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Single-plate kinome screening in live-cells to enable highly cost-efficient kinase inhibitor profiling 活细胞单片激酶组筛选,使高成本效益的激酶抑制剂分析。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-16 DOI: 10.1016/j.slasd.2025.100214
Martin P. Schwalm , Stefan Knapp
Cancer research, cancer treatment, and the field of chemical biology are examples which heavily rely on the discovery of selective kinase inhibitors. While determining on-target potency is often feasible for most laboratories, the equally critical but frequently neglected selectivity screening remains less accessible to the broader scientific community. This limitation can stem from various factors, such as the unavailability of a large number of purified kinases or the costs associated with commercial screening systems. To address these challenges and enable a wider range of scientists, this protocol leverages a commercial kinome selectivity screen to facilitate a low-cost, two-day, single-plate selectivity screen against 192 kinases.
癌症研究、癌症治疗和化学生物学领域都是严重依赖于选择性激酶抑制剂发现的例子。虽然确定靶效价对大多数实验室来说通常是可行的,但同样重要但经常被忽视的选择性筛选仍然很少被广泛的科学界所接受。这种限制可能源于各种因素,例如无法获得大量纯化的激酶或与商业筛选系统相关的成本。为了应对这些挑战,并使更广泛的科学家,该方案利用商业激酶组选择性筛选来促进低成本,两天,针对192个激酶的单板选择性筛选。
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引用次数: 0
Green chemistry: Modern therapies using nanocarriers for treating rare brain cancer metastasis from colon cancer 绿色化学:利用纳米载体治疗结肠癌转移性脑癌的现代疗法。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-16 DOI: 10.1016/j.slasd.2025.100213
Doaa S․R․ Khafaga , Ghazala Muteeb , Darin․W․ Aswa , Mohammad Aatif , Mohd Farhan , Salma Allam
Brain metastasis (BM) from colon cancer is associated with a poor prognosis and restricted treatment alternatives, largely due to issues related to blood-brain barrier (BBB) permeability and the negative effects of standard chemotherapy. Nanotechnology improves treatment efficacy by enabling targeted and controlled drug delivery. This review article evaluates the potential of nanotechnology-based therapies for treating colon cancer BM, emphasizing their capacity to cross the BBB, diminish metastatic growth, and enhance overall survival rates. A review of multiple studies evaluated nanoparticles (NPs) as carriers for chemotherapy, focusing on parameters including particle size, surface charge, and drug-loading capacity. The study also reviewed studies that examined BBB penetration, in vitro tumor accumulation, and in vivo tumor growth inhibition. In vitro findings indicated that NPs accumulate more efficiently in BM tissue than in healthy brain tissue and show significant BBB penetration. In vivo, nanotherapy markedly inhibited tumor growth and prolonged survival relative to conventional chemotherapy or control treatments while also exhibiting reduced side effects. Recent studies demonstrated that plant extracts can effectively and safely synthesize nanomaterials, positioning them as a viable and environmentally friendly precursor for nanomaterial production. Nanotechnology-based therapies demonstrate significant potential in the treatment of colon cancer BM by minimizing systemic toxicity, enhancing therapeutic efficacy, and facilitating more targeted drug delivery. Further research is required to confirm these findings and implement them in clinical practice.
