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Current Protocols in Pharmacology最新文献

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Binding Assays for Bromodomain Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory Disease 溴结构域蛋白的结合分析:在肿瘤和炎症性疾病药物发现中的应用
Current Protocols in Pharmacology Pub Date : 2018-04-02 DOI: 10.1002/cpph.35
Nina I. Zolotarjova, Richard Wynn
{"title":"Binding Assays for Bromodomain Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory Disease","authors":"Nina I. Zolotarjova,&nbsp;Richard Wynn","doi":"10.1002/cpph.35","DOIUrl":"10.1002/cpph.35","url":null,"abstract":"<p>Bromodomains are protein domains that recognize acetylated lysine residues and are important for recruiting a large number of protein and multiprotein complexes to sites of lysine acetylation. They play an important role in chromatin biology and are popular targets for drug discovery. Compound screening in this area requires the use of biochemical assays to assess the binding potency of potential drug candidates. Foremost among the efforts to target bromodomains are those aimed at identifying compounds that interact with the bromodomain and extra-terminal domain (BET) family of bromodomain-containing proteins (BRD2, BRD3, BRD4, and BRDT). Inhibitors of these proteins are under clinical development for a large variety of oncologic indications. Described in this unit are several assays to assess the binding potency and selectivity within the BET protein family. Included are AlphaScreen, fluorescence polarization, and thermal shift assays. The strengths and weaknesses of each assay are discussed. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.35","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36339293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Three Decades of Chloride Intracellular Channel Proteins: From Organelle to Organ Physiology 三十年的氯离子胞内通道蛋白:从细胞器到器官生理学
Current Protocols in Pharmacology Pub Date : 2018-04-02 DOI: 10.1002/cpph.36
Shubha Gururaja Rao, Devasena Ponnalagu, Neel J. Patel, Harpreet Singh
{"title":"Three Decades of Chloride Intracellular Channel Proteins: From Organelle to Organ Physiology","authors":"Shubha Gururaja Rao,&nbsp;Devasena Ponnalagu,&nbsp;Neel J. Patel,&nbsp;Harpreet Singh","doi":"10.1002/cpph.36","DOIUrl":"10.1002/cpph.36","url":null,"abstract":"<p>Intracellular organelles are membranous structures central for maintaining cellular physiology and the overall health of the cell. To maintain cellular function, intracellular organelles are required to tightly regulate their ionic homeostasis. Any imbalance in ionic concentrations can disrupt energy production (mitochondria), protein degradation (lysosomes), DNA replication (nucleus), or cellular signaling (endoplasmic reticulum). Ionic homeostasis is also important for volume regulation of intracellular organelles and is maintained by cation and anion channels as well as transporters. One of the major classes of ion channels predominantly localized to intracellular membranes is chloride intracellular channel proteins (CLICs). They are non-canonical ion channels with six homologs in mammals, existing as either soluble or integral membrane protein forms, with dual functions as enzymes and channels. Provided in this overview is a brief introduction to CLICs, and a summary of recent information on their localization, biophysical properties, and physiological roles. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.36","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36338198","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 39
Quantitative Intracerebral Microdialysis Studies to Determine Unbound Extracellular Fluid Drug Concentrations in Discrete Areas of the Brain 定量脑内微透析研究以确定脑离散区域未结合的细胞外液药物浓度
Current Protocols in Pharmacology Pub Date : 2018-04-02 DOI: 10.1002/cpph.38
Matthew R. Durk
{"title":"Quantitative Intracerebral Microdialysis Studies to Determine Unbound Extracellular Fluid Drug Concentrations in Discrete Areas of the Brain","authors":"Matthew R. Durk","doi":"10.1002/cpph.38","DOIUrl":"10.1002/cpph.38","url":null,"abstract":"<p>Intracerebral microdialysis is a powerful technique for quantifying unbound brain extracellular fluid concentrations of drugs and drug candidates over time in live, conscious, freely moving animals. The results provide crucial information on the brain penetrance of the administered agent. This unit details the surgical procedures, study planning and execution, and data analysis required for a brain microdialysis study following intravenous infusion of a test substance to steady state. The accumulated data makes possible the determination of the area under the concentration–time curve ratio and steady-state concentration ratio for unbound compound in rat brain extracellular fluid as compared to that in plasma (K<sub>pu,u</sub>). In vitro determination of the compound recovery from the probe, a critical parameter in a microdialysis study, is considered as well. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.38","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36338201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
In Vitro Enzyme Assays for JmjC-Domain-Containing Lysine Histone Demethylases (JmjC-KDMs) 含jmjc结构域赖氨酸组蛋白去甲基化酶(JmjC-KDMs)的体外酶分析
Current Protocols in Pharmacology Pub Date : 2018-04-02 DOI: 10.1002/cpph.34
Hanna Tarhonskaya, Anthony Tumber, Akane Kawamura, Christopher J. Schofield
