SLAS Discovery最新文献

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

SLAS Discovery

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

SLAS Discovery

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

SLAS Discovery

Pub Date : 2024-11-08 DOI: 10.1016/j.slasd.2024.100194

Elijah Harbut , Yiorgos Makris , Alexander Pertsemlidis , Leonidas Bleris

引用次数: 0

The development of a novel high-throughput membrane potential assay and a solid-supported membrane (SSM)-based electrophysiological assay to study the pharmacological inhibition of GLUT9/SLC2A9 isoforms in a drug discovery program 开发新型高通量膜电位测定法和基于固体支撑膜（SSM）的电生理学测定法，以研究药物发现计划中对 GLUT9/SLC2A9 同工酶的药理抑制。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2024-11-08 DOI: 10.1016/j.slasd.2024.100193

Antje Pommereau , Francesca Sassone , Alessandro Poli , Marcella De Silvestris , Lia Scarabottolo , Yasmin Zuschlag , Thomas Licher , Felix Bärenz

GLUT9/SLC2A9 is a urate transporter and takes a fundamental role in the maintenance of normal serum urate levels. GLUT9 is the sole transporter of reabsorbed urate from renal epithelial cells to blood, thus making it an ideal pharmacological target for the development of urate-lowering drugs. None of the three currently available assays for studying GLUT9 pharmacological inhibition can support a high throughput drug discovery screening campaign. In this manuscript we present two novel assay technologies which can be used in a drug discovery screening cascade for GLUT9: a GLUT9 membrane potential assay for primary screening; and a solid-supported membrane (SSM)-based supported electrophysiological assay for secondary screening.

GLUT9/SLC2A9 是一种尿酸盐转运体，在维持血清尿酸盐正常水平方面发挥着重要作用。GLUT9 是将肾上皮细胞重吸收的尿酸盐转运到血液中的唯一转运体，因此是开发降尿酸药物的理想药理靶点。目前用于研究 GLUT9 药理抑制的三种检测方法都无法支持高通量药物筛选活动。在本手稿中，我们介绍了两种可用于 GLUT9 药物发现筛选级联的新型检测技术：一种是用于初筛的 GLUT9 膜电位检测法；另一种是用于复筛的基于固体支撑膜 (SSM) 的支撑电生理学检测法。

引用次数: 0

Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ 开发实时监测 Hippo 通路成分 14-3-3 和 TAZ 之间相互作用的活细胞检测方法。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2024-11-05 DOI: 10.1016/j.slasd.2024.100191

Blaž Andlovic , Alexander Wolf , Malgorzata Hiltmann , Bert M. Klebl , Jan Eickhoff , Christian Ottmann

The Hippo pathway plays an important role in organ size control and tissue homeostasis. Dysregulation is involved in many pathologies, including cancer, which has attracted interest in targeting the Hippo pathway. Since the upstream components are bona fide tumor suppressors, it is feasible to target oncogenic downstream targets such as TAZ, a key downstream effector in the Hippo pathway. Its activity is regulated by phosphorylation on multiple sites, with Ser89 playing a critical role in regulation of TAZ activity. Phosphorylation of TAZ at Ser89 promotes binding to 14–3–3 scaffolding proteins, preventing nuclear translocation and abolishing target gene transcription. Here we describe the development of a cell-based assay suitable for high-throughput screening, based on a split NanoLuc luciferase, for monitoring interactions between 14 3–3 and TAZ in living cells. We have validated the assay by screening of a kinase-biased library. The assay can be quickly adapted for higher throughput and thus offers a valuable tool to study new signal inputs involved in regulation of TAZ activity as well as for identification of molecules that modulate the Hippo pathway.

