SLAS Discovery最新文献_第4页

Orthogonal temperature-related intensity change (TRIC) and TR-FRET as a high-throughput screening platform for the discovery of SLIT2 binders: A proof-of-concept approach 正交温度相关强度变化（TRIC）和TR-FRET作为发现SLIT2结合物的高通量筛选平台：概念验证方法

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-08-15 DOI: 10.1016/j.slasd.2025.100264

Nelson García-Vázquez, Moustafa T. Gabr

SLIT2, a secreted glycoprotein involved in axon guidance, immune modulation, and tumor progression, remains largely unexplored as a pharmacological target due to the absence of small-molecule modulators. Here, we present a proof-of-concept high-throughput screening platform that integrates Temperature-Related Intensity Change (TRIC) technology with time-resolved Förster resonance energy transfer (TR-FRET) to identify small molecules capable of disrupting the SLIT2/ROBO1 interaction. Screening a lipid metabolism–focused compound library (653 molecules) yielded bexarotene, as the most potent small molecule SLIT2 binder reported to date, with a dissociation constant (K_D) of 2.62 µM. Follow-up TR-FRET assays demonstrated dose-dependent inhibition of SLIT2/ROBO1 interaction, with relative half-maximal inhibitory concentration (relative IC₅₀) = 77.27 ± 17.32 µM, with a maximal inhibition (Imax) of ∼40 % at 400 µM. These findings suggest a novel extracellular activity of bexarotene and validate the combined use of TRIC and TR-FRET as a scalable screening strategy for SLIT2-targeted small molecules. This platform lays the groundwork for future high-throughput discovery efforts against SLIT2 and its signaling axis.

SLIT2是一种参与轴突引导、免疫调节和肿瘤进展的分泌糖蛋白，由于缺乏小分子调节剂，SLIT2作为药理学靶点在很大程度上尚未被探索。在这里，我们提出了一个概念验证的高通量筛选平台，该平台集成了温度相关强度变化（TRIC）技术和时间分辨Förster共振能量转移（TR-FRET），以识别能够破坏SLIT2/ROBO1相互作用的小分子。筛选以脂质代谢为重点的化合物文库（653个分子）得到bexarotene，作为迄今为止报道的最有效的SLIT2小分子粘合剂，其解离常数（KD）为2.62µM。后续的TR-FRET试验显示SLIT2/ROBO1相互作用的抑制呈剂量依赖性，相对半最大抑制浓度（相对IC50） = 77.27±17.32µM，在400µM时最大抑制（Imax）为~ 40%。这些发现表明贝沙罗汀具有一种新的细胞外活性，并验证了TRIC和TR-FRET联合使用作为slit2靶向小分子的可扩展筛选策略。该平台为未来针对SLIT2及其信号轴的高通量发现工作奠定了基础。

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引用次数: 0

Cell based high-throughput screening for small molecule inhibitors of ATE1 基于细胞的高通量筛选ATE1小分子抑制剂。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-08-13 DOI: 10.1016/j.slasd.2025.100263

Claudia McCown , Evan A. Ambrose , Devang M. Patel , Hassan Al-Ali , Louis Scampavia , Fangliang Zhang , Timothy P. Spicer

Arginyltransferase 1 (ATE1) catalyzes post-translational arginylation, a process implicated in protein stability, cellular function, and disease pathology. Dysregulated arginylation is associated with neurodegenerative disorders, cancer, and inflammation. Particularly, the increase of ATE1 activity has been shown to cause cell death in response to acute stress, highlighting ATE1 as a promising therapeutic target. Despite its therapeutic relevance, no selective small-molecule inhibitors of ATE1 have been FDA-approved at this time, with previous screening efforts yielding compounds with high promiscuity and toxicity. This, in part, is due to the lack of assays that would accommodate large-scale screening for effective and safe ATE1-inhibitors. To address this challenge, we developed a cell-based high-throughput screening (HTS) assay utilizing a fluorescent reporter system based on an ATE1 substrate peptide fused to a fluorescence protein and co-expressed alongside another fluorescence protein for normalization. The assay enables real-time quantification of ATE1 activity by monitoring arginylation-dependent protein degradation within intact cells, measured by the ratio of the two fluorescence signals. We validated the assay in 96-well and 1536-well plate formats, demonstrating its scalability and robustness through key performance metrics, including Z'-factor and signal-to-background ratio. A pilot screen of a Library of Pharmacologically Active Compounds (LOPAC®1280) was performed to evaluate this approach. This study establishes a scalable and selective platform for discovering ATE1 inhibitors, paving the way for future therapeutic development targeting ATE1-mediated disease pathways.

