SLAS Discovery最新文献

AI-based analysis of label-free live cell imaging of T-cell mediated tumor killing assay enables competitive and robust hit calling 基于人工智能的无标记活细胞成像分析t细胞介导的肿瘤杀伤实验使竞争和强大的打击呼叫。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2026-02-03 DOI: 10.1016/j.slasd.2026.100297

Josefa dela Cruz-Chuh , Daniel Siegismund , John Moffat , Stephan Heyse , Katherine R. Kozak , Stephan Steigele

For the discovery and optimization of personalized cancer treatments using immune cell therapeutics, such as T-cell receptor (TCR-T) therapy and bispecific antibodies (BsAbs), robust functional activity of candidates must be confirmed in immune-mediated killing assays. In these assays, co-cultures of several cell lines and patient-derived primary cancer cells often are imaged live using automated microscopy. Conventionally, such assays use fluorescent dyes or specifically expressed nuclear proteins for labeling, followed by classical image analysis reliant on cell segmentation. They are therefore subject to artifacts like phototoxicity and bleaching, inaccurate segmentation due to the typical variations in visual phenotype with time as well as requiring the constant adaptation of analysis parameters for experiments across different human tissue types or donors.

Here we present a new approach utilizing brightfield images in combination with a hands-free, scalable artificial intelligence (AI)-based analysis workflow, requiring no fluorescent markers at all. We have applied this new workflow to a T-cell mediated killing assay and benchmarked it against current semi-manual, cell segmentation-based analysis of fluorescent images. We found that the new workflow performs well on phenotypically diverse cancer cells, with greater efficiency though elimination of manual adjustment steps, and produces results of equivalent consistency.

We conclude that this AI-based analysis workflow has the potential to substantially simplify T-cell mediated live cell killing assays, eliminating the need for labeling, and allows their efficient analysis, operating on brightfield images and thus avoiding time-consuming and difficult analysis of labeled images using classical segmentation-based analysis.

为了发现和优化使用免疫细胞疗法(如t细胞受体（TCR-T）疗法和双特异性抗体（BsAbs）的个性化癌症治疗，候选药物的强大功能活性必须在免疫介导的杀伤试验中得到证实。在这些检测中，通常使用自动显微镜对几种细胞系和患者来源的原发癌细胞的共培养物进行实时成像。通常，这种检测使用荧光染料或特异性表达的核蛋白进行标记，然后进行依赖于细胞分割的经典图像分析。因此，它们受到诸如光毒性和漂白等人工制品的影响，由于视觉表型随时间的典型变化而导致的不准确分割，以及需要不断适应不同人体组织类型或供体的实验分析参数。在这里，我们提出了一种新的方法，利用明场图像结合免提，可扩展的基于人工智能（AI）的分析工作流程，根本不需要荧光标记。我们已经将这种新的工作流程应用于t细胞介导的杀伤试验，并将其与当前基于荧光图像的半手动细胞分段分析进行了基准测试。我们发现，新的工作流程在表型多样化的癌细胞上表现良好，通过消除手动调整步骤，效率更高，并产生等效一致性的结果。我们得出的结论是，这种基于人工智能的分析工作流程有可能大大简化t细胞介导的活细胞杀伤试验，消除了标记的需要，并允许其高效分析，在明场图像上操作，从而避免使用经典的基于分割的分析对标记图像进行耗时和困难的分析。

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引用次数: 0

PCIM: Learning pixel attributions via pixel-wise channel isolation mixing in high content imaging PCIM：在高内容成像中通过像素通道隔离混合学习像素属性。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-12-01 DOI: 10.1016/j.slasd.2025.100287

Daniel Siegismund, Mario Wieser, Stephan Heyse, Stephan Steigele

Deep Neural Networks (DNNs) have shown remarkable success in various computer vision tasks. However, their black-box nature often leads to difficulty in interpreting their decisions, creating an unfilled need for methods to explain the decisions, and ultimately forming a barrier to their wide acceptance especially in biomedical applications. This work introduces a novel method, Pixel-wise Channel Isolation Mixing (PCIM), to calculate pixel attribution maps, highlighting the image parts most crucial for a classification decision but without the need to extract internal network states or gradients. Unlike existing methods, PCIM treats each pixel as a distinct input channel and trains a blending layer to mix these pixels, reflecting specific classifications. This unique approach allows the generation of pixel attribution maps for each image, but agnostic to the choice of the underlying classification network. Benchmark testing on three application relevant, diverse high content Imaging datasets show state-of-the-art performance, particularly for model fidelity and localization ability in both, fluorescence and bright field High Content Imaging. PCIM contributes as a unique and effective method for creating pixel-level attribution maps from arbitrary DNNs, enabling interpretability and trust.

