, proteases and enzyme class analysis were enriched in molecular functions of proteases, protease inhibitors, c-type lectin and hydrolases which are strongly associated with typical functions of ES proteins. We identified a total of 452 ES proteins from human and plant parasitic nematodes, homologues to DrugBank-approved targets and C. elegans RNA interference phenotype genes which could represent potential targets for parasite control and provide valuable resource for further experimental studies to understand host-pathogen interactions.     

The KDEL receptor: New functions for an old protein

The KDEL receptor is a seven-transmembrane-domain protein that was first described about 20 years ago. Its well-known function is to retrotransport chaperones from the Golgi complex to the endoplasmic reticulum. Recent studies, however, have suggested that the KDEL receptor has additional functions. Indeed, we have demonstrated that chaperone-bound KDEL receptor triggers the activation of Src family kinases on the Golgi complex. This activity is essential in the regulation of Golgi-to-plasma membrane transport. However, the identification of different KDEL receptor interactors that are inconsistent with these established functions opens the possibility of further receptor activities.