SLAS Discovery最新文献

{"title":"AI-based analysis of label-free live cell imaging of T-cell mediated tumor killing assay enables competitive and robust hit calling","authors":"Josefa dela Cruz-Chuh ,&nbsp;Daniel Siegismund ,&nbsp;John Moffat ,&nbsp;Stephan Heyse ,&nbsp;Katherine R. Kozak ,&nbsp;Stephan Steigele","doi":"10.1016/j.slasd.2026.100297","DOIUrl":"10.1016/j.slasd.2026.100297","url":null,"abstract":"<div><div>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.</div><div>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.</div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"39 ","pages":"Article 100297"},"PeriodicalIF":2.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146127845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Streamlining cellular thermal shift assay for ultra-high throughput screening","authors":"Pascal Lambertz,&nbsp;Loretta Hamacher,&nbsp;Jana Flegel,&nbsp;Philipp Pflüger,&nbsp;Torsten Feller,&nbsp;Mike Küster,&nbsp;Tom Stockter,&nbsp;Tommaso Mari,&nbsp;Yousef Morcos,&nbsp;Martin Adamczewski","doi":"10.1016/j.slasd.2025.100293","DOIUrl":"10.1016/j.slasd.2025.100293","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"38 ","pages":"Article 100293"},"PeriodicalIF":2.7,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145656180","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An ELISA for discovering protein-protein interaction inhibitors: Blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook as a test case","authors":"Maithili Deshpande ,&nbsp;Michael J. Lynch ,&nbsp;Kurni Kurniyati ,&nbsp;Chunhao Li ,&nbsp;Brian R. Crane","doi":"10.1016/j.slasd.2025.100292","DOIUrl":"10.1016/j.slasd.2025.100292","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"38 ","pages":"Article 100292"},"PeriodicalIF":2.7,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145643982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protocols and research articles in 3D biology: technologies and methodologies reshaping 3D cell culture","authors":"Glauco R. Souza,&nbsp;Evan Cromwell,&nbsp;Madhu Nag","doi":"10.1016/j.slasd.2025.100288","DOIUrl":"10.1016/j.slasd.2025.100288","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"38 ","pages":"Article 100288"},"PeriodicalIF":