深度神经网络（dnn）在各种计算机视觉任务中取得了显著的成功。然而，它们的黑箱性质往往导致难以解释它们的决定，对解释这些决定的方法产生了未填补的需求，并最终形成了它们被广泛接受的障碍，特别是在生物医学应用中。这项工作引入了一种新的方法，像素通道隔离混合（PCIM），用于计算像素属性图，突出显示对分类决策最关键的图像部分，但不需要提取内部网络状态或梯度。与现有的方法不同，PCIM将每个像素视为一个不同的输入通道，并训练一个混合层来混合这些像素，反映特定的分类。这种独特的方法允许为每个图像生成像素属性图，但与底层分类网络的选择无关。在三个应用相关的、不同的高内容成像数据集上的基准测试显示了最先进的性能，特别是在荧光和亮场高内容成像的模型保真度和定位能力方面。PCIM作为一种独特而有效的方法，可以从任意dnn中创建像素级属性图，从而实现可解释性和信任度。

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引用次数: 0

Fluorescent probe-based detection of outer membrane damage of Gram-negative bacteria 基于荧光探针的革兰氏阴性菌外膜损伤检测。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-12-01 DOI: 10.1016/j.slasd.2025.100290

Bingchen Li, Qionglu Duan, Wenjing Shi, Yinghong Li, Yuanjuan Wei, Shuyi Si, Yucheng Wang, Minghua Wang, Yan Li

Infections caused by drug-resistant bacteria, particularly Gram-negative species, represent one of the most significant global public health challenges. The outer membrane (OM) is a crucial target for the development of drugs against Gram-negative bacteria. For the confirmation of the mechanism of OM-targeted drugs, the evaluation of OM damage is necessary. In this study, we optimized the method for detecting OM damage employing N-phenyl-1-naphthylamine (NPN) and ethidium bromide (EtBr) as probes in Escherichia coli (E. coli), including the bacterial loading, probe concentration, and incubation time. In addition, three OM-targeted compounds with distinctive mechanisms: polymyxin B, ACHN-975, and IMB-0042, were used to investigate the advantages and problems of two fluorescent probes. The compound fluorescence and quenching effect on the probes were detected. It was found that IMB-0042 caused fluorescence quenching on NPN. The treatment time of compounds with different OM damage mechanisms had a significant impact on the detection. For polymyxin B-treated E. coli cells, which directly disrupts OM, significant fluorescence changes were observed with both probes at short (30 min) and long durations (1–5 h). In contrast, compounds ACHN-975 and IMB-0042, which inhibit OM biosynthesis, showed detectable fluorescence only at long durations. In summary, this study presents a detailed scheme for the detection of OM damage induced by different antibiotics based on NPN and EtBr.

耐药细菌，特别是革兰氏阴性菌引起的感染是全球公共卫生面临的最重大挑战之一。外膜（OM）是开发抗革兰氏阴性菌药物的重要靶点。为了确认OM靶向药物的作用机制，OM损伤的评估是至关重要的。本研究以n -苯基-1-萘胺（NPN）和溴化乙啶（EtBr）为探针，对大肠杆菌（E. coli）中OM损伤的检测方法进行了优化，包括细菌载量、探针浓度和培养时间。此外，我们还利用多粘菌素B （polymyxin B）、ACHN-975和IMB-0042这三种具有不同机制的om靶向化合物，研究了两种荧光探针的优势和存在的问题。检测了探针的复合荧光和猝灭效应。发现IMB-0042在NPN上引起荧光猝灭。不同OM损伤机制的化合物处理时间对检测结果有显著影响。对于直接破坏OM的多粘菌素b处理的大肠杆菌细胞，两种探针在短时间（30分钟）和长时间（1-5小时）都观察到显著的荧光变化。相比之下，抑制OM生物合成的化合物ACHN-975和IMB-0042仅在长时间内显示可检测的荧光。综上所述，本研究提出了基于NPN和EtBr检测不同抗生素引起的OM损伤的详细方案。