{"title":"In Vitro Enzyme Assays for JmjC-Domain-Containing Lysine Histone Demethylases (JmjC-KDMs)","authors":"Hanna Tarhonskaya,&nbsp;Anthony Tumber,&nbsp;Akane Kawamura,&nbsp;Christopher J. Schofield","doi":"10.1002/cpph.34","DOIUrl":"10.1002/cpph.34","url":null,"abstract":"<p>Histone modifications, including lysine methylation marks on histone tails, modulate the accessibility of genes for transcription. Changes in histone tail methylation patterns can cause transcriptional activation or repression. The dynamic regulation of lysine methylation patterns is enabled by two distinct groups of enzymes: histone methyltransferases (KMTs) and demethylases (KDMs). The Jumonji C (JmjC) domain–containing lysine histone demethylases (JmjC-KDMs) alter the methylation levels of histone tails by removing tri-, di-, or mono-methylation marks. Because JmjC-KDMs activities are dysfunctional in cancer and other clinical conditions, they are targets for drug discovery. Efforts are underway to develop high-throughput assays capable of identifying selective, small-molecule inhibitors of KDMs. Detailed in this unit are protocols for mass spectrometry–based and formaldehyde dehydrogenase–coupled enzyme-based assays that can be used to identify inhibitors of JmjC-KDMs. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.34","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36339292","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Overview of Experimental Models of the Blood-Brain Barrier in CNS Drug Discovery 中枢神经系统药物开发中血脑屏障实验模型综述
Current Protocols in Pharmacology Pub Date : 2018-02-16 DOI: 10.1002/0471141755.ph0715s62
Alan M. Palmer, Mohammad S. Alavijeh
{"title":"Overview of Experimental Models of the Blood-Brain Barrier in CNS Drug Discovery","authors":"Alan M. Palmer,&nbsp;Mohammad S. Alavijeh","doi":"10.1002/0471141755.ph0715s62","DOIUrl":"10.1002/0471141755.ph0715s62","url":null,"abstract":"<p>The blood-brain barrier (BBB) is a physical and metabolic entity that isolates the brain from the systemic circulation. The barrier consists of tight junctions between endothelial cells that contain egress transporters and catabolic enzymes. To cross the BBB, a drug must possess the appropriate physicochemical properties to achieve a sufficient time-concentration profile in brain interstitial fluid (ISF). In this overview, we review techniques to measure BBB permeation, which is evidenced by the free concentration of compound in brain ISF over time. We consider a number of measurement techniques, including in vivo microdialysis and brain receptor occupancy following perfusion. Consideration is also given to the endothelial and nonendothelial cell systems used to assess both the BBB permeation of a test compound and its interactions with egress transporters, and computer models employed for predicting passive permeation and the probability of interactions with BBB transporters. <i>Curr. Protoc. Pharmacol</i>. 62:7.15.1-7.15.30. © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"62 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471141755.ph0715s62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32103706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 21
Characterization of P1 (Adenosine) Purinoceptors P1(腺苷)嘌呤受体的表征
Current Protocols in Pharmacology Pub Date : 2018-02-16 DOI: 10.1002/0471141755.ph0109s62
Michael F. Jarvis
{"title":"Characterization of P1 (Adenosine) Purinoceptors","authors":"Michael F. Jarvis","doi":"10.1002/0471141755.ph0109s62","DOIUrl":"10.1002/0471141755.ph0109s62","url":null,"abstract":"<p>The purine nucleoside adenosine (ADO) is an important modulator of cellular function in mammalian tissues, modulating cellular function and neuronal excitability via interactions with different cell surface receptor subtypes that are heterogeneously distributed in both the mammalian CNS and peripheral tissues. Four ADO receptor subtypes have been cloned and characterized. Described in this unit are three radioligand binding assays for pharmacological characterization of the high-affinity ADO receptor subtypes A<sub>1</sub>, A<sub>2A</sub>, and A<sub>3</sub> receptors. Pharmacological characterization of the low-affinity A<sub>2B</sub> receptor has been enabled by the use of tritiated xanthine PSB-603. Because receptor localization is an important criterion for differentiation of receptor subtypes, a support protocol that describes the methodology for the localization of ADO receptors in rat brain tissue using autoradiography is also included. <i>Curr. Protoc. Pharmacol</i>. 62:1.9.1-1.9.16. © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"62 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471141755.ph0109s62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32103702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 4
Utility and Applications of Orthotopic Models of Human Non-Small Cell Lung Cancer (NSCLC) for the Evaluation of Novel and Emerging Cancer Therapeutics 人类非小细胞肺癌(NSCLC)原位模型在评估新型和新兴癌症治疗方法中的效用和应用
Current Protocols in Pharmacology Pub Date : 2018-02-16 DOI: 10.1002/0471141755.ph1427s62
Verline Justilien, Alan P. Fields