精氨酸基转移酶1 （ATE1）催化翻译后精氨酸化，这一过程涉及蛋白质稳定性、细胞功能和疾病病理。精氨酸化失调与神经退行性疾病、癌症和炎症有关。特别是，ATE1活性的增加已被证明会导致细胞在急性应激反应中死亡，这表明ATE1是一个有希望的治疗靶点。尽管ATE1具有治疗意义，但目前fda还没有批准ATE1的选择性小分子抑制剂，之前的筛选工作产生了高混杂性和毒性的化合物。这在一定程度上是由于缺乏适合大规模筛选有效和安全的ate1抑制剂的检测方法。为了解决这一挑战，我们开发了一种基于细胞的高通量筛选（HTS）试验，利用荧光报告系统，该系统基于ATE1底物肽与荧光蛋白融合，并与另一种荧光蛋白共同表达以进行标准化。该分析通过监测完整细胞内精氨酸依赖蛋白的降解，通过两个荧光信号的比率来测量ATE1活性的实时定量。我们在96孔和1536孔平板格式中验证了该分析方法，通过关键性能指标（包括Z因子和信本比）证明了其可扩展性和稳健性。对药理学活性化合物库（LOPAC®1280）进行了试点筛选以评估该方法。该研究为发现ATE1抑制剂建立了一个可扩展和选择性的平台，为未来针对ATE1介导的疾病途径的治疗开发铺平了道路。

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引用次数: 0

Development of a BRET based chloride biosensor for high throughput screening of KCC2 modulators 用于高通量筛选KCC2调节剂的BRET氯化物生物传感器的研制

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-06-24 DOI: 10.1016/j.slasd.2025.100245

Charles S. Lay, Elvira Diamantopoulou, Katharina L. Dürr, Idlir Liko, Steven J. Charlton

The generation of action potentials in neuronal cells and many other physiological processes involve the transport of chloride ions. Whilst there have been advances in chloride imaging techniques utilizing FRET biosensors, there is a lack of methodologies that are amenable to high-throughput screening for drug discovery. In this study, we developed a novel BRET-based biosensor (Glorider), utilizing a chloride-sensitive GFP variant fused to NanoLuciferase. The Glorider biosensor was then used to kinetically measure the effect of WNK, KCC2 and NKCC1 modulators in real time in living cells, including recently reported KCC2 agonists.

神经细胞中动作电位的产生和许多其他生理过程都涉及氯离子的转运。虽然利用FRET生物传感器的氯化物成像技术取得了进展，但缺乏适用于药物发现的高通量筛选的方法。在这项研究中，我们开发了一种新型的基于bret的生物传感器（Glorider），利用氯化物敏感的GFP变体与NanoLuciferase融合。然后使用Glorider生物传感器实时动态测量WNK、KCC2和NKCC1调节剂在活细胞中的作用，包括最近报道的KCC2激动剂。

引用次数: 0

An overview of drugging the bacterial cytoskeleton, rod, and divisome systems 细菌细胞骨架、杆状细胞和分裂系统的药物研究综述。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-08-08 DOI: 10.1016/j.slasd.2025.100261