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引用次数: 0

High-throughput screening identifies non-nucleoside inhibitors of the SARS-CoV-2 polymerase with novel mechanisms 高通量筛选鉴定具有新机制的SARS-CoV-2聚合酶非核苷类抑制剂

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-12-01 DOI: 10.1016/j.slasd.2025.100289

Colin R. Woodford, Kristine E. Frank, Haizhong Zhu, Gilman Dionne, Michael R. Schrimpf, Sujatha M. Gopalakrishnan, Nathaniel L. Elsen

The RNA-dependent RNA polymerase (RdRp) of coronaviruses, comprising highly conserved non-structural proteins, is a critical player in the viral lifecycle and represents a promising target for developing pan-coronavirus antivirals. Despite substantial efforts to identify RdRp inhibitors through drug repurposing and novel compound discovery campaigns, potent in vitro non-nucleoside inhibitors remain elusive. In this study, we detail the development of a robust PicoGreen assay, which facilitated the screening of AbbVie's extensive chemical library, encompassing over 900,000 small molecules, against the SARS-CoV-2 RdRp. Through a combination of biochemical and biophysical assays, we identified two potent non-nucleoside compounds with activity against our PicoGreen beta-coronavirus panel. Mechanism of action investigations revealed these compounds bind exclusively to the nsp12–8 complex, unveiling a potentially unique inhibitory mechanism. These compounds serve as valuable starting points for structure-activity relationship (SAR) explorations and potential therapeutic leads.

冠状病毒的RNA依赖性RNA聚合酶（RdRp）由高度保守的非结构蛋白组成，在病毒生命周期中起着关键作用，是开发泛冠状病毒抗病毒药物的一个有希望的靶点。尽管通过药物再利用和新化合物发现活动来鉴定RdRp抑制剂做出了大量努力，但有效的体外非核苷类抑制剂仍然难以捉摸。在本研究中，我们详细介绍了一种强大的PicoGreen检测方法的开发，该方法有助于筛选艾伯维广泛的化学文库，包括超过900,000个小分子，以对抗SARS-CoV-2 RdRp。通过结合生化和生物物理检测，我们发现了两种有效的非核苷类化合物，它们对我们的PicoGreen β -冠状病毒抗体具有活性。作用机制研究表明，这些化合物仅与nsp12-8复合物结合，揭示了一种潜在的独特抑制机制。这些化合物为结构-活性关系（SAR）的探索和潜在的治疗线索提供了有价值的起点。

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引用次数: 0

Streamlining cellular thermal shift assay for ultra-high throughput screening 精简细胞热移分析超高通量筛选。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-11-29 DOI: 10.1016/j.slasd.2025.100293

Pascal Lambertz, Loretta Hamacher, Jana Flegel, Philipp Pflüger, Torsten Feller, Mike Küster, Tom Stockter, Tommaso Mari, Yousef Morcos, Martin Adamczewski

The Cellular Thermal Shift Assay (CETSA) has emerged as a powerful tool for evaluating drug-target interactions in live cells, yet its application in ultra-high throughput screening (uHTS) has been limited by technical constraints. In this study, we present significant advancements in CETSA methodology, focusing on the development of an innovative isothermal CETSA platform for primary uHTS screen in 1536 well plates and a Gradient Peltier Device (GPD) for retesting hits in full melting curve CETSA. Our optimized isothermal CETSA allows for the evaluation of adherent cells in their physiological state, enhancing assay performance through a controlled thermal ramp-up instead of traditional heat shock methods and utilizing highly sensitive luminescence detection. The GPD enables all steps of a full melt curve CETSA to be conducted in one single flat bottom microtiter plate, improving data quality by reducing handling and pipetting steps and improving temperature control. We benchmarked both methods against an established fluorescence polarization assay using the androgen receptor as a model target. Results demonstrated a strong correlation between both CETSA methods and the fluorescence polarization assay, indicating the potential for identifying true binders while minimizing false positives. Our findings highlight the utility of this optimized CETSA platform for high throughput drug discovery, paving the way for more effective screening of true binders in live cells.

