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Current protocols in chemical biology最新文献

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Early-Stage Formulation Considerations 前期配方考虑
Current protocols in chemical biology Pub Date : 2018-02-13 DOI: 10.1002/cpch.32
Robert W. Lee, Mark Mitchnick
{"title":"Early-Stage Formulation Considerations","authors":"Robert W. Lee,&nbsp;Mark Mitchnick","doi":"10.1002/cpch.32","DOIUrl":"10.1002/cpch.32","url":null,"abstract":"<p>When a drug candidate—i.e., a new chemical entity (NCE) or new molecular entity (NME)—is discovered, there is a requirement to identify a vehicle for <i>in vitro</i> and/or <i>in vivo</i> evaluation to assess the activity and/or toxicity of the compound (here we refer to the biologically active compound as the active pharmaceutical ingredient: API). Ideally, this vehicle will not impart any biological activity or any toxicity that would mask or confound the effects of the API. At this early stage in development, and given the high attrition rates of drug candidates in discovery, it does not make sense to fully characterize the API—speed and cost are generally the driving factors. This chapter provides guidance for the development of early-stage test articles (i.e., drug products containing APIs intended to be used for the <i>in vitro</i> and/or <i>in vivo</i> evaluation) and not necessarily formulations that are intended to progress into clinical evaluation. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.32","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35657096","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
The In Situ Enzymatic Screening (ISES) Approach to Reaction Discovery and Catalyst Identification 原位酶筛选(ISES)方法用于反应发现和催化剂鉴定。
Current protocols in chemical biology Pub Date : 2018-02-13 DOI: 10.1002/cpch.30
Robert A. Swyka, David B. Berkowitz
{"title":"The In Situ Enzymatic Screening (ISES) Approach to Reaction Discovery and Catalyst Identification","authors":"Robert A. Swyka,&nbsp;David B. Berkowitz","doi":"10.1002/cpch.30","DOIUrl":"10.1002/cpch.30","url":null,"abstract":"<p>The importance of discovering new chemical transformations and/or optimizing catalytic combinations has led to a flurry of activity in reaction screening. The <i>in situ</i> enzymatic screening (ISES) approach described here utilizes biological tools (enzymes/cofactors) to advance chemistry. The protocol interfaces an organic reaction layer with an adjacent aqueous layer containing reporting enzymes that act upon the organic reaction product, giving rise to a spectroscopic signal. ISES allows the experimentalist to rapidly glean information on the relative rates of a set of parallel organic/organometallic reactions under investigation, without the need to quench the reactions or draw aliquots. In certain cases, the real-time enzymatic readout also provides information on sense and magnitude of enantioselectivity and substrate specificity. This article contains protocols for single-well (relative rate) and double-well (relative rate/enantiomeric excess) ISES, in addition to a colorimetric ISES protocol and a miniaturized double-well procedure. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.30","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35657094","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
In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target 小分子靶标DNA适体的体外选择与鉴定
Current protocols in chemical biology Pub Date : 2018-02-13 DOI: 10.1002/cpch.28
Annamaria Ruscito, Erin M. McConnell, Anna Koudrina, Ranganathan Velu, Christopher Mattice, Vernon Hunt, Maureen McKeague, Maria C. DeRosa
{"title":"In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target","authors":"Annamaria Ruscito,&nbsp;Erin M. McConnell,&nbsp;Anna Koudrina,&nbsp;Ranganathan Velu,&nbsp;Christopher Mattice,&nbsp;Vernon Hunt,&nbsp;Maureen McKeague,&nbsp;Maria C. DeRosa","doi":"10.1002/cpch.28","DOIUrl":"10.1002/cpch.28","url":null,"abstract":"<p>Aptamers, synthetic oligonucleotide-based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization of aptamers for small molecule targets have resulted in their underrepresentation, despite the need for small molecule detection in fields such as medicine, the environment, and agriculture. This protocol describes the steps in the selection, sequencing, affinity characterization, and truncation of DNA aptamers that are specific for small molecule targets. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.28","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35657009","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: 18
In Vivo Delivery of Nanoparticles into Plant Leaves 纳米颗粒在植物叶片中的体内传递
Current protocols in chemical biology Pub Date : 2018-02-13 DOI: 10.1002/cpch.29
Honghong Wu, Israel Santana, Joshua Dansie, Juan P. Giraldo
