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Protocol to develop A 3D tumor model for drug testing applications 开发用于药物测试应用的3D肿瘤模型的协议。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-01 DOI: 10.1016/j.slasd.2024.100206
Prasiddha Guragain, Sunil Singh, Hossein Tavana
Three-dimensional (3D) tumor models provide physiologically relevant tumor environments and have become a major tool in cancer research and drug discovery. This article presents a protocol for creating a 3D organotypic tumor model by embedding a cancer cell spheroid within a collagen matrix containing dispersed fibroblasts. This model offers significant advantages over the conventional monolayer cell culture, monoculture spheroids of cancer cells, and intermixed co-culture of cancer and stromal cells by mimicking the spatial organization and mechanical properties of a solid tumor. Compatible with robotic automation, our protocol significantly enhances reproducibility and scalability of creating a tumor model to study tumor-stromal interactions and test therapeutic compounds
三维(3D)肿瘤模型提供了与生理相关的肿瘤环境,已成为癌症研究和药物发现的主要工具。本文提出了一种通过在含有分散成纤维细胞的胶原基质中嵌入癌细胞球体来创建三维器官型肿瘤模型的方案。该模型通过模拟实体肿瘤的空间组织和力学特性,与传统的单层细胞培养、单一培养的癌细胞球形细胞以及癌细胞和基质细胞混合共培养相比,具有显著的优势。与机器人自动化兼容,我们的方案显着提高了创建肿瘤模型的可重复性和可扩展性,以研究肿瘤-基质相互作用和测试治疗性化合物。
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引用次数: 0
Absolute quantification of Neuron-specific enolase based on surface plasmon resonance 基于表面等离子体共振的神经元特异性烯醇化酶的绝对定量。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-01 DOI: 10.1016/j.slasd.2024.100205
Cui Lin , Yijie Wang , Tao Peng , Pengpeng Liu , Yuanyuan Liang , Wencheng Kang , Xiaoping Yu , Yang Song , Xuping Shentu
Neuron-specific enolase (NSE) is currently the most reliable biomarker for small cell lung cancer (SCLC), which is important for disease monitoring, clinical evaluation and diagnosis. However, traditional methods suffer from various disadvantages, including instability, complexity, time-consuming operations, and the necessity for standards. In this study, we developed a calibration-free concentration analysis (CFCA) method based on surface plasmon resonance (SPR) technology, to accurately quantify the active concentration of NSE without relying on any standards. Based on the principle of CFCA, the active concentration of NSE can be calculated by observing binding rate variations at two flow rates under partial mass transport limitation and combining it with the known diffusion coefficient of the NSE. Using the method of CFCA, the active concentration of NSE was determined was only 0.48 mg/mL with an intra-day repeatability of 4.75%. The method has the advantages of simplicity, rapidity, realistic analysis and ease of implementation of high-throughput automated detection. Therefore, the method is expected to become the main measurement method for protein active concentration, which will be beneficial for the development of active protein standards.
神经元特异性烯醇化酶(Neuron-specific enolase, NSE)是目前小细胞肺癌(small cell lung cancer, SCLC)最可靠的生物标志物,对疾病监测、临床评价和诊断具有重要意义。然而,传统的方法有各种缺点,包括不稳定、复杂、耗时的操作和需要标准。在本研究中,我们开发了一种基于表面等离子体共振(SPR)技术的无校准浓度分析(CFCA)方法,可以在不依赖任何标准的情况下准确量化NSE的活性浓度。基于CFCA原理,通过观察在部分质量输运限制下两种流速下的结合速率变化,结合已知的NSE扩散系数,可以计算出NSE的活性浓度。CFCA法测定NSE活性浓度仅为0.48 mg/mL,日内重复性为4.75%。该方法具有简单、快速、分析真实、易于实现高通量自动化检测等优点。因此,该方法有望成为蛋白质活性浓度的主要测量方法,有利于活性蛋白质标准的制定。
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引用次数: 0
A novel platform for oral epithelia sheet biofabrication via magnetic 3D bioprinting 磁性3D生物打印口腔上皮片生物制造的新平台。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-01 DOI: 10.1016/j.slasd.2024.100203
Tien T.T. Truong , Toan V. Phan , Yamin Oo , Oranart Matangkasombut , João N. Ferreira
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引用次数: 0
Development of a DNA-encoded library screening method “DEL Zipper” to empower the study of RNA-targeted chemical matter 开发一种dna编码文库筛选方法“DEL Zipper”,使rna靶向化学物质的研究成为可能。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-12-21 DOI: 10.1016/j.slasd.2024.100204
Zhongyao Ma , Bin Zou , Jiannan Zhao , Rui Zhang , Qiaoqiao Zhu , Xiaofeng Wang , Linan Xu , Xiang Gao , Xinyue Hu , Wei Feng , Wen Luo , Min Wang , Yunyun He , Zhifeng Yu , Weiren Cui , Qi Zhang , Letian Kuai , Wenji Su
To date, RNA-targeted chemical matter is under explored due to a lack of robust screening assays. In this study, we present a novel RNA-targeted small molecule screening approach using a specialized DNA-encoded library (DEL). Our findings reveal that the specialized DEL library, called “DEL Zipper”, can significantly reduce single-stranded DNA-RNA region interaction signals during various kinds of RNA selection. By performing the selection against both G-quadruplex, we have identified novel hits that interact with RNA targets and the results are validated through binding. This study demonstrates that the “DEL Zipper” method is a robust screening assay that has potential for discovering small molecule ligands for diverse RNA targets.