细胞热移测定（CETSA）已成为评估活细胞中药物-靶标相互作用的有力工具，但其在超高通量筛选（uHTS）中的应用受到技术限制。在这项研究中，我们介绍了CETSA方法的重大进展，重点是开发了一种创新的等温CETSA平台，用于1536个井板的初级uHTS筛检，以及一种梯度Peltier设备（GPD），用于重新测试全熔化曲线CETSA中的hit。我们优化的等温CETSA允许在其生理状态下评估贴壁细胞，通过控制热上升而不是传统的热休克方法和利用高灵敏度的发光检测来提高检测性能。GPD使全熔体曲线CETSA的所有步骤都能在一个平底微量滴定板上进行，通过减少处理和移液步骤以及改善温度控制来提高数据质量。我们用雄激素受体作为模型靶标，对这两种方法进行基准测试。结果表明，在CETSA方法和荧光偏振法之间存在很强的相关性，表明在最大限度地减少假阳性的同时识别真正的结合物的潜力。我们的发现强调了这种优化的CETSA平台在高通量药物发现中的实用性，为更有效地筛选活细胞中的真正结合物铺平了道路。

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引用次数: 0

An ELISA for discovering protein-protein interaction inhibitors: Blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook as a test case 用于发现蛋白质相互作用抑制剂的ELISA：阻断螺旋体鞭毛钩亚基之间的赖氨酸交联作为测试案例。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-11-25 DOI: 10.1016/j.slasd.2025.100292

Maithili Deshpande , Michael J. Lynch , Kurni Kurniyati , Chunhao Li , Brian R. Crane

The inhibition of a specific protein-protein interaction is often difficult to achieve in targeted drug design. We report the development and optimization of a general-purpose, readily implemented enzyme-linked immunosorbent assay (ELISA) for high-throughput screening to identify small-molecule inhibitors of protein interactions. This ELISA does not involve the use of any capture antibodies, probes, or compounds coated on the plate and represents a general strategy to identify inhibitors of a given protein-protein interaction. We demonstrate its utility in blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook by targeting the native form of the FlgE protein, which differs from the strategies used in previous assays. The flagellar hook protein FlgE self-catalyzes the formation of a lysinoalanine (Lal) inter-subunit crosslink that is essential for the motility, and thus, infectivity of spirochetes. Prevention of Lal crosslinking through inhibition with small molecules thus represents an avenue for therapeutic development against spirochete-related diseases, such as Lyme and syphilis. Screening a library of ∼700 compounds with the ELISA confirmed that hexachlorophene, currently the only known inhibitor of Lal crosslinking in FlgE, effectively inhibits the crosslinking reaction. In addition, the assay identified two new potential inhibitors, honokiol and zafirlukast, and several activators which belong to well-known classes of antibiotics.

在靶向药物设计中，抑制特定蛋白质-蛋白质相互作用通常是很难实现的。我们报告了一种通用的、易于实施的酶联免疫吸附试验（ELISA）的开发和优化，用于高通量筛选，以鉴定蛋白质相互作用的小分子抑制剂。该ELISA不涉及使用任何捕获抗体，探针或涂覆在板上的化合物，代表了识别给定蛋白质-蛋白质相互作用抑制剂的一般策略。我们证明了它在阻断螺旋体鞭毛钩亚基之间赖氨酸丙氨酸交联的效用，通过靶向FlgE蛋白的天然形式，这与以前的分析中使用的策略不同。鞭毛钩蛋白FlgE自催化赖氨酸丙氨酸（Lal）亚基间交联的形成，这对螺旋体的运动性和感染性至关重要。因此，通过抑制小分子来预防Lal交联是开发治疗螺旋体相关疾病（如莱姆病和梅毒）的途径。用ELISA筛选了约700个化合物的文库，证实了目前已知的FlgE中唯一的Lal交联抑制剂六氯苯（hexachlorophene）可以有效抑制交联反应。此外，该检测还发现了两种新的潜在抑制剂，honokiol和zafirlukast，以及几种属于知名抗生素类别的激活剂。

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引用次数: 0

MICRO-TAG enzyme complementation enables quantification of cellular drug-target engagement in temperature series MICRO-TAG酶互补可以在温度序列中定量细胞药物靶标参与。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-11-22 DOI: 10.1016/j.slasd.2025.100291

Ivan Babic , Nikolas Bryan , Claire Cunningham , Avery Sampson , Daniel Starczynowski , Elmar Nurmemmedov