Elvis Awuni

Bacterial infections and antibiotic resistance remain significant threats to global health, with millions of related deaths recorded annually. Projections that antibacterial resistance-related deaths could reach alarming proportions in the coming years, along with the shortcomings of current interventions, highlight the need for new drug targets, novel antibiotics, and revised strategies and policy actions. The bacterial cytoskeleton, rod, and divisome systems (BCRDs) perform vital cellular roles and serve as a reserve of numerous potential therapeutic targets. The components of the BCRDs play different roles but share some relationships, suggesting the possibility of exploiting synergistic, polytherapeutic, and polypharmacological effects with antibiotics to mitigate bacterial resistance. Unfortunately, few drug targets within the BCRDs have been validated, and bacterial resistance to the inhibitors and approved antibiotics poses a challenge to the health and pharmaceutical industries. This review provides a concise but comprehensive overview of drugging the BCRDs, emphasizing the relationships and druggable potentials, validated targets, inhibitors, challenges, interventions, prospects, perspectives, and future directions geared toward reinvigorating research and overcoming bottlenecks in the sector. Overall, the material presented and discussed could facilitate the identification and validation of new therapeutic targets, the discovery and development of novel clinical drugs, and the revision of strategies and policy interventions to augment the fight against antibiotic resistance.

细菌感染和抗生素耐药性仍然对全球健康构成重大威胁，每年有数百万人因此死亡。预测与抗菌药物耐药性有关的死亡在未来几年可能达到惊人的比例，加上目前干预措施的缺点，突出表明需要新的药物靶点、新型抗生素以及修订战略和政策行动。细菌细胞骨架、杆状细胞和分裂系统（BCRDs）在细胞中起着至关重要的作用，并作为许多潜在治疗靶点的储备。bcrd的组成部分具有不同的功能，但具有一些共同的蛋白质和相互关联的作用，这表明利用抗生素的协同、多治疗和多药理作用来减轻细菌耐药性的可能性。不幸的是，bcrd中的药物靶点很少得到验证，细菌对抑制剂和已批准的抗生素的耐药性对卫生和制药行业构成了挑战。本文简要而全面地概述了bcrd的药物治疗，强调了功能关系和药物潜力，验证的靶点，抑制剂，挑战，干预措施，前景，观点和未来的方向，旨在重振研究和克服该领域的瓶颈。总的来说，所提出和讨论的材料可以促进新的治疗靶点的识别和验证，新的临床药物的发现和开发，以及战略和政策干预的修订，以加强对抗生素耐药性的斗争。

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引用次数: 0

Cytotoxic Profiling of Korea Chemical Bank Diversity Library 韩国化学银行多样性库的细胞毒性分析

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-08-23 DOI: 10.1016/j.slasd.2025.100267

Jinhee Kim , Kwang-Eun Choi , Yuno Lee , Daeyoung Jeong , Hyun Young Kim , Jung-In Lee , Heeyeong Cho , Nam-Chul Cho

Cytotoxicity profiling of screening libraries is a critical component in early-stage drug discovery to identify compounds with undesirable toxic effects. Here, we report the cytotoxicity profiling of the Korea Chemical Bank (KCB) diversity library, comprising 7040 compounds curated via virtual screening, clustering, and druggability assessment. A subset of 5181 compounds was randomly selected and screened using the WST-1 assay in five mammalian cell lines (HEK293, HFL1, HepG2, NIH3T3, and CHOK1) at concentrations of 30 µM and 10 µM, following 24 h and 48 h incubation periods. Cytotoxic compounds were defined as those exhibiting >50 % inhibition at 30 µM after 48 h. A total of 17 compounds showed consistent cytotoxicity across all five cell lines. Comparative analysis of physicochemical properties revealed that cytotoxic compounds exhibited higher lipophilicity (ALogP/ LogD) and the number of aromatic rings (AR) relative to non-cytotoxic compounds. These results indicate that the majority of the KCB diversity library comprises non-cytotoxic compounds, reflecting effective pre-filtering of toxic physicochemical properties during library design.