希波通路在器官大小控制和组织稳态中发挥着重要作用。失调涉及包括癌症在内的多种病症，这引起了人们对靶向 Hippo 通路的兴趣。由于上游成分是真正的肿瘤抑制因子，因此以致癌的下游靶点为目标是可行的，例如希波通路的关键下游效应物 TAZ。它的活性受多个位点的磷酸化调控，其中 Ser89 在 TAZ 活性调控中起着关键作用。TAZ在Ser89上的磷酸化会促进与14-3-3支架蛋白的结合，阻止核转位并取消靶基因转录。在此，我们介绍了一种基于细胞的检测方法的开发情况，该方法适用于高通量筛选，它基于分裂的 NanoLuc 荧光素酶，用于监测活细胞中 14-3-3 和 TAZ 之间的相互作用。我们通过筛选激酶偏倚库验证了该检测方法。该检测方法可以快速适应更高的通量，因此为研究参与 TAZ 活性调控的新信号输入以及鉴定调节 Hippo 通路的分子提供了一种有价值的工具。

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引用次数: 0

Rapid-response RNA-fluorescence in situ hybridization (FISH) assay platform for coronavirus antiviral high-throughput screening 用于冠状病毒抗病毒高通量筛选的快速反应 RNA 荧光原位杂交 (FISH) 检测平台

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2024-11-04 DOI: 10.1016/j.slasd.2024.100189

Ryan Chan , Christian Shema Mugisha , Vorada Chuenchob , Stephanie A. Moquin , Ujjini H. Manjunatha , Nadine Jarrousse , Vineet D. Menachery , Xuping Xie , Erika L. Flannery , Richard T. Eastman

Over the past 25 years, the global community has faced challenges posed by three distinct outbreaks of coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of a novel alphacoronavirus canine CoV (CCoV-HuPn2018) in human patients in Malaysia underscores the potential for crossover infections to humans. The threat of the ever-evolving nature of viral infections as well as the lingering health and socioeconomic effects of the recent SARS-CoV-2 pandemic emphasize the urgent need for advanced antiviral drug screening tools that can be quickly implemented to strengthen preparedness and preventive measures against future outbreaks. Here, we present the development and validation of a novel RNA-fluorescence in situ hybridization (FISH) imaging assay as a 384-well, high-throughput rapid response platform for antiviral drug discovery. RNA-FISH is a powerful tool to visualize specific mRNA in cultured cells using a high-content imaging platform. The flexibility of RNA-FISH probe sets allows for the rapid design of viral genome-specific probes, enabling in vitro assay development to test for inhibition of viral replication by either biologic or small molecule inhibitors. Screening of 170 antiviral compounds in concentration-response demonstrates a strong correlation between the RNA-FISH assay and an immunofluorescence assay (IFA) for both human coronaviruses HCoV-OC43 and HCoV-229E. Additionally, we successfully applied this methodology in the context of CCoV strain 1–71, proving rapid development and deployment, opening new avenues for the evaluation of antiviral drugs to potential future emerging threats.

在过去的 25 年中，全球社会面临着严重急性呼吸系统综合征冠状病毒（SARS-CoV）、中东呼吸系统综合征冠状病毒（MERS-CoV）和严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）等三种不同冠状病毒爆发所带来的挑战。在马来西亚的人类患者中发现了一种新型α-冠状病毒犬科 CoV（CCoV-HuPn2018），这凸显了人类受到交叉感染的可能性。病毒感染不断演变的威胁，以及最近 SARS-CoV-2 大流行对健康和社会经济造成的持久影响，都强调了对先进抗病毒药物筛选工具的迫切需要，这些工具可以快速实施，以加强对未来疫情爆发的准备和预防措施。在此，我们介绍了一种新型 RNA 荧光原位杂交（FISH）成像检测方法的开发和验证情况，该方法可作为 384 孔高通量快速反应平台用于抗病毒药物的发现。RNA-FISH 是一种功能强大的工具，可利用高含量成像平台观察培养细胞中的特定 mRNA。RNA-FISH 探针组的灵活性使其能够快速设计病毒基因组特异性探针，从而实现体外检测开发，以测试生物或小分子抑制剂对病毒复制的抑制作用。对 170 种抗病毒化合物进行的浓度反应筛选表明，RNA-FISH 检测和免疫荧光检测 (IFA) 对人类冠状病毒 HCoV-OC43 和 HCoV-229E 都有很强的相关性。此外，我们还成功地将该方法应用于 CCoV 1-71 株，证明了该方法的快速开发和部署，为评估抗病毒药物以应对未来潜在的新威胁开辟了新途径。