结肠癌脑转移(BM)与预后不良和治疗方案受限相关,主要是由于血脑屏障(BBB)通透性和标准化疗的负面影响。纳米技术通过实现靶向和受控的药物递送来提高治疗效果。这篇综述文章评估了纳米技术治疗结肠癌转移瘤的潜力,强调了它们穿越血脑屏障、减少转移性生长和提高总体生存率的能力。对纳米颗粒(NPs)作为化疗载体的多项研究进行了回顾,重点关注颗粒大小、表面电荷和载药能力等参数。该研究还回顾了血脑屏障渗透、体外肿瘤积累和体内肿瘤生长抑制的研究。体外研究结果表明,NPs在脑梗死组织中比在健康脑组织中更有效地积累,并表现出明显的血脑屏障渗透。在体内,与常规化疗或对照治疗相比,纳米疗法显著抑制肿瘤生长,延长生存期,同时也显示出更少的副作用。最近的研究表明,植物提取物可以有效和安全地合成纳米材料,使其成为一种可行的、环保的纳米材料前体。基于纳米技术的治疗方法通过最小化全身毒性、提高治疗效果和促进更有针对性的药物传递,在结肠癌BM的治疗中显示出巨大的潜力。需要进一步的研究来证实这些发现并将其应用于临床实践。
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引用次数: 0
Super-resolution microscopy as a drug discovery tool 超分辨率显微镜作为药物发现工具。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-15 DOI: 10.1016/j.slasd.2025.100209
Lauren Toms, Lorna FitzPatrick, Philip Auckland
At the turn of the century a fundamental resolution barrier in fluorescence microscopy known as the diffraction limit was broken, giving rise to the field of super-resolution microscopy. Subsequent nanoscopic investigation with visible light revolutionised our understanding of how previously unknown molecular features give rise to the emergent behaviour of cells. It transpires that the devil is in these fine molecular details, and essential nanoscale processes were found everywhere researchers chose to look. Now, after nearly two decades, super-resolution microscopy has begun to address previously unmet challenges in the study of human disease and is poised to become a pivotal tool in drug discovery.
在世纪之交,荧光显微镜中被称为衍射极限的基本分辨率障碍被打破,从而产生了超分辨率显微镜领域。随后的可见光纳米研究彻底改变了我们对以前未知的分子特征如何引起细胞涌现行为的理解。事实证明,关键就在这些细微的分子细节中,研究人员在任何地方都能找到必要的纳米尺度过程。现在,经过近二十年,超分辨率显微镜已经开始解决人类疾病研究中以前未遇到的挑战,并准备成为药物发现的关键工具。
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引用次数: 0
A high throughput assay for phosphoribosylformylglycinamidine synthase 磷酸核糖基甲酰基甘氨酸合成酶的高通量测定。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-15 DOI: 10.1016/j.slasd.2025.100212
Nandini Sharma , Yuka Otsuka , Louis Scampavia , Timothy P. Spicer , Jarrod B. French
Metabolic reprogramming of purine biosynthesis is a hallmark of cancer metabolism and represents a critical vulnerability. The enzyme phosphoribosylformylglycinamidine synthase (PFAS) catalyzes the fourth step in de novo purine biosynthesis and has been demonstrated to be prognostic for survival of liver cancer. Despite the importance of this protein as a drug target, there are no known specific inhibitors of PFAS activity. Here, we describe a new continuous, spectrophotometric assay for the synthase domain of PFAS that is amenable to high-throughput screening (HTS). This mechanism-based fluorescent assay makes use of the acid phosphatase substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). PFAS catalyzes the turnover of DiFMUP with a KM of 108 ± 7 µM. After optimization and miniaturization of the assay for 1,536-well format, we conducted a pilot HTS using the LOPAC1280 library. The assay performed extremely well, with an average Z′ of 0.94 ± 0.02, average signal to noise of 5.01 ± 0.06, excellent inter plate correlation, and a hit rate of 1.18 %. This assay provides a critically needed tool to advance the study of PFAS enzymology and will be foundational for the discovery of small molecule inhibitors both as functional probes and for the basis of new drug development.