筛选文库的细胞毒性分析是早期药物发现中识别具有不良毒性作用的化合物的关键组成部分。在这里，我们报告了韩国化学银行（KCB）多样性文库的细胞毒性分析，该文库包括7040种化合物，通过虚拟筛选、聚类和药物性评估。在5种哺乳动物细胞系（HEK293、HFL1、HepG2、NIH3T3和CHOK1）中，随机选择5181个化合物，在浓度为30µM和10µM的条件下，经过24 h和48 h的孵育，使用WST-1试验进行筛选。细胞毒性化合物被定义为48小时后在30µM下表现出50%的抑制作用。共有17种化合物在所有5种细胞系中表现出一致的细胞毒性。理化性质对比分析表明，细胞毒性化合物的亲脂性（ALogP/ LogD）和芳香环数（AR）均高于非细胞毒性化合物。这些结果表明，大多数KCB多样性文库包含非细胞毒性化合物，反映了在文库设计过程中对毒性物理化学性质进行了有效的预过滤。

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引用次数: 0

High-throughput screening for the identification of dual inhibitors of BRD4 and RIPK3 toward the development of small-molecule medical countermeasure agents against arsenicals 高通量筛选鉴定BRD4和RIPK3双抑制剂，开发小分子抗砷医学对策剂。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-07-02 DOI: 10.1016/j.slasd.2025.100247

Marina Fosso Yatchang , Ling Zhai , Omar Moukha-Chafiq , Bini Mathew , Fuad Al Abir , Sixue Zhang , Pedro Ruiz , Sara McKellip , Miranda Nebane , Jake Y. Chen , Anupam Agarwal , James R. Bostwick , Mark J. Suto , Mohammad Athar , Corinne E. Augelli-Szafran

Warfare arsenicals are potent blistering agents and cause severe inflammation following their skin exposure. Data from our group (unpublished) show that these chemicals act by activating bromodomain-4 and RIPK signaling. To develop a dual inhibitor of the bromodomain-containing protein 4 (BRD4) and the receptor-interacting serine/threonine-protein kinase 3 (RIPK3), we conducted a high-throughput screening (HTS) campaign for inhibitors of BRD4 and RIPK3 activity to identify anti-inflammatory agent candidates that could alleviate arsenicals-induced injury. Our primary assays were adapted to 384-well microplates and used to screen a collection of 4074 compounds consisting of FDA-approved drugs and other bioactive compounds. The BRD4 primary screen had an average Z' value of 0.93 and a signal-to-background (S/B) ratio of 3018, while the RIPK3 primary screen had an average Z' value of 0.86 and S/B = 12.6. A counter screen assay was used to ensure activity was due to target engagement and not assay interference. Hits that inhibited BRD4 binding by > 54.6 % and kinase activity by > 22.4 % in the primary screen and were not statistical outliers in the counter screen assays, were confirmed in concentration-response format. Hits were also tested in a cell-based IL-6 assay to determine corresponding inflammatory inhibitory activity. Eighteen compounds were active in both BRD4 and RIPK3 assays, of which three displayed IC₅₀ values < 10 μM with promising IL-6 inhibition. These compounds could serve as good candidates for further chemical optimization for the development of small-molecule medical counter measure agents against arsenicals.

战争砷是强效的起泡剂，皮肤接触后会引起严重的炎症。我们小组的数据（未发表）表明，这些化学物质通过激活溴域-4和RIPK信号发挥作用。为了开发含溴结构域蛋白4 （BRD4）和受体相互作用丝氨酸/苏氨酸蛋白激酶3 （RIPK3）的双重抑制剂，我们对BRD4和RIPK3活性抑制剂进行了高通量筛选（HTS），以确定可以减轻砷诱导损伤的抗炎药候选物。我们的主要分析适用于384孔微孔板，用于筛选由fda批准的药物和其他生物活性化合物组成的4,074种化合物。BRD4主筛的平均Z′值为0.93，S/B比为3018；RIPK3主筛的平均Z′值为0.86,S/B = 12.6。使用反筛测定来确保活性是由于目标接触而不是测定干扰。在初级筛选中，hit抑制BRD4结合的>为54.6%，激酶活性的>为22.4%，在反筛试验中没有统计学异常值，在浓度-反应格式中得到证实。hit还通过基于细胞的IL-6检测来确定相应的炎症抑制活性。18种化合物在BRD4和RIPK3检测中均有活性，其中3种化合物的IC50值< 10 μM，具有良好的IL-6抑制作用。这些化合物可以作为进一步化学优化的良好候选者，用于开发小分子抗砷药物。