Drug discovery for challenging drug targets necessitates the proteomic complexities of the cellular milieu for contextual target folding and function. Conventional biophysical methods for assessing drug interaction with a target are often not sufficiently suited for drug discovery as they impose acellular environment on the target and rely on recombinant purified protein material. In contrast, cell target engagement offers a powerful paradigm for drug discovery, through measurement of transitions in the thermodynamic state of a target protein, as it engages with drug molecules in the cell. Split-enzyme cell target engagement methods offer scaled utility during early drug discovery. Here, we describe a novel highly sensitive and scalable fluorescence-based cell target engagement method that leverages complementation of split-RNase S. This offers a unique combination of procedural and biophysical advantages, enabling its seamless integration with various instruments and applications designed for fluorescence detection. Most importantly, this new method allows for quantitation of cell target engagement in programmable temperature series format, consistent with conventional thermal shift assays, rather than at a single melting temperature. We demonstrate the sensitivity and versatility of this approach for drug discovery using targets MAPK1, KRAS, and UBE2N.

具有挑战性的药物靶点的药物发现需要细胞环境的蛋白质组学复杂性来进行上下文靶点折叠和功能。用于评估药物与靶标相互作用的传统生物物理方法通常不适合用于药物发现，因为它们对靶标施加了非细胞环境，并且依赖于重组纯化的蛋白质材料。相比之下，细胞靶标结合为药物发现提供了一个强有力的范例，通过测量靶蛋白与细胞内药物分子结合时热力学状态的转变。分裂酶细胞靶标接合方法在早期药物发现中提供了规模效用。在这里，我们描述了一种新颖的高灵敏度和可扩展的基于荧光的细胞靶标接合方法，该方法利用了分裂rna酶s的互补。这提供了程序和生物物理优势的独特组合，使其能够与各种为荧光检测设计的仪器和应用程序无缝集成。最重要的是，这种新方法允许以可编程温度序列格式定量细胞靶标接合，与传统的热移测定法一致，而不是在单一的熔化温度下。我们证明了这种方法在使用靶点MAPK1、KRAS和UBE2N的药物发现中的敏感性和通用性。

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引用次数: 0

Protocols and research articles in 3D biology: technologies and methodologies reshaping 3D cell culture 协议和研究文章在3D生物学：技术和方法重塑3D细胞培养。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-11-19 DOI: 10.1016/j.slasd.2025.100288

Glauco R. Souza, Evan Cromwell, Madhu Nag

引用次数: 0

High-content immunofluorescence assay detecting PD-L1 expression changes in head and neck cancer patient-derived cultures 高含量免疫荧光法检测头颈癌患者来源培养物中PD-L1表达变化。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-11-12 DOI: 10.1016/j.slasd.2025.100286

Yuen-Keng Ng , Stacy Magdalene Abbang , Jishi Ye , Wenying Piao , Yu-Xiong Su , Jason Ying Kuen Chan , Chin Wang Lau , Cecilia Pik Yuk Lau , Hui Li , Vivian Wai Yan Lui

Clinical uses of monoclonal antibodies against immune checkpoint molecules, such as the Program Death-Ligand 1 (PD-L1) or Program Death Protein-1 (PD-1), has transformed cancer therapy across pan-cancers. In addition to antibody therapies, there is a growing interest in identifying small molecules that could modulate PD-L1 levels in cancer cells. Yet, most current PD-L1 assays are not robust enough to be developed for drug screening purposes. Here, we report the development of a sensitive PD-L1 immunofluorescence assay that can capture PD-L1 expression heterogeneity in HNC patient tumor cultures and allows relative quantification of PD-L1 levels in cells in a streamlined and robust manner. Furthermore, this imaging-based assay can capture additional spatial or subcellular localization information of PD-L1 expression in patient cultures and has the potential to be combined with other image-based assays for future drug development purposes. Importantly, we demonstrated that this assay was robust enough to evaluate dose-dependent PD-L1-modulatory effects of drugs in patient-derived tumor cultures and demonstrated patient-to-patient variability of drug responses for PD-L1 modulation. This assay has the potential to be adopted for high-throughput drug screening for identifying small molecules modulators of PD-L1 using individual patient tumor cultures of various cancer types.