{"title":"Utility and Applications of Orthotopic Models of Human Non-Small Cell Lung Cancer (NSCLC) for the Evaluation of Novel and Emerging Cancer Therapeutics","authors":"Verline Justilien,&nbsp;Alan P. Fields","doi":"10.1002/0471141755.ph1427s62","DOIUrl":"10.1002/0471141755.ph1427s62","url":null,"abstract":"<p>Lung cancer is a leading cause of cancer deaths worldwide. Despite advances in chemotherapy, radiation therapy, and surgery, lung cancer continues to have a low 5-year survival rate, highlighting a dire need for more effective means of prevention, diagnosis, prognosis, and treatment. Mouse models that recapitulate the clinical features of advanced human lung cancer are critical for testing novel therapeutic approaches. This unit describes a highly reproducible, easy-to-establish orthotopic murine model of lung cancer, provides methods for in vivo imaging and monitoring of tumor growth, and discusses the usefulness of this model for translational lung cancer research and the development of therapeutic strategies. <i>Curr. Protoc. Pharmacol</i>. 62:14.27.1-14.27.17. © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"62 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471141755.ph1427s62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32103705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 23
Harvesting, Isolation, and Functional Assessment of Primary Vagal Ganglia Cells 初代迷走神经节细胞的收集、分离和功能评估
Current Protocols in Pharmacology Pub Date : 2018-02-16 DOI: 10.1002/0471141755.ph1215s62
Eric Dubuis, Megan Grace, Michael A. Wortley, Mark A. Birrell, Maria G. Belvisi
{"title":"Harvesting, Isolation, and Functional Assessment of Primary Vagal Ganglia Cells","authors":"Eric Dubuis,&nbsp;Megan Grace,&nbsp;Michael A. Wortley,&nbsp;Mark A. Birrell,&nbsp;Maria G. Belvisi","doi":"10.1002/0471141755.ph1215s62","DOIUrl":"10.1002/0471141755.ph1215s62","url":null,"abstract":"<p>Airway sensory nerves play an important defensive role in the lungs, being central in mediating protective responses like cough and bronchoconstriction. In some cases, these responses become excessive, hypersensitive, and deleterious. Understanding the normal function of airway nerves and phenotype changes associated with disease will help in developing new therapeutics for treating chronic obstructive pulmonary disease and chronic cough. Guinea pigs, and to a lesser extent ferrets, are commonly employed for studying the cough reflex because they have a cough response similar to humans. While rats and mice do not exhibit a cough response, they do possess sensory nerves that respond to the same range of tussive stimuli as guinea pigs and humans. Described in this unit are protocols for harvesting guinea pig, mouse, and rat sensory nerve cell bodies to assess molecular and functional changes associated with pulmonary disease, and to identify new targets for therapeutic intervention. <i>Curr. Protoc. Pharmacol</i>. 62:12.15.1-12.15.27. © 2013 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"62 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471141755.ph1215s62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32103704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT) 多巴胺转运体(DAT)功能表征的体外实验
Current Protocols in Pharmacology Pub Date : 2018-02-13 DOI: 10.1002/cpph.33
Shaili Aggarwal, Ole V. Mortensen
{"title":"In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT)","authors":"Shaili Aggarwal,&nbsp;Ole V. Mortensen","doi":"10.1002/cpph.33","DOIUrl":"10.1002/cpph.33","url":null,"abstract":"<p>Detailed in this unit are protocols for studying the <i>in vitro</i> uptake of dopamine (DA) as a means for defining the functional characteristics of dopamine transporters. All assays are performed using commercially available cell lines that transiently express the transporter under investigation. The three main assays provided are: a kinetic assay to calculate the affinity (<i>K</i><sub>M</sub>) and maximal velocity (<i>V</i><sub>max</sub>) of radiolabeled DA uptake into cells; concentration-response assays to measure the potencies (IC<sub>50</sub>/<i>K</i><sub>i</sub> values) of test compounds as transport inhibitors; and an efflux assay to assess the ability and potency (EC<sub>50</sub>) of a ligand to elicit reverse transport of DA accumulated in the cell. Although the methods are described using DAT and its ligands, the same procedure can be employed for studying serotonin and norepinephrine transporters as well. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.33","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35674748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
In Vitro Histone Acetylation Assay 体外组蛋白乙酰化试验
Current Protocols in Pharmacology Pub Date : 2018-02-13 DOI: 10.1002/cpph.31
James A.L. Brown
{"title":"In Vitro Histone Acetylation Assay","authors":"James A.L. Brown","doi":"10.1002/cpph.31","DOIUrl":"10.1002/cpph.31","url":null,"abstract":"<p>Acetylation is a core cellular process involved in maintaining genomic integrity, gene regulation, and metabolism. Histone acetyltransferases (HATs) are an enzyme family that regulates these processes by catalyzing the transfer of an acetyl moiety onto target proteins. Perturbations of cellular acetylation profiles have been associated with a variety of disease states, including cancer. Changes in acetylation profiles can be achieved by mechanisms associated with acetyltransferases, such as gene down-regulation or alterations in the activity of key acetyltransferase enzymes. An important set of tools for quantifying enzyme activity are in vitro histone acetylation assays, using either endogenous or tagged overexpressed proteins. Detailed in this unit is an in vitro acetylation assay used to quantify HAT activity. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.31","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35674751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
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