{"title":"In Vivo Delivery of Nanoparticles into Plant Leaves","authors":"Honghong Wu,&nbsp;Israel Santana,&nbsp;Joshua Dansie,&nbsp;Juan P. Giraldo","doi":"10.1002/cpch.29","DOIUrl":"10.1002/cpch.29","url":null,"abstract":"<p>Plant nanobiotechnology is an interdisciplinary field at the interface of nanotechnology and plant biology that aims to utilize nanomaterials as tools to study, augment or impart novel plant functions. The delivery of nanoparticles to plants <i>in vivo</i> is a key initial step to investigate plant nanoparticle interactions and the impact of nanoparticles on plant function. Quantum dots are smaller than plant cell wall pores, have versatile surface chemistry, bright fluorescence and do not photobleach, making them ideal for the study of nanoparticle uptake, transport, and distribution in plants by widely available confocal microscopy tools. Herein, we describe three different methods for quantum dot delivery into leaves of living plants: leaf lamina infiltration, whole shoot vacuum infiltration, and root to leaf translocation. These methods can be potentially extended to other nanoparticles, including nanosensors and drug delivery nanoparticles. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.29","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35657095","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
Evolving Aptamers with Unnatural Base Pairs 进化具有非自然碱基对的适体
Current protocols in chemical biology Pub Date : 2018-02-13 DOI: 10.1002/cpch.31
Michiko Kimoto, Ken-ichiro Matsunaga, Ichiro Hirao
{"title":"Evolving Aptamers with Unnatural Base Pairs","authors":"Michiko Kimoto,&nbsp;Ken-ichiro Matsunaga,&nbsp;Ichiro Hirao","doi":"10.1002/cpch.31","DOIUrl":"10.1002/cpch.31","url":null,"abstract":"<p>A novel technology, genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technology. A hydrophobic unnatural base pair, Ds–Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (<span>S</span>ystematic <span>E</span>volution of <span>L</span>igands by <span>EX</span>ponential enrichment) to generate DNA aptamers that bind to targets. A few Ds bases in DNA aptamers significantly increase the binding affinity to targets, enabling the use of DNA aptamers as an alternative to antibodies. This protocol describes the ExSELEX (genetic alphabet <span>Ex</span>pansion for <span>SELEX</span>) method to generate Ds-containing DNA aptamers. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.31","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35657010","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: 9
Characterizing the Nano-Bio Interface Using Microscopic Techniques: Imaging the Cell System is Just as Important as Imaging the Nanoparticle System. 利用显微技术表征纳米生物界面:细胞系统成像与纳米颗粒系统成像同样重要。
Current protocols in chemical biology Pub Date : 2017-09-14 DOI: 10.1002/cpch.26
Christie M Sayes, Henry Lujan
{"title":"Characterizing the Nano-Bio Interface Using Microscopic Techniques: Imaging the Cell System is Just as Important as Imaging the Nanoparticle System.","authors":"Christie M Sayes,&nbsp;Henry Lujan","doi":"10.1002/cpch.26","DOIUrl":"https://doi.org/10.1002/cpch.26","url":null,"abstract":"<p><p>The rapid growth of nanotechnology and its industries has elevated the need to understand the risks associated with handling, using, and disposing of nanomaterials. These risks can be assessed through exposure measurement and hazard identification. One of the common challenges associated with quantifying nanomaterials in products, waste, humans, or the environment is the lack of tools available to measure concentration. The ability of refined tools and techniques to qualitatively detect nanoparticles in complex matrices has been demonstrated. For biological and ecological tests systems, dose can be represented as initial concentration in the applied matrix, concentration administered during the route of exposure, concentration at the target organ, and intake concentration at the cellular level. Each of these concentration measurements requires different sets of tools to perform accurate analyses. Advances in microscopy techniques provide new opportunities for reporting observations occurring at the interaction of a nanoparticle with a biomolecular entity of similar size within a biological test(s) system. This protocol outlines the steps to image nanomaterials within cell-based systems. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 3","pages":"213-231"},"PeriodicalIF":0.0,"publicationDate":"2017-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.26","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35352411","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
A Guide to Quantitative Biomarker Assay Development using Whispering Gallery Mode Biosensors. 使用低语画廊模式生物传感器的定量生物标志物测定开发指南。
Current protocols in chemical biology Pub Date : 2017-09-14 DOI: 10.1002/cpch.23
Heather M Robison, Ryan C Bailey