嘌呤生物合成的代谢重编程是癌症代谢的一个标志,代表了一个关键的脆弱性。磷酸核糖基甲酰基甘氨酸合成酶(PFAS)催化嘌呤新生生物合成的第四步,并已被证明是肝癌生存的预后因素。尽管这种蛋白作为药物靶点很重要,但目前还没有已知的特异性PFAS活性抑制剂。在这里,我们描述了一种新的连续分光光度法测定PFAS合成酶结构域,适用于高通量筛选(HTS)。这种基于机制的荧光分析利用了酸性磷酸酶底物,6,8-二氟-4-甲基伞花酰磷酸(DiFMUP)。PFAS催化DiFMUP的周转,KM为108±7µM。在优化和小型化1536孔格式的分析后,我们使用LOPAC1280文库进行了试点HTS。该方法的检测效果非常好,平均Z´为0.94±0.02,平均信噪比为5.01±0.06,板间相关性良好,准确率为1.18%。该分析为推进PFAS酶学研究提供了一个急需的工具,并将为发现小分子抑制剂作为功能探针和新药开发的基础奠定基础。
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引用次数: 0
Rapid luminescence-based screening method for SARS- CoV-2 inhibitors discovery 基于荧光快速筛选SARS- CoV-2抑制剂的方法。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-15 DOI: 10.1016/j.slasd.2025.100211
Abdeldjalil Madani, Nadine Alvarez, Steven Park, Madhuvika Murugan, David S. Perlin
The COVID-19 pandemic has emphasized the necessity for rapid and adaptable drug screening platforms against live pathogenic viruses that require high levels of biosafety containment. Conventional antiviral testing is time-consuming and labor-intensive. Here, we outline the design and validation of a semi-automated drug-screening platform for SARS-CoV-2 that utilizes multiple liquid handlers, a stable A549 cell line expressing ACE2 and TMPRSS2 receptors, and a recombinant SARS-CoV-2 strain harboring the nano-luciferase gene. This platform allows for accelerated low-, mid-, and high-throughput screenings by bypassing the virus inactivation and the staining steps compared to assays utilizing fluorescent reporter viruses or immunofluorescence. First, we demonstrated that the luminescence signal obtained at 24 h post-infection is robust and can be used as a surrogate for fluorescent reporter viruses and immunofluorescence assays that require 48 h incubation post infection. We confirmed the susceptibility of the reporter virus to a panel of reference drugs and validated the luminescence signal in 96- and 384-well plates in accordance with NIH criteria for high-throughput screening. The validation assays showed reproducible results, robust Z factor of ≥0.5, and a coefficient of variation of <20% achieved in both 96 and 384-well plate formats. Lastly, we assessed the assay's performance by screening 240 compounds from the MMV Global Health Library, using the 384-well plate format and remdesivir as a control compound. The single point screening resulted in the identification of 48 hits that inhibited more than 50% of the viral growth. We selected the 15 most active compounds to evaluate their inhibitory concentration and their cytotoxicity, which resulted in the confirmation of the 3 most potent and least toxic compounds that were never reported as antivirals. These results confirm that our platform can be reliably employed for rapid drug screening against SARS-CoV-2 and can be easily adapted to other nano-luciferase reporter viruses.
COVID-19大流行强调了针对活致病性病毒的快速和适应性强的药物筛选平台的必要性,这些平台需要高水平的生物安全控制。传统的抗病毒检测既耗时又费力。在这里,我们概述了一个半自动化的SARS-CoV-2药物筛选平台的设计和验证,该平台利用多个液体处理器、表达ACE2和TMPRSS2受体的稳定A549细胞系和含有纳米荧光素酶基因的重组SARS-CoV-2菌株。与使用荧光报告病毒或免疫荧光的检测相比,该平台可以通过绕过病毒灭活和染色步骤来加速低、中、高通量筛选。首先,我们证明了在感染后24小时获得的发光信号是稳健的,可以用作荧光报告病毒和免疫荧光检测的替代品,这些检测需要在感染后48小时孵育。我们确认了报告病毒对一组参考药物的敏感性,并根据NIH高通量筛选标准在96孔和384孔板上验证了发光信号。验证试验结果重复性好,稳健性Z因子≥0.5,变异系数为
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引用次数: 0
Heterotypic spheroids as a strategy for 3D culture of cryopreserved primary human hepatocytes in stirred-tank systems 异型球体作为在搅拌槽系统中低温保存的人原代肝细胞三维培养的策略。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-11 DOI: 10.1016/j.slasd.2025.100210
Francisca Arez , Lena Preiss , Isabella Ramella Gal , Sofia P. Rebelo , Lassina Badolo , Catarina Brito , Thomas Spangenberg , Paula M. Alves
Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability in vitro in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications.
Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.
原代人肝细胞(PHHs)是解决肝功能的首选细胞来源。尽管来源于原生组织,但使用原生材料的瓶颈之一是供体与供体之间的差异。低温保存的phh提供了来自同一供体的大量细胞,并且标准化了细胞分离和低温保存程序,减轻了一些供体间的差异。尽管如此,来自不同商业来源的phh在体外的几个参数上存在差异,包括解冻后的活力、镀敷能力、聚集潜力和培养寿命。在这里,我们结合了搅拌罐培养系统,它允许强大的聚集过程,以及HepaRG细胞系的共培养方法,从冷冻保存的phh中产生球体。采用小型搅拌槽培养系统可以解决原料稀缺和成本高的问题。在优化的共培养条件下,我们可以从4个不同的商业来源获得的12个供体中产生PHH:HepaRG球体。所有phh均表现出相似的聚集特征,早在共培养的第3天就形成了紧凑的小异型球体,并在培养中维持了至少5周。异型球体维持了肝细胞的极化和特性,并表现出对CYP3A4、CYP2C9、CYP1A2、CYP2D6和CYP2C8这5种主要I期代谢酶的代谢能力。此外,异型球体在临床开发中显示出代谢新化合物的能力,显示出它们在药物发现应用中的潜力。总的来说,我们提出了一种强大的聚合策略,适用于来自不同供应商的冷冻保存phh,用于体外药理学和毒理学研究。
{"title":"Heterotypic spheroids as a strategy for 3D culture of cryopreserved primary human hepatocytes in stirred-tank systems","authors":"Francisca Arez ,&nbsp;Lena Preiss ,&nbsp;Isabella Ramella Gal ,&nbsp;Sofia P. Rebelo ,&nbsp;Lassina Badolo ,&nbsp;Catarina Brito ,&nbsp;Thomas Spangenberg ,&nbsp;Paula M. Alves","doi":"10.1016/j.slasd.2025.100210","DOIUrl":"10.1016/j.slasd.2025.100210","url":null,"abstract":"<div><div>Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability <em>in vitro</em> in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications.</div><div>Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100210"},"PeriodicalIF":2.7,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142980832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models 用于分析神经细胞模型中线粒体形态的新型高含量和开源图像分析工具。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-06 DOI: 10.1016/j.slasd.2025.100208
Marcus Y. Chin , David A. Joy , Madhuja Samaddar, Anil Rana, Johann Chow, Takashi Miyamoto, Meredith Calvert
Mitochondria undergo dynamic morphological changes depending on cellular cues, stress, genetic factors, or disease. The structural complexity and disease-relevance of mitochondria have stimulated efforts to generate image analysis tools for describing mitochondrial morphology for therapeutic development. Using high-content analysis, we measured multiple morphological parameters and employed unbiased feature clustering to identify the most robust pair of texture metrics that described mitochondrial state. Here, we introduce a novel image analysis pipeline to enable rapid and accurate profiling of mitochondrial morphology in various cell types and pharmacological perturbations. We applied a high-content adapted implementation of our tool, MitoProfilerHC, to quantify mitochondrial morphology changes in i) a mammalian cell dose response study and ii) compartment-specific drug effects in primary neurons. Next, we expanded the usability of our pipeline by using napari, a Python-powered image analysis tool, to build an open-source version of MitoProfiler and validated its performance and applicability. In conclusion, we introduce MitoProfiler as both a high-content-based and an open-source method to accurately quantify mitochondrial morphology in cells, which we anticipate to greatly facilitate mechanistic discoveries in mitochondrial biology and disease.