迄今为止,由于缺乏可靠的筛选分析,rna靶向化学物质尚处于探索阶段。在这项研究中,我们提出了一种新的rna靶向小分子筛选方法,使用专门的dna编码文库(DEL)。我们的研究结果表明,在各种RNA选择过程中,称为“DEL Zipper”的特殊DEL文库可以显著减少单链DNA-RNA区域相互作用信号。通过对g -四重体进行选择,我们发现了与RNA靶点相互作用的新靶点,并通过结合验证了结果。这项研究表明,“DEL Zipper”方法是一种强大的筛选试验,具有发现不同RNA靶标的小分子配体的潜力。
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引用次数: 0
Corrigendum to “Varieties of Interactions of anti-CD133 Aptamers with Cell Cultures from Patient Glioblastoma” [SLAS Discov. 2024 Nov 16;29(8):100195] “抗cd133适配体与胶质母细胞瘤患者细胞培养物的各种相互作用”的更正[SLAS发现,2024年11月16日;29(8):100195]。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-12-01 DOI: 10.1016/j.slasd.2024.100199
Olga Antipova , Valeria Moiseenko , Fatima Dzarieva , Ekaterina Savchenko , Igor Pronin , Galina Pavlova , Alexey Kopylov
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引用次数: 0
Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors 开源亲电片段筛选平台确定UCHL1共价抑制剂的化学起点。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-12-01 DOI: 10.1016/j.slasd.2024.100198
Scott B. Ficarro , Zachary H. Marto , Nicholas M. Girardi , Dingyu Deng , Isabella Jaen Maisonet , Guillaume Adelmant , Laura E. Fleming , Mona Sharafi , Isidoro Tavares , Andrew Zhao , HyoJeon Kim , Hyuk-Soo Seo , Sirano Dhe-Paganon , Sara J. Buhrlage , Jarrod A. Marto
Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple Python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 < 5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.
基于靶标的共价片段文库筛选与质谱已经成为一种强大的策略,以确定小分子抑制剂的化学起点或在感兴趣的蛋白质上找到新的结合口袋。这些文库跨越不同的化学空间,包含少量的化合物。针对纯化蛋白目标筛选共价片段减少了对质谱仪在绝对通量、检测限和动态范围方面的需求。鉴于这些宽松的分析要求,我们寻求开发一种开源的、中等通量的质谱系统,用于基于靶标的共价片段筛选。我们的平台包括自动化,双LC脱盐柱集成电喷雾电离快速样品导入和质谱检测。该系统通过一个简单的python图形用户界面运行在商品微控制器板上,允许与各种液相色谱和质谱仪器集成。我们提供了用于片段池、构建样本批次的脚本,以及用于数据处理和可视化的例程。该系统能够以混合格式每天对~ 10,000个共价片段进行初步筛选。在一项概念验证研究中,我们进行了一次和二次筛选,以确定27个针对UCHL1的命中片段,UCHL1是一种去泛素化酶,正在成为多种临床适应症中感兴趣的药物靶点。我们通过一系列正交生化和化学蛋白质组学分析验证和分类这些共价化合物。最有希望的氯乙酰胺共价片段体外抑制UCHL1活性(IC50)
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引用次数: 0
High throughput application of the NanoBiT Biochemical Assay for the discovery of selective inhibitors of the interaction of PI3K-p110α with KRAS 高通量应用NanoBiT生化检测发现PI3K-p110α与KRAS相互作用的选择性抑制剂。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-12-01 DOI: 10.1016/j.slasd.2024.100197
Mohamed Ismail , Gareth Davies , Graham Sproat , Tiziana Monteverde , Jonathan Tart , Marta Acebrón-García-de-Eulate , Andrea Gohlke , David Hancock , Santosh Adhikari , Sandra Stefanovic-Barrett , David M Smith , Vikki Flemington , Emma S. Gleave-Hanford , Geoffrey A. Holdgate , Jason G. Kettle , Julian Downward
The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target. Despite the weak binding affinity of the p110α/KRAS interaction (KD = 3 μM), the NBBA proved to be robust and displayed excellent Z’-factor statistics during the HTS primary screening of 726,000 compounds, which led to the identification of 8,000 active compounds. A concentration response screen comparing KRAS/p110α with two closely related PI3K isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of an IC50 for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC50 < 2 μM. By using Differential Scanning Fluorimetry (DSF) we confirmed several compounds that bind directly to purified p110α. The most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds.