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引用次数: 0

Development of novel high-throughput biochemical competition assays to identify ligands of human asialoglycoprotein receptor 1 新型高通量生物化学竞争法鉴定人asialglyprotein receptor 1配体的建立

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-08-21 DOI: 10.1016/j.slasd.2025.100265

Jianming Liu , Bradley Peter , Lauren Rhodes , Mats Ormö , Bo Peng , Pia Hansson , Anders Gunnarsson , Laurent Knerr , Filip Miljković , Maria Ölwegård-Halvarsson , Mahya Dezfouli , Niek Dekker , Helena Lindmark , Shalini Andersson

Hepatocyte-specific Asialoglycoprotein receptor (ASGPR) and its native ligand N-acetylgalactosamine (GalNAc) have been actively exploited for targeted delivery of therapeutic and diagnostic agents to the liver. Identification of new potent ligands of ASGPR is of high interest to advance this field and expand to new applications in drug discovery. However, success of novel potent ASGPR ligand discovery has been limited due to the lack of robust high-throughput assays amenable to High-Throughput Screening (HTS). Here, we describe the design and development of two novel biochemical competition binding assays using recombinant human trimeric ASGR1 protein (ASGPR subunit 1) as a mimic of the native multimeric complex and a reference Alexa-647 fluorophore-labelled tri-GalNAc ligand as a tracer. Both ASGR1 TR-FRET and fluorescence polarization (FP) assays are in 384-well microplate format and have a large detection range (IC₅₀ of 2.5 nM - 100 µM), suitable for both monovalent and multivalent ASGPR ligands as well as oligonucleotide conjugates. The ASGR1 FP assay was miniaturized into a 1536-well assay format and a pilot screen of a small molecule library of about 7500 compounds was conducted, identifying 23 positive hits with IC₅₀ values between 12 - 100 µM. Five of the primary hits were validated in orthogonal TR-FRET and SPR binding assays and one of them was successfully docked into the ASGPR, with the docking pose closely matching the binding mode of structurally analogous compound found to be co-crystalized with ASGR1. The successful development of these new ASGR1 biochemical assays provides a new platform for an HTS campaign on small molecule collections to discover novel ASGPR ligands for liver-targeted delivery of efficient therapeutic agents, LYTACs or as potential drugs.

肝细胞特异性亚洲糖蛋白受体（ASGPR）及其天然配体n -乙酰半乳糖胺（GalNAc）已被积极用于靶向递送治疗和诊断药物到肝脏。发现新的ASGPR有效配体对该领域的发展和药物开发具有重要意义。然而，由于缺乏适用于高通量筛选（HTS）的强大的高通量分析，新型有效ASGPR配体发现的成功受到限制。在这里，我们描述了两种新的生化竞争结合试验的设计和开发，使用重组人三聚体ASGR1蛋白（ASGPR亚基1）作为天然多聚体复合物的模拟物和参考Alexa-647荧光团标记的三galnac配体作为示踪剂。ASGR1 TR-FRET和荧光偏振（FP）检测均采用384孔微孔板格式，具有较大的检测范围（IC50为2.5 nM - 100µM），适用于单价和多价ASGPR配体以及寡核苷酸偶联物。ASGR1 FP分析被缩小为1536孔的分析格式，并对大约7500个化合物的小分子文库进行了先导筛选，鉴定出23个阳性点，IC50值在12 - 100µM之间。通过正交TR-FRET和SPR结合实验验证了5个主要命中位点，其中1个成功对接ASGPR，对接姿态与ASGR1共晶结构类似化合物的结合模式非常接近。这些新的ASGR1生化检测方法的成功开发为小分子收集的HTS运动提供了一个新的平台，以发现新的ASGPR配体，用于肝脏靶向递送有效的治疗药物lytac或潜在药物。

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引用次数: 0

MT1G activation in dopaminergic neurons identifies chelators and their relationships to cytotoxicity 多巴胺能神经元MT1G激活鉴定螯合剂及其与细胞毒性的关系。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-06-21 DOI: 10.1016/j.slasd.2025.100244