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引用次数: 0

Standards for reporting optical biosensor experiments (STROBE): Improving standards in the reporting of optical biosensor-based data in the literature 光学生物传感器实验报告标准（STROBE）：改进文献中基于光学生物传感器的数据报告标准。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2024-10-31 DOI: 10.1016/j.slasd.2024.100192

Paul E. Belcher , Anna Moberg , Michael B. Murphy

The number of peer-reviewed publications that feature biosensor data increases every year. A search of PubMed using common technique terminology, including bio-layer interferometry (BLI), surface plasmon resonance (SPR) and grating-coupled interferometry (GCI) generated more than 2500 scientific papers from 2022. Compared to 2009, when David Myszka and Rebecca Rich presented their most recent review of biosensor literature (Rich and Myszka, 2011), this number has nearly doubled. With this increasing number of publications comes an increasing need for standardization of the way biosensor data is reported in journals to allow for replication of the experiments that were performed. Biosensor data is often poorly described in papers which makes it difficult, if not impossible, to replicate the experiment. Critical information typically missing includes sample preparation, method settings, and data evaluation details. We have also found published work in which the authors have failed to report the type of sensor that was used, or which biosensor instrumentation was used. To come to terms with this growing problem, we propose a standardization of the way biosensor data is reported in scientific journals. We call this standard STROBE, standards for reporting optical biosensor experiments.

以生物传感器数据为特色的同行评审出版物数量逐年增加。使用常见的技术术语，包括生物层干涉测量法（BLI）、表面等离子体共振（SPR）和光栅耦合干涉测量法（GCI），在PubMed上进行搜索，结果显示2022年以来发表了2500多篇科学论文。与 2009 年大卫-麦兹卡（David Myszka）和丽贝卡-里奇（Rebecca Rich）发表的最新生物传感器文献综述[1]相比，这一数字几乎翻了一番。随着论文数量的不断增加，人们越来越需要对期刊中生物传感器数据的报告方式进行标准化，以便对所进行的实验进行复制。论文中对生物传感器数据的描述往往很差，这使得复制实验变得很困难，甚至是不可能。通常缺少的关键信息包括样品制备、方法设置和数据评估细节。我们还发现，在已发表的论文中，作者没有报告所使用的传感器类型，也没有报告所使用的生物传感器仪器。为了解决这个日益严重的问题，我们建议对科学杂志中生物传感器数据的报告方式进行标准化。我们将这一标准称为 STROBE，即光学生物传感器实验报告标准。

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引用次数: 0

Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay 磁性骨骼肌球体三维生物打印，用于基于球体的筛选测定。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2024-10-28 DOI: 10.1016/j.slasd.2024.100190

Chayanit Chaweewannakorn , Khin The Nu Aye , Joao N. Ferreira

Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.

在过去十年中，基于三维（3D）的磁性细胞培养系统得到了快速发展。关于骨骼肌，三维培养系统可为肌肉生理学和新陈代谢领域的转化临床研究提供生物学见解。由于三维培养系统能促进细胞与细胞外基质（ECM）之间的强烈相互作用，因此能通过改善空间导向的细胞集结和形态特征来模拟体内组织/器官，从而改善细胞培养环境。然而，三维传统培养技术的耗时和复杂性对三维系统的广泛应用构成了挑战。本文介绍了一种工作台方案，用于创建一个创新、快速组装和用户友好的培养平台，用于骨骼肌球体的磁性三维生物打印。我们的方案研究结果表明，骨骼肌组织的形态学结果和功能发育是一致的，肌肉特异性收缩蛋白和肌管的表达以及对胆碱能神经递质刺激的反应都证明了这一点。这一概念验证方案证实了未来在人体骨骼肌研究中进一步验证和应用基于球蛋白的检测方法的潜力。