NanoBiT生化实验(NBBA)是NanoBiT细胞实验的生化形式,旨在筛选哺乳动物细胞裂解物中的弱蛋白-蛋白相互作用(PPIs)。在这里,我们提出了一个高通量筛选(HTS)应用NBBA筛选小分子和片段文库,以确定阻断KRAS-G12D与磷脂酰肌醇3-激酶(PI3K) p110α相互作用的化合物。这种相互作用促进PI3K活性,从而促进细胞生长、增殖和存活,是小鼠肺癌模型中肿瘤发生和生长所必需的,而对正常成年小鼠的健康影响很小,这表明p110α/KRAS相互作用作为肿瘤药物靶点的意义。尽管p110α/KRAS相互作用的结合亲和力较弱(KD = 3 μM),但在HTS对726,000个化合物的初步筛选中,NBBA被证明是稳健的,并表现出良好的Z′因子统计,最终鉴定出8,000个活性化合物。通过比较KRAS/p110α与两个密切相关的PI3K亚型(p110δ和p110γ)的浓度响应筛选,鉴定出选择性的p110α特异性化合物,并能够推导出这些命中的IC50。我们发现了大约30种化合物对p110α的选择性比p110δ和p110γ高20倍以上,IC50 < 2 μM。通过差示扫描荧光法(DSF),我们确认了几个直接与纯化的p110α结合的化合物。最有效的命中将随后与p110α共结晶,以帮助进一步阐明相互作用的性质和扩展优化这些化合物。
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引用次数: 0
Varieties of interactions of anti-CD133 aptamers with cell cultures from patient glioblastoma 抗 CD133 嵌合体与胶质母细胞瘤患者细胞培养物之间的各种相互作用。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-11-17 DOI: 10.1016/j.slasd.2024.100195
Olga Antipova , Valeria Moiseenko , Fatima Dzarieva , Ekaterina Savchenko , Igor Pronin , Galina Pavlova , Alexey Kopylov
Development of aptatheranostics for glioblastoma (GB) requires investigating aptamer interactions with cells. The paper has described flow cytometry (FC) assessment of direct interactions of fluorescent anti-CD133 aptamers with cells, focusing on cell cultures derived from patient GB (CCPGB). Conventional cell lines with different levels of CD133 mRNA, Caco-2 and HCT116, were used to compare interactions with known 2′FY-RNA aptamer A15 and DNA aptamers of Ap and Cs series, labeled with FAM and Cy5. In addition, interactions of certain non-aptameric oligonucleotides were studied. In the case of antibody interactions with cells, FC signals, mean fluorescence intensities (MFIs), correlated with sizable amounts of CD133 mRNA in Caco-2 cells, and CCPGBs 107 and G01. Unexpectedly, MFI per se could not be the solid indicator of specific interactions of aptamer - CD133/cell. Instead, two types of interactions, target CD133-driven and off-target membrane-associated ones, contribute to MFI. The latter was notably observed for CCPGB Sus/fP2 with tiny CD133 mRNA amount. To prove specificity of aptamer - CD133/cell interactions, titration experiments have been performed, revealing half-saturation concentrations of 120±27 for 2′FY-RNA A15 and 180±12 for DNA Cs5 with Caco-2 cells. This knowledge is an essential step to develop aptatheranostics for GB.