Zhi-Bin Tong, Srilatha Sakamuru, James Travers, Tuan Xu, Shu Yang, Menghang Xia, Anton Simeonov, Ruili Huang, David Gerhold

Although multiple pesticides and solvents are risk factors for Parkinson’s disease [1] and other neurodegenerative diseases, most risk factors remain undiscovered. We previously identified the metallothionein gene MT1G as a biomarker for neurotoxicity induced by all seven neurotoxicants tested in LUHMES dopaminergic neurons. Here we used CRISP/R technology to insert a HiBiT tag into the MT1G gene of the LUHMES cell line. The engineered LUHMES MT1G::HiBiT cell lines were used to develop a quantitative high throughput screening [2] assay in a 3D-suspension culture platform with 1536 well microplates. We validated this qHTS assay by screening the LOPAC (Library of Pharmacologically Active Compounds) collection composed of 1280 compounds plus 88 selected Tox21 chemicals, demonstrating high signal-to-noise and reproducibility. In screening this library, 49 compounds were confirmed to significantly increase MT1G-HiBiT activity, including 35 compounds that exhibited cytotoxicity below 50 μM, and 14 noncytotoxic compounds. Most of these MT1G-HiBiT inducers killed cells at concentrations moderately higher than their MT1G-HiBiT activation potencies (AC₅₀), however 14 showed MT1G-HiBiT AC₅₀ values more than 3-fold lower than cytotoxicity IC₅₀ values, and two showed higher values. Among the 49 MT1G-HiBiT inducers, 45 compounds resembled chelators. To test this apparent association, 27 known chelators were gathered and tested. Of these, 23 were active in the MT1G-HiBiT activity assay, confirming the propensity of chelators to activate MT1G transcription. Screening chemical libraries with this validated assay and characterizing the effects of active chemicals on cultured neurons may enable the identification of neurotoxicants or neurotoxic chemotypes that may cause neurodegenerative diseases.

虽然多种农药和溶剂是帕金森病[1]和其他神经退行性疾病的危险因素，但大多数危险因素仍未被发现。我们之前确定金属硫蛋白基因MT1G是LUHMES多巴胺能神经元中所有7种神经毒物诱导的神经毒性的生物标志物。我们使用CRISP/R技术将HiBiT标签插入到LUHMES细胞系的MT1G基因中。利用LUHMES MT1G::HiBiT细胞系在1536孔微孔板的3d悬浮培养平台上进行定量高通量筛选[2]实验。我们通过筛选LOPAC（药理学活性化合物库）收集的1280种化合物和88种选定的Tox21化学物质来验证该qHTS方法，显示出高信噪比和重复性。在筛选该文库时，49个化合物被证实显著提高MT1G-HiBiT活性，其中35个化合物表现出50 μM以下的细胞毒性，14个化合物表现出非细胞毒性。大多数MT1G-HiBiT诱导剂在浓度高于其MT1G-HiBiT激活能力（AC50）时杀死细胞，但有14种MT1G-HiBiT诱导剂的AC50值低于细胞毒性IC50值的3倍以上，有2种诱导剂的AC50值高于细胞毒性IC50值。在49个MT1G-HiBiT诱导剂中，45个化合物与螯合剂相似。为了验证这种明显的联系，收集并测试了27种已知的螯合剂。其中，23个在MT1G- hibit活性测定中具有活性，证实了螯合剂激活MT1G转录的倾向。筛选化学文库与这种验证试验和表征活性化学物质对培养的神经元的影响可能使识别神经毒物或神经毒性化学型，可能导致神经退行性疾病。

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引用次数: 0

An enzyme activity-based workflow for the identification and characterization of covalent inhibitors 基于酶活性的共价抑制剂鉴定和表征工作流程。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-07-18 DOI: 10.1016/j.slasd.2025.100250

Buyun Tang, Tatiana Gladysheva, Paul Lang

The field of covalent drug development has advanced rapidly, offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins, resulting in prolonged pharmacodynamic effects. This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas, including oncology, neurological disorders, and infectious diseases. However, characterization of covalent inhibitors poses unique challenges, highlighting the need for simplified and robust assay methods. This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently. By streamlining the evaluation process, this approach enhances the reliability and reproducibility of covalent inhibitor assessment, ultimately accelerating the discovery and optimization of novel covalent therapeutics.

