Name: drug repurposing hypothesis generation using the "re:fine drugs" system
Uploaded: 2016-12-11T15:00:00.000+00:00
Duration: 5 min 10 s
Description: Watch this Scientific Journal Video about Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System at JoVE.com

This work was partially supported by the National Institutes of Health (NIH) Clinical and Translational Science Awards (CTSA) Grant (UL1TR001070) to the Ohio State University's Center for Clinical and Translational Science (CCTS) and the National Library Of Medicine under Award Number T15LM011270.

1.基因查询发起，药物或疾病条款<ol><li>通过以下链接:http://drug-repurposing.nationwidechildrens.org:访问为"精药品RE"的主页。药（仿制药名），疾病（新病指示）或基因（官方HGNC基因符号）:由从任何以下三类进入搜索栏查询词开头。 </li><li>过滤搜索栏的功能，只包括"药品名称"，"新疾病适应症"，"基因符号"或"全部"类别。搜索栏包括查询条目自动填充功能。 </li><li>放在一个关键字，然后点击"搜索"按钮。排序通过以下任一列的结果表:"药品"，"注册指示"，"P值"，"P-值来调整"，"胜算比"，"研究"，"新标识"，"药品银行指示"，"MEDLINE文摘＃"，"CLI命令＃nical试验注册中心"，"势"，"SNP"，"基因"或"行动"。 </li><li>导航到高级搜索选项，以使药品信息功能。在"信息"栏中点击图标为一个特定的药物。观察一个网页，列出所有相应的信息，包括对价值关联，疾病名称，药品名称，基因信息（NCBI基因链接）药品的详细信息（DrugBank链接）。 </li></ol> 2.高级选项的探索<ol><li>点击位于页面的右侧的"高级"按钮，并提供多种选择，以进一步缩小的结果。高级搜索选项包括修改如下:药物，协会，疾病，潜在的基因和行动。 </li><li>出口通过点击页面右侧的"导出"按钮，结果表。点击以向下折叠高级搜索窗口中的"简单"按钮。&lt;/ LI&gt; <li>在高级选项"毒品"选项卡中，指定特定的药物适应症或额外的药名来筛选结果。 </li><li>在"关联"选项卡中，筛选的显着性水平P值，调整后的P值与FDR，影响大小（比值比），和/或研究型（GWAS，PheWAS或两者）。 </li><li>在"治未病"选项卡中，指定新的预测使用某种疾病的描述。 </li><li>下的"潜在"标签中，根据以下任一条件过滤结果:（i）药物指示是否被包含在DrugBank数据库，（ⅱ）数量MEDLINE摘要与药物的共现和疾病中，（ ⅲ）数量与药物的共现和疾病以及（iv）重新利用潜在ClinicalTrials.gov数据库条目。 注:重新为潜在选项描述了发现的新颖性:（一）已知:关系已存在于数据库DrugBank，（二）大力支持:一些支持在临床试验注册和MEDLINE摘要;（三）可能是:一些支持在任何临床试验注册表或MEDLINE摘要及（iv）小说:在临床试验注册也不在MEDLINE摘要没有证据。 </li><li>在"基因"标签，输入SNP标识符或基因符号，以滤除特定的药物靶基因的结果。 </li><li>在"操作"选项卡，指定打击毒品目标（S），其中包括激动剂，拮抗剂等，未知或全部（来源:DrugBank数据库）药物作用的类型。 </li></ol>

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The authors declare that they have no competing financial interests.

The protocol described here for the RE:fine Drugs interactive dashboard can be modified in different ways according to the user's preferences. This method uniquely integrates GWAS and PheWAS data as a novel paradigm underlying drug repurposing hypothesis generation. Specifically, this system provides access to both 52,966 PheWAS associations and 7,945 GWAS associations with advanced options to filter the results by the study type, effect size and/or significance level. Another advantage of this method over existing computational drug repurposing tools is that queries may be made from drug, gene or disease perspectives. 
There are several limitations to this method. Currently, the PheWAS data is limited to primarily adult patient population from five institutions contained in the Electronic Medical Records and Genomics (eMERGE) network with a mean age of 69.5 years 12. Additionally, the "repurposing potential" feature uses co-occurrence of search terms in Medline abstracts as one of its criteria. It is well known that text mining methods using co-occurrence have limitations with respect to syntactical structure and literature bias. Thus, we recommend this feature be used as a starting point to explore the potential novelty and/or evidence supporting specific drug repurposing hypotheses and recommend additional investigation into the biomedical literature and clinical trial databases. 
Future directions for this work not described here would be to extend this database to additional sources of GWAS and PheWAS data as they become available. Similar efforts to systematically translate results from large-scale GWAS studies into drug repurposing hypotheses have been previously published 9,13-14. It may be useful to compare these different workflows to predict drug candidates from GWAS data in future studies. Additionally, several other methods exist to computationally generate drug repurposing hypotheses from different data sources, including: genomics, transcriptomics, chemical structures, drug side effect profiles, as previously summarized 6,11. Future methodological advancements could also include automating drug combination predictions and providing information on drug toxicity to guide follow up studies for drug candidates. 
Furthermore, the hypotheses generated from RE:fine Drugs may be further validated using electronic health records, before initiating clinical trials 15. Finally, future studies will be needed to compare this system to other target-based drug repurposing methods.

与传统药物发现方法，包括高通量药物和先导化合物筛选相关的昂贵和低效的过程，是造成延误翻译研究发现转化为治疗病人1,2。 1十亿美元和15 - 20年的平均时需携带从板凳上一个新的药物床边3。此外，药品52％的第一阶段临床试验在开发过程中失败了，只有25％即进入第二阶段的化合物进行充分三期临床研究4。再利用药物或药物重新定位的目标是续约失败的药物和/或找到批准的药品适应症新颖，以提供新的治疗方法给患者更快的速度和更高的成功率。药品再利用可能会降低时间表，使现有的药物在患者中使用，以3-12岁5。药品再利用的重要医疗应用包括:与差PROGNOS疾病是低存活率，抗药性疾病，资金不足的疾病的研究领域和贫困和缺医少药的病人群体。 计算药物再利用被定义为设计和验证自动化的工作流程，可以生成用于为候选药物6新适应症假设的过程。现有的计算药物再利用的方法已被归类目标为基础，以知识为基础的，基于签名的，基于网络和基于目标的机制，并可以从基因，疾病或药物的角度来导向。此外，计算方法可能会进一步加速证明了概念验证性实验和小规模临床研究为改变用途的候选药物7。我们曾报道的"RE:精药品"，一个免费的，互动式的网上药品再利用产生假设基于药物基因与疾病的关系8的传递理论工具。整体摹这种方法的OAL是系统集成不同类型的药物，遗传和临床资料，以使药物再利用从不同的社区，包括临床，工业和监管社区用户。该系统的基本方法先前已经报道了利用全基因组关联分析（GWAS），并在药物phenome范围内的关联研究（PheWAS）数据再利用的研究9,10。这些类型的数据的新组合，我们区别于其他基于目标的方法6,11 webtool。 该RE:精药物制度目前包含60911药物再利用假设覆盖916药品，567个基因和1,770疾病。该webtool为研究人员提供交互式搜索药物重复利用的假设和使用不同的标准优先考虑他们一个友好的用户界面。例如，用户可以过滤药物再利用假设用在生物医学文献支持和临床试验databaSE，显著p值，关联比值比或具体指示。此系统的唯一要求是上网。

<table><tbody><tr><td>Access the homepage for &amp;ldquo;RE:fine Drugs&amp;rdquo; at the following link: http://drug-repurposing.nationwidechildrens.org.&amp;nbsp;</td><td>n/a</td><td>n/a</td><td>The only requirement for this system is Internet access</td></tr></tbody></table>

drug repurposing hypothesis generation using the "re:fine drugs" system

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

在这个例子中，基因"IL2RB"被输入为基于基因的查询，以及由自动填充功能（ 图1）中的自动识别。对于IL2RB基因十二药物重新利用假设被返回， 如图2。对于特定的药物再利用假说"，达珠单抗"在这种情况下，详细信息页，从"信息"栏（ 图3）提供。结果被过滤的药物标签是通过对应于"赛尼哌"药物的所有结果， 如图4。 图5显示了只有这些药物具有已知的指示，为"移植"长期疾病（来源:DrugBank数据库）。关联选项卡允许用户过滤通过统计学意义（P值）和遗传效应大小（比值比），SNP-表型的关系，限定为公关的几率的比率在与特定基因型（SNP等位基因）以上的个体疾病的存在的可能性，而不与SNP等位基因的个体的疾病的esence。 图6示出的P值范围内的关联标签下过滤的0.000001至0.05的结果。 图7显示了具体的基础上，我们在研究中发现的新适应症"哮喘"药品再利用假设。 图8表示的电位标签下的结果通过含有的药物和疾病方面共现5 MEDLINE摘要的最小数目进行过滤。在这个例子中，基因标签下所有药物的结果针对所述"IL2RB"基因，对应于原始查询字词（ 图9）。最后，"行动"选项卡下过滤， 如图 10所示的成绩来回报充当对IL2RB基因兴奋剂所有药物。 <img alt="图1"SRC ="/文件/ ftp_upload / 54948 / 54948fig1.jpg"/&gt; 图1:RE:精药品的交互式仪表板主页。用户可以通过输入药品名称，新的疾病指示或基因符号开头的查询。还提供了用于说明用于产生药物再利用假设方法的GWAS和PheWAS参考文件的链接。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54948/54948fig2.jpg" /> 图 2: 查询条目自动填充功能。作为一个例子，所述基因查询词"IL2RB"被自动识别为一个基因术语。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO: - together.within页保留="1"&gt; <img alt="图3" src="/files/ftp_upload/54948/54948fig3.jpg" /> 图3: 从基于基因的查询（ 例如，IL2RB）产生药物再利用的结果的表。对于IL2RB基因药物十二假设再利用产生。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54948/54948fig4.jpg" /> 图4:从结果页个别药物信息页面。点击从"信息"列中的图标显示为药物达珠单抗的详细信息。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5: 药品标签下的高级搜索选项通过特定的药物进行筛选。在此示例中，三个结果示为药物达珠单抗。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54948/54948fig6.jpg" /> 图6: 药品标签下的高级搜索选项从DrugBank数据库中的特定疾病的指示进行过滤。与用于疾病的术语"移植"公知的指示所有药物被示出。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图7:关联选项卡下的高级搜索选项，通过显着性水平进行筛选。在这种情况下，设置八个结果其关联显着性水平的0.000001至0.05的P值范围内。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54948/54948fig8.jpg" /> 图8: 疾病标签下高级搜索选项，通过我们在本研究中提取一个特定的疾病的指示进行筛选。在这个例子中，四结果示哮喘作为新使用的疾病的指示。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">请点击此处查看大VERSI</a>这个数字的。 <img alt="图9" src="/files/ftp_upload/54948/54948fig9.jpg" /> 图9: 潜在选项卡下的高级搜索选项，通过在MEDLINE摘要药物和疾病方面共现过滤。在这个例子中，四结果示由含有的药物和疾病方面共现5 MEDLINE摘要的最小数目的支持。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/54948/54948fig10.jpg" /> 图10: 基因标签下高级搜索选项由特定基因符号，其中所有结果对应于用于原始查询IL2RB基因进行筛选。在这个例子中，所有的药物的结果该基因标签下被定位为"IL2RB"基因，对应于原始查询字词。操作选项卡下的高级搜索选项只受体激动剂的药物筛选。在这个例子中，六结果返回为充当对IL2RB基因激动剂所有药物。 <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System at JoVE.com

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

药品再利用产生假设使用"RE:精药品"系统

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

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9.2K 次观看.  The Ohio State University. Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options. 

视频: Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

科研

生物化学

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

促进药物发现：斑马鱼在一个自动化的高含量检测炎症

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

免疫与感染

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

发育生物学

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

基于核磁共振的片段筛选，样品最少，自动化程度最高

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

实施<em&gt;体外</em&gt;耐药性检测:最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

医学

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

基于生物传感器的高通量生物跨越和生物信息学分析战略，用于全球药物-蛋白质相互作用验证

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

生物工程

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换:应用程序的设计和开发非ATP竞争性CDK抑制剂

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

生物学

Chemogenetic Regulation in Reprogrammed Stem Cell-derived Precursor Cells in Treating Neurodegenerative Diseases

The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for precise neuronal modulation. Here, we utilized CRISPR-engineered human reprogrammed stem cells expressing excitatory (hM3Dq) or inhibitory (hM4Di) DREADD receptors to evaluate the functional integration and modulation of transplanted dopaminergic precursors in a murine model of Parkinson&#39;s disease (PD). Key steps included generating non-fusion DREADD-expressing stem cell lines, differentiating them into midbrain dopaminergic precursors, and transplanting these cells into the striatum of 6-hydroxydopamine (6-OHDA)-lesioned mice. We conducted behavioral assessments and electrophysiological recordings to analyze the effects of the transplanted cells. Behavioral tests, such as the cylinder test, demonstrated significant modulation of motor function following clozapine-N-oxide (CNO) administration. Specifically, activation of hM4Di reduced contralateral forelimb movement, whereas activation of hM3Dq was associated with enhanced motor behavior. Electrophysiological recordings revealed distinct synaptic responses. hM4Di activation increased interevent intervals and decreased peak amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs), whereas hM3Dq activation decreased interevent intervals and increased peak amplitudes, reflecting enhanced excitatory signaling. In summary, the integration of behavioral and electrophysiological assessments validates the precise functional incorporation of engineered chemically reprogrammed stem cells into host neural circuits.

Here, we describe a protocol for engineering chemically reprogrammed stem cells to achieve precise neuronal modulation by differentiating these cells into dopaminergic precursor cells, transplanting them into mouse models of Parkinson's disease, and evaluating behavioral and electrophysiological outcomes to confirm the successful integration and functional effectiveness of the transplanted cells.

重编程干细胞衍生前体细胞的化学遗传学调控治疗神经退行性疾病

The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for ...

神经科学

Drug Discovery: Overview

Drug discovery is a multifaceted process involving extensive screening, testing, and optimization of lead compounds to identify potential new drugs for therapeutic use. It combines several approaches, including screening large numbers of natural products, chemical modification of known active molecules, identification of new drug targets, and rational design based on biological mechanisms and drug-receptor structure. These approaches are carried out in both academic research laboratories and the pharmaceutical industry.
The process begins with drug screening, which involves various assays to evaluate the pharmacological profile and activity of the drug at different levels, such as molecular, cellular, organ system, and whole animal levels. The candidate molecule undergoes extensive testing to determine its mechanism of action, selectivity, and potential toxic effects. Animal models of human diseases are used to select compounds for further development, especially in cases where preclinical models are well-established.
The candidate compound is screened for its activity on the target protein at the molecular level using techniques like receptor binding assays. Cell function studies determine whether the drug acts as an agonist, partial agonist, inverse agonist, or antagonist at relevant receptors. Whole animal studies assess the drug&#39;s effects on organ systems and disease models.
Lead compounds with the desired pharmacological activity and selectivity are identified during the screening and lead generation processes. These lead compounds serve as the basis for further chemical modification and optimization in the lead optimization phase. Lead optimization involves a range of tests and assays to increase the potency of the compound, optimize selectivity, and evaluate other characteristics, such as pharmacokinetic properties. Animal studies assess the compound&#39;s activity in vivo, potential adverse effects, genotoxicity, and oral availability.
Only a small percentage of drug discovery projects succeed in generating a drug candidate, and the lead optimization phase can be time-consuming and challenging. Many factors can hinder progress, including the inability to optimize lead compounds or the failure to produce desired effects in animal models. Successful drug candidates proceed to the next phase, preclinical development, before entering human clinical trials.

药物发现:概述

Drug discovery is a multifaceted process involving extensive screening, testing, and optimization of lead compounds to identify potential new drugs for ...