针对免疫检查点分子的单克隆抗体的临床应用，如程序死亡配体1 （PD-L1）或程序死亡蛋白1 (PD-1)，已经改变了泛癌症的癌症治疗。除了抗体疗法外，人们对确定可以调节癌细胞中PD-L1水平的小分子也越来越感兴趣。然而，目前大多数PD-L1检测方法还不够强大，无法用于药物筛选。在这里，我们报告了一种敏感的PD-L1免疫荧光测定方法的发展，该方法可以捕获HNC患者肿瘤培养物中PD-L1表达的异质性，并允许以流线型和稳健的方式对细胞中PD-L1水平进行相对量化。此外，这种基于成像的检测可以捕获患者培养物中PD-L1表达的额外空间或亚细胞定位信息，并有可能与其他基于图像的检测相结合，用于未来的药物开发目的。重要的是，我们证明了该分析足够稳健，可以评估药物在患者来源的肿瘤培养物中剂量依赖性的PD-L1调节作用，并证明了PD-L1调节药物反应的患者对患者的可变性。该试验有可能被用于高通量药物筛选，以识别PD-L1的小分子调节剂，使用不同癌症类型的个体患者肿瘤培养物。

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引用次数: 0

Validation, optimization, and quality assessment of a neuro-behavioral in vivo assay for phenotypic high-content evaluation of anesthetics 麻醉药物表型高含量评价的神经行为体内试验的验证、优化和质量评估

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-10-25 DOI: 10.1016/j.slasd.2025.100285

Günther K.H. Zupanc, Mariam Ahmed

In vivo assays based on aquatic model organisms have played a pivotal role in the development of anesthetics and their pharmacological and physiological characterization. They have been designed primarily as cost-effective tools for initial screening of compounds, prioritizing high throughput and simple measurement of a behavioral readout. Common limitations include manual recoding and analysis of behavioral readouts, thereby introducing potential bias; focus on the binary endpoints of a single behavior; restriction of the assessment of drug effects to the behavioral level, thus being agnostic of neural and/or molecular targets; and low content overall. The Neuro-Behavioral Assay presented here overcomes these restrictions. It is based on the non-invasive recording of the electric organ discharge of an electric fish, serving as a proxy of the neural activity of an endogenous brainstem oscillator, the pacemaker nucleus. From the single recording of the discharge, three behavioral readouts (reflecting the fish’s locomotor activity as well as the frequency and the rate of amplitude-frequency modulations of the synchronized neural pacemaker oscillations) are extracted and analyzed automatically, thereby eliminating the operator’s bias. The behavioral output is recorded on a continuous scale, thus enabling assessment of gradual changes in behavior induced by anesthetics and during recovery from anesthesia. By testing the effects of three anesthetics (tricaine methanesulfonate, eugenol, and urethane), protocols for running the assay have been optimized and validated through in vitro electrophysiology. Assessment of the results by statistical analysis and measures of assay performance, such as the Signal-to-Noise Ratio and the Z-Factor, have demonstrated a high quality of the Neuro-Behavioral Assay. Overall, while keeping the costs at a moderate level, the Neuro-Behavioral Assay generates high content and offers the opportunity for target deconvolution. It is, therefore, uniquely suited for bridging the current gap in anesthetic drug discovery between the identification of candidate molecules through high-throughput screening assays and the preclinical testing of lead compounds through assays employing mammalian model systems.

基于水生模式生物的体内试验在麻醉药的发展及其药理学和生理学表征中发挥了关键作用。它们主要被设计为具有成本效益的工具，用于化合物的初始筛选，优先考虑高通量和简单的行为读数测量。常见的限制包括手动重新编码和分析行为读数，从而引入潜在的偏差；关注单一行为的二元端点；将药物作用的评估限制在行为水平，从而无法确定神经和/或分子靶点；整体内容也很低。这里提出的神经行为试验克服了这些限制。它是基于对电鱼的电器官放电的非侵入性记录，作为内源性脑干振荡器（起搏器核）的神经活动的代理。从单次放电记录中，提取并自动分析三个行为读数（反映鱼的运动活动以及同步神经起搏器振荡的幅度频率调制的频率和速率），从而消除操作员的偏差。行为输出记录在一个连续的尺度上，从而可以评估麻醉引起的行为的逐渐变化和麻醉恢复期间。通过测试三种麻醉剂（三卡因甲磺酸盐、丁香酚和氨基甲酸乙酯）的效果，优化并通过体外电生理学验证了运行该分析的方案。通过统计分析和检测性能测量（如信噪比和z因子）对结果进行评估，证明了神经行为检测的高质量。总的来说，在将成本保持在中等水平的同时，神经行为分析产生高含量，并提供目标反卷积的机会。因此，它非常适合填补目前在麻醉药物发现方面的空白，即通过高通量筛选测定候选分子和通过采用哺乳动物模型系统测定先导化合物的临床前测试。

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引用次数: 0