线粒体经历动态形态变化取决于细胞线索,压力,遗传因素,或疾病。线粒体的结构复杂性和疾病相关性促使人们努力生成图像分析工具,用于描述治疗开发的线粒体形态。通过高含量分析,我们测量了多个形态参数,并采用无偏特征聚类来识别描述线粒体状态的最稳健的纹理指标对。在这里,我们引入了一种新的图像分析管道,可以快速准确地分析各种细胞类型和药理扰动下的线粒体形态。我们应用了我们的工具MitoProfilerHC的高含量适应性实现,量化了i)哺乳动物细胞剂量反应研究和ii)初级神经元中室特异性药物效应的线粒体形态学变化。接下来,我们通过使用napari(一个python驱动的图像分析工具)来扩展管道的可用性,构建开源版本的MitoProfiler,并验证其性能和适用性。总之,我们介绍MitoProfiler作为一种基于高含量和开源的方法来准确量化细胞中的线粒体形态,我们预计这将极大地促进线粒体生物学和疾病的机制发现。
{"title":"Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models","authors":"Marcus Y. Chin ,&nbsp;David A. Joy ,&nbsp;Madhuja Samaddar,&nbsp;Anil Rana,&nbsp;Johann Chow,&nbsp;Takashi Miyamoto,&nbsp;Meredith Calvert","doi":"10.1016/j.slasd.2025.100208","DOIUrl":"10.1016/j.slasd.2025.100208","url":null,"abstract":"<div><div>Mitochondria undergo dynamic morphological changes depending on cellular cues, stress, genetic factors, or disease. The structural complexity and disease-relevance of mitochondria have stimulated efforts to generate image analysis tools for describing mitochondrial morphology for therapeutic development. Using high-content analysis, we measured multiple morphological parameters and employed unbiased feature clustering to identify the most robust pair of texture metrics that described mitochondrial state. Here, we introduce a novel image analysis pipeline to enable rapid and accurate profiling of mitochondrial morphology in various cell types and pharmacological perturbations. We applied a high-content adapted implementation of our tool, MitoProfilerHC, to quantify mitochondrial morphology changes in i) a mammalian cell dose response study and ii) compartment-specific drug effects in primary neurons. Next, we expanded the usability of our pipeline by using napari, a Python-powered image analysis tool, to build an open-source version of MitoProfiler and validated its performance and applicability. In conclusion, we introduce MitoProfiler as both a high-content-based and an open-source method to accurately quantify mitochondrial morphology in cells, which we anticipate to greatly facilitate mechanistic discoveries in mitochondrial biology and disease.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100208"},"PeriodicalIF":2.7,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 人瞬时受体电位阳离子通道TRPM8、TRPV1和TRPA1 384孔动态Ca2+动员实验的优化和校准
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-01 DOI: 10.1016/j.slasd.2024.100207
David A. Close , V. Blair Journigan , Paul A. Johnston
Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca2+ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca2+ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca2+ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca2+ mobilization kinetic data to plot curves and calculate EC50 and IC50 values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC50 and IC50 ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC50 or IC50 values required to distinguish compound potencies. EC50 and IC50 values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC50 and IC50 values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.