共价药物开发领域发展迅速，由于这些药物能够与靶蛋白形成缓慢可逆或不可逆的键，从而产生持久的药效学效应，因此具有广阔的治疗潜力。这种独特的作用机制激发了人们对共价抑制剂在各种疾病领域的兴趣，包括肿瘤学、神经系统疾病和传染病。然而，共价抑制剂的表征提出了独特的挑战，强调需要简化和强大的分析方法。本协议描述了一个基于酶活性的工作流程，旨在有效地识别和表征共价抑制剂。通过简化评估过程，该方法提高了共价抑制剂评估的可靠性和可重复性，最终加速了新型共价疗法的发现和优化。

引用次数: 0

A scalable human gut-immune co-culture model for evaluating inflammatory bowel disease anti-inflammatory therapies 评估炎症性肠病抗炎治疗的可扩展人类肠道免疫共培养模型。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-09-01 Epub Date: 2025-07-09 DOI: 10.1016/j.slasd.2025.100248

Swetha Peddibhotla , Lauren A. Boone , Earnest L. Taylor, Bryan E. McQueen, Elizabeth M. Boazak

Current treatments for inflammatory bowel disease (IBD) are often ineffective long-term, as many patients ultimately become unresponsive to anti-inflammatory drugs. The need for improved therapeutics is urgent. Animal models utilized for drug development are limited by interspecies variability and poor translatability. However, most in vitro models lack the sophistication to model the key interplay of the immune system with the intestinal epithelium in line with the known role of the immune system in the etiology of the disease.

To address this gap, we developed a primary intestinal epithelial cell co-culture system to incorporate elements of innate immune signaling. This system models immune-epithelial interactions using RepliGut^Ⓡ - Planar Transverse Colon cultured on a Transwell™ system with THP-1 derived macrophages in a receiver compartment of a 96-well plate format, compatible with high-throughput screening (HTS) workflows. Epithelial barrier integrity and cell viability were maintained in co-culture with unstimulated macrophages. However, similar to the pathology associated with IBD, epithelial integrity was compromised in co-culture with LPS + IFN-γ pre-stimulated macrophages as evidenced by declining TEER and cell viability and increased inflammatory cytokine release. Cotreatment with anti-inflammatory IBD therapeutics adalimumab or tofacitinib mitigated these effects, demonstrating the model’s ability to replicate key inflammatory responses and prevention.

Reproducibility and scalability of the model system further position the model for screening and/or mechanistic interrogation of anti-inflammatory drugs, improving drug discovery, and accelerating the translation of new IBD therapies into clinical practice.

目前对炎症性肠病（IBD）的治疗通常是长期无效的，因为许多患者最终对消炎药没有反应。迫切需要改进治疗方法。用于药物开发的动物模型受到物种间可变性和可翻译性差的限制。然而，大多数体外模型缺乏复杂性来模拟免疫系统与肠上皮的关键相互作用，以符合免疫系统在疾病病因学中的已知作用。为了解决这一空白，我们开发了一个原代肠上皮细胞共培养系统，以纳入先天免疫信号的元素。该系统使用在Transwell™系统上培养的RepliGut®- Planar横结肠，在96孔板格式的受体室中培养THP-1衍生巨噬细胞，模拟免疫-上皮相互作用，与高通量筛选（HTS）工作流程兼容。上皮屏障的完整性和细胞活力与未刺激的巨噬细胞共培养。然而，与IBD相关的病理类似，与LPS + IFN-γ预刺激的巨噬细胞共培养时，上皮完整性受到损害，TEER和细胞活力下降，炎症细胞因子释放增加。与抗炎IBD治疗药物阿达木单抗或托法替尼联合治疗减轻了这些影响，证明了该模型复制关键炎症反应和预防的能力。该模型系统的可重复性和可扩展性进一步为抗炎药物的筛选和/或机制询问奠定了基础，改善了药物发现，并加速了新的IBD治疗方法转化为临床实践。

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引用次数: 0