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引用次数: 0

Bioassay protocol metadata annotation: Proposed standards adoption 生物测定协议元数据注释：建议采用的标准。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2024-10-19 DOI: 10.1016/j.slasd.2024.100188

Rama Balakrishnan , Ellen L. Berg , Christopher C. Butler , Alex M. Clark , Sheryl P. Denker , Isabella Feierberg , Jason Harris , Timothy P. Ikeda , Samantha Jeschonek , Vladimir A. Makarov , Christopher Southan , Dana Vanderwall , Peter Winstanley

We present a standardized metadata template for assays used in pharmaceutical drug discovery research, according to the FAIR principles. We also describe the use of an automated tool for annotating assays from a variety of sources, including PubChem, commercial assay providers, and the peer-reviewed literature, to this metadata template. Adoption of a standardized metadata template will allow drug discovery scientists to better understand and compare the increasing amounts of assay data becoming available, and will facilitate the use of artificial intelligence tools and other computational methods for analysis and prediction. Since bioassays drive advances in biomedical research, improvements in assay metadata can improve productivity in discovery of new therapeutics, platform technologies, and assay methods.

我们根据 FAIR 原则为药物发现研究中使用的检测方法提供了一个标准化元数据模板。我们还介绍了一种自动工具的使用情况，该工具可根据该元数据模板对各种来源的化验结果进行注释，包括 PubChem、商业化验提供商和同行评议文献。采用标准化元数据模板将使药物发现科学家能够更好地理解和比较日益增多的化验数据，并促进人工智能工具和其他计算方法在分析和预测中的应用。由于生物检测推动了生物医学研究的进步，因此改进检测元数据可以提高发现新疗法、平台技术和检测方法的效率。

引用次数: 0

A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies 反映作用机制的双细胞生物测定法，用于确定硬骨蛋白中和抗体的生物活性。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2024-10-01 DOI: 10.1016/j.slasd.2024.100187

Suzhen Wei , Qiang Wu , Chunlai Cao , Zhuoni Yang , Jianrui Shi , Jingqun Huang , Hua He , Yongjie Lai , Jing Li

Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs.

骨质疏松症是全球老年人面临的主要威胁。Wnt 信号通路在骨骼发育和稳态中起着至关重要的作用。硬骨素是一种 Wnt 配体抑制剂，能与 Wnt 配体竞争成骨细胞上的低密度脂蛋白受体相关蛋白 5 或 6（LRP5/6），从而抑制骨形成。硬骨素中和单克隆抗体（mAbs）已成为治疗骨质疏松症的潜在骨形成疗法。以细胞为基础的生物测定可确定产品的相对活性（与其作用机制有关），从药物发现到质量控制和批量生产都非常重要。目前用于硬骨素中和 mAbs 的细胞生物测定通常使用 Wnt1 或 Wnt3a 来刺激 Wnt 通路；硬骨素是 Wnt1 的直接抑制剂，但不是 Wnt3a 的直接抑制剂。Wnt1 是一种高度疏水性蛋白质，它能与产生细胞的细胞膜结合，并以共刺激方式刺激邻近细胞的 Wnt 通路。对诱导 Wnt1 信号传导的药物进行生物测定时，应采用并列方式。在这里，我们提出了一种反映作用机制的双细胞报告基因检测方法。在这种检测方法中，Wnt1 生产者细胞与含有 Wnt 报告基因的细胞共同培养，生产者细胞上的 Wnt1 会激活细胞间直接接触的报告基因细胞中的 Wnt 信号通路，而硬骨蛋白中和 mAbs 能特异性地有效拮抗硬骨蛋白介导的 Wnt 报告基因抑制。该生物测定方法具有良好的特异性、准确性、线性和精密度，适用于硬骨蛋白中和 mAbs 的质量控制、稳定性测试、批量放行和生物相似性评估。

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引用次数: 0