本文描述了TRPM8、TRPV1和TRPA1的人类瞬时受体电位(TRP)通道Ca2+动员测定的开发、优化和校准。HEK293T细胞需要异源表达hTRPM8,以获得抗TRPM8抗体染色和TRPM8激动剂诱导的Ca2+动员信号,两者都用于优化转染效率。优化FLIPR钙6染料浓度、加载时间、TRPM8转染细胞播种密度,设定DMSO耐受性≤0.2%。TRPM8 Ca2+动员试验的静息基线相对荧光单位(RFUs)信号显示出大量的井间变异性,尽管这种差异相对于最大激动剂诱导的反应来说很小。从Ca2+动员动力学数据中提取最大RFU、累计RFU总和或曲线下面积,绘制曲线并计算EC50和IC50值。折叠基线(FOB)比率数据处理消除了井与井之间静息基线信号的差异,减少了误差条,改善了曲线拟合,降低了95%置信区间EC50和IC50范围。FOB比率数据处理降低了可变性,提高了单次实验中重复测量的精度,从而降低了区分化合物效价所需的EC50或IC50值的最小阈值差异。单次实验测定的TRPM8激动剂和拮抗剂的EC50和IC50值与多个独立实验的结果高度一致。基准TRPM8、TRPV1和TRPA1的EC50和IC50值均在先前报道的激动剂和拮抗剂标准范围内。FOB比率数据处理提高了TRP Ca2+动员测定的精度和准确性,提高了它们在研究结构活性关系方面的实用性。
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引用次数: 0
Drug repurposing screen for the rare disease ataxia-telangiectasia 罕见疾病共济失调-毛细血管扩张的药物再利用筛选。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-01 DOI: 10.1016/j.slasd.2024.100200
Namrata Jayanth , Gurvan Mahé , Matthew Campbell , Mike Lipkin , Shushant Jain , Rhea van de Bospoort , Jennifer Thornton , Brad Margus , David F. Fischer
Ataxia Telangiectasia (A-T) is a rare, autosomal recessive genetic disorder characterized by a variety of symptoms, including progressive neurodegeneration, telangiectasia, immunodeficiency, and an increased susceptibility to cancer. It is caused by bi-allelic mutations impacting a gene encoding a serine/threonine kinase ATM (Ataxia Telangiectasia Mutated), which plays a crucial role in DNA repair and maintenance of genomic stability. The disorder primarily affects the nervous system, leading to a range of neurological issues, including cerebellar ataxia. The cause of neurodegeneration due to mutations in ATM is still an area of investigation, and currently there is no known treatment to slow down or stop the progression of the neurological problems.
In this collaboration of the A-T Children's Project (ATCP) with Charles River Discovery, we successfully developed a high-throughput assay using induced pluripotent stem cells (iPSC) from A-T donors to measure DNA damage response (DDR). By measuring the changes in levels of activated phosphorylated CHK2 (p-CHK2), which is a downstream signaling event of ATM, we were able to identify compounds that restore this response in the DDR pathway in A-T derived patient cells. Over 6,000 compounds from small molecule drug repurposing libraries were subsequently screened in the assay developed, leading to identification of several promising in vitro hits.
Using the assay developed and the identified hits opens avenues to investigate potential therapeutics for A-T.
共济失调毛细血管扩张症(a - t)是一种罕见的常染色体隐性遗传病,其特征是多种症状,包括进行性神经变性、毛细血管扩张、免疫缺陷和对癌症的易感性增加。它是由双等位基因突变影响编码丝氨酸/苏氨酸激酶ATM(共济失调毛细血管扩张突变)的基因引起的,该基因在DNA修复和基因组稳定性的维持中起着至关重要的作用。这种疾病主要影响神经系统,导致一系列神经问题,包括小脑性共济失调。由ATM突变引起的神经退行性变的原因仍是一个研究领域,目前还没有已知的治疗方法来减缓或阻止神经问题的进展。在a -t儿童项目(ATCP)与Charles River Discovery的合作中,我们成功开发了一种使用来自a -t供体的诱导多能干细胞(iPSC)来测量DNA损伤反应(DDR)的高通量测定方法。通过测量活化磷酸化CHK2 (p-CHK2)水平的变化,这是ATM的下游信号事件,我们能够识别在a - t衍生的患者细胞中恢复DDR途径中这种反应的化合物。随后,从小分子药物再利用文库中筛选了6000多种化合物,并确定了几种有前景的体外命中。使用开发的检测方法和确定的命中点为研究A-T的潜在治疗方法开辟了道路。
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引用次数: 0