开发治疗胶质母细胞瘤(GB)的适配体疗法需要研究适配体与细胞的相互作用。本文介绍了流式细胞术(FC)对荧光抗 CD133 合体与细胞直接相互作用的评估,重点是来自 GB 患者(CCPGB)的细胞培养物。研究人员利用具有不同 CD133 mRNA 水平的传统细胞系 Caco-2 和 HCT116,比较了它们与已知的 2'FY-RNA 嵌合体 A15 以及用 FAM 和 Cy5 标记的 Ap 和 Cs 系列 DNA 嵌合体的相互作用。 此外,还研究了某些非嵌合体寡核苷酸的相互作用。在抗体与细胞相互作用的情况下,FC 信号、平均荧光强度(MFIs)与 Caco-2 细胞、CCPGB 107 和 G01 中大量的 CD133 mRNA 相关。出乎意料的是,MFI 本身并不能作为灵媒-CD133/细胞特异性相互作用的可靠指标。相反,两种类型的相互作用(目标 CD133 驱动的相互作用和非目标膜相关的相互作用)对 MFI 有贡献。在 CD133 mRNA 含量极低的 CCPGB Sus/fP2 中明显观察到了后者。为了证明适配体-CD133/细胞相互作用的特异性,进行了滴定实验,结果显示,2'FY-RNA A15 和 DNA Cs5 与 Caco-2 细胞的半饱和浓度分别为 120±27 和 180±12。这一知识是开发用于 GB 的合体止吐药的重要一步。
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引用次数: 0
TGF-β receptor-specific NanoBRET Target Engagement in living cells for high-throughput kinase inhibitor screens 活细胞中的 TGF-β 受体特异性 NanoBRET 靶标参与,用于高通量激酶抑制剂筛选。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-11-12 DOI: 10.1016/j.slasd.2024.100196
Marius Wits , Nicole Haarmans , Gonzalo Sanchez-Duffhues , Marie-José Goumans
Targeting transforming growth factor-β (TGF-β) receptors is a promising pharmacological approach to normalize aberrant signaling in genetic and non-genetic TGF-β associated diseases including fibrosis, cancer, cardiovascular and musculoskeletal disorders. To identify novel TGF-β receptor kinase inhibitors, methods like in vitro kinase assays, western blot or transcriptional reporter assays are often used for screening purposes. While these methods may have certain advantages, the lack of integration of key features such as receptor specificity, high-throughput capability, and cellular context resemblance remains a major disadvantage. This deficiency could ultimately hinder the translation of study outcomes into later (clinical) stages of drug development. In this study, we introduce an adjusted and optimized live cell NanoBRET Target Engagement (TE)-based method to identify TGF-β receptor specific kinase inhibitors. This comprehensive toolkit contains various TGF-β type I and type II receptors, with corresponding nanoBRET tracers, and disease-related cell lines, including novel non-commercially available materials. The nanoBRET capacity and kinase inhibitory window can be significantly enhanced for functional measurements when stable expression cell lines and substantially low tracer concentrations are used. In addition, this system can be tailored to study TGF-β associated genetic disorders and possibly be used to screen for disease-specific therapeutics. Therefore, the use of this optimized, live cell, antibody-independent nanoBRET Target Engagement assay is highly encouraged for future high-throughput compound screens targeting TGF-β/BMP receptors.
靶向转化生长因子-β(TGF-β)受体是一种很有前景的药理学方法,可使与 TGF-β 相关的遗传性和非遗传性疾病(包括纤维化、癌症、心血管疾病和肌肉骨骼疾病)中的异常信号转导正常化。为鉴定新型 TGF-β 受体激酶抑制剂,通常采用体外激酶测定、Western 印迹或转录报告测定等方法进行筛选。虽然这些方法有一定的优势,但缺乏对受体特异性、高通量能力和细胞环境相似性等关键特征的整合仍是其主要缺点。这一缺陷最终会阻碍研究成果转化到药物开发的后期(临床)阶段。在本研究中,我们介绍了一种经过调整和优化的基于活细胞 NanoBRET Target Engagement (TE) 的方法,用于鉴定 TGF-β 受体特异性激酶抑制剂。这个综合工具包包含各种 TGF-β I 型和 II 型受体、相应的 nanoBRET 示踪剂以及与疾病相关的细胞系,包括新型非商业性材料。当使用稳定表达的细胞系和低浓度示踪剂时,纳米BRET能力和激酶抑制窗口可显著增强功能测量。此外,该系统还可定制用于研究与 TGF-β 相关的遗传疾病,并可能用于筛选疾病特异性疗法。因此,在未来针对 TGF-β/BMP 受体的高通量化合物筛选中,我们非常鼓励使用这种优化的、活细胞的、不依赖抗体的 nanoBRET 靶点接合测定。
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引用次数: 0
The history, landscape, and outlook of human cell line authentication and security 人类细胞系鉴定与安全的历史、现状和前景。
IF 2.7 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-11-08 DOI: 10.1016/j.slasd.2024.100194
Elijah Harbut , Yiorgos Makris , Alexander Pertsemlidis , Leonidas Bleris
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引用次数: 0
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