{"title":"A Guide to Quantitative Biomarker Assay Development using Whispering Gallery Mode Biosensors.","authors":"Heather M Robison,&nbsp;Ryan C Bailey","doi":"10.1002/cpch.23","DOIUrl":"https://doi.org/10.1002/cpch.23","url":null,"abstract":"<p><p>Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target-specific capture agents, analyte binding can be measured with very low limits of detection. There are many geometric manifestations of WGM sensors, with chip-integrated silicon photonic devices first commercialized because of the robust, wafer-scale device fabrication, facile optical interrogation, and amenability to the creation of multiplexed sensor arrays. Using these arrays, a number of biomolecular targets have been detected in both label-free and label-enhanced assay formats. For example, sub-picomolar detection limits for multiple cytokines were achieved using an enzymatically enhanced sandwich immunoassay that showed high analyte specificity suitable for detection in complex, clinical matrices. This protocol describes a generalizable approach for the development of quantitative, multiplexed immunoassays using silicon photonic microrings as an example WGM platform. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 3","pages":"158-173"},"PeriodicalIF":0.0,"publicationDate":"2017-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.23","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35352416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 16
High-Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor. 使用UbFluor高通量筛选HECT E3泛素连接酶。
Current protocols in chemical biology Pub Date : 2017-09-14 DOI: 10.1002/cpch.24
Peter K Foote, David T Krist, Alexander V Statsyuk
{"title":"High-Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor.","authors":"Peter K Foote,&nbsp;David T Krist,&nbsp;Alexander V Statsyuk","doi":"10.1002/cpch.24","DOIUrl":"https://doi.org/10.1002/cpch.24","url":null,"abstract":"<p><p>HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases. The release of fluorophore upon transthiolation allows fluorescence polarization detection of HECT E3 activity. In the presence of inhibitors, HECT E3 activity is ablated, and thus no reaction and no change in FP are observed. This assay has been adapted for high-throughput screening of small molecules against HECT E3 ligases, and its utility has been proven in the discovery of HECT E3 ligase inhibitors. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 3","pages":"174-195"},"PeriodicalIF":0.0,"publicationDate":"2017-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.24","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35352415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
Optimized PEI-based Transfection Method for Transient Transfection and Lentiviral Production. 基于pei的瞬时转染和慢病毒生产方法优化。
Current protocols in chemical biology Pub Date : 2017-09-14 DOI: 10.1002/cpch.25
Shaozhe Yang, Xiaoling Zhou, Rongxiang Li, Xiuhong Fu, Pingnan Sun
{"title":"Optimized PEI-based Transfection Method for Transient Transfection and Lentiviral Production.","authors":"Shaozhe Yang,&nbsp;Xiaoling Zhou,&nbsp;Rongxiang Li,&nbsp;Xiuhong Fu,&nbsp;Pingnan Sun","doi":"10.1002/cpch.25","DOIUrl":"https://doi.org/10.1002/cpch.25","url":null,"abstract":"<p><p>Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI-based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI-based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI-based transfection has been optimized to improve its efficiency, reproducibility, and consistency. This protocol outlines the steps for ordinary transient transfection and lentiviral production using PEI. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 3","pages":"147-157"},"PeriodicalIF":0.0,"publicationDate":"2017-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.25","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35352413","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: 28
Overview of Methods and Strategies for Conducting Virtual Small Molecule Screening 虚拟小分子筛选方法与策略综述
Current protocols in chemical biology Pub Date : 2017-09-14 DOI: 10.1002/cpch.27
Xavier Fradera, Kerim Babaoglu
{"title":"Overview of Methods and Strategies for Conducting Virtual Small Molecule Screening","authors":"Xavier Fradera,&nbsp;Kerim Babaoglu","doi":"10.1002/cpch.27","DOIUrl":"10.1002/cpch.27","url":null,"abstract":"<p>Virtual screening (VS) in the context of drug discovery is the use of computational methods to discover novel ligands with a desired biological activity from within a larger collection of molecules. These techniques have been in use for many years, there is a wide range of methodologies available, and many successful applications have been reported in the literature. VS is often used as an alternative or a complement to High-throughput screening (HTS) or other methods to identify ligands for target validation or medicinal chemistry projects. This unit does not present an exhaustive review of available methods, or document specific instructions on use of individual software packages. Rather, a general overview of the methods available are presented and general strategies are described for VS based on accepted practices and the authors’ experience as computational chemists in an industrial research laboratory. First, the most common methods available for VS are reviewed, categorized as either receptor- or ligand-based. Subsequently, strategic considerations are presented for choosing a VS method, or a combination of methods, as well as the necessary steps to prepare, run, and analyze a VS campaign. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.27","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35352417","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: 40
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