Miniaturization and characterization of patient derived hepatocellular carcinoma tumor organoid cultures for cancer drug discovery applications 用于癌症药物发现应用的病人来源的肝细胞癌肿瘤类器官培养的小型化和特性。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-01 DOI: 10.1016/j.slasd.2024.100201
David A. Close , Paul A. Johnston
Patient derived tumor organoid (PDTO) models retain the structural, morphological, genetic, and clonal heterogeneity of the original tumors. The ability to efficiently generate, expand, and biobank PDTOs has the potential to make the clinical diversity of cancer accessible for personalized medicine assay guided therapeutic drug selection and drug discovery. We describe the miniaturization and growth in 96- and 384-well formats of a single non-tumor liver and two Hepatocellular carcinoma (HCC) organoids derived from cryopreserved PDTO cells and the application of high content imaging (HCI) to characterize the models and enhance drug sensitivity testing. Non-invasive sequentially acquired transmitted light images showed that seeding cryopreserved cells from non-tumoral and HCC PDTOs into 96- or 384-well plates in reduced growth factor Matrigel (rgf-MG) that were fed with growth medium every 3 days supported organoid growth up to 15 days. The number and sizes of organoids increased with longer times in culture. HCC PDTO's had more heterogeneous morphologies than non-tumor organoids with respect to size, shape, and optical density. Organoids cultured in rgf-MG could be stained in situ with HCI reagents without mechanical, chemical or enzymatic disruption of the hydrogel matrices and quantitative data extracted by image analysis. Hoechst and live/dead reagents provided organoid numbers and viability comparisons. HCC PDTO's stained with phalloidin or immuno-stained with α-tubulin antibodies revealed F-actin and microtubule cytoskeleton organization. HCC PDTO's stained with antibodies to signaling pathway proteins and their phosphorylation status allowed comparisons of relative expression levels and inference of pathway activation. Images of HCC PDTO's exposed to ellipticine showed that drugs penetrate Matrigel hydrogels and accumulate in organoid cells. 9-day 384-well HCC organoid cultures exhibited robust and reproducible growth signals suitable for cancer drug testing. Complimenting cell viability readouts with multiple HCI parameters including morphological features and dead cell staining improved the analysis of drug impact and enhanced the value that could be extracted from these more physiologically relevant three-dimensional HCC organoid cultures.
患者源性肿瘤类器官(PDTO)模型保留了原始肿瘤的结构、形态、遗传和克隆异质性。有效地生成、扩展和生物库pdto的能力有可能使癌症的临床多样性可用于个性化医学检测指导的治疗药物选择和药物发现。我们描述了96孔和384孔格式的单个非肿瘤肝脏和两个肝细胞癌(HCC)类器官的小型化和生长,这些器官来自冷冻保存的PDTO细胞,并应用高含量成像(HCI)来表征模型并增强药物敏感性测试。非侵入性序列获得的透射光图像显示,将非肿瘤和HCC pto中冷冻保存的细胞接种到96孔或384孔的rgf-MG培养皿中,每3天添加一次生长培养基,可支持类器官生长长达15天。类器官的数量和大小随培养时间的延长而增加。HCC的PDTO在大小、形状和光密度方面比非肿瘤类器官具有更多的异质形态。在rgf-MG中培养的类器官可以用HCI试剂原位染色,而不会对水凝胶基质造成机械、化学或酶的破坏,也不会通过图像分析提取定量数据。赫斯特试剂和活/死试剂提供类器官数量和活力比较。phalloidin染色或α-微管蛋白抗体免疫染色显示F-actin和微管细胞骨架组织。肝癌PDTO的信号通路蛋白抗体染色及其磷酸化状态可以比较相对表达水平和途径激活的推断。肝细胞癌PDTO暴露于椭圆线的图像显示药物穿透基质水凝胶并在类器官细胞中积累。9天384孔肝细胞癌类器官培养显示出适合癌症药物测试的稳健且可重复的生长信号。用多种HCI参数(包括形态学特征和死细胞染色)补充细胞活力数据,改善了药物影响的分析,并增强了从这些更具有生理学相关性的三维HCC类器官培养中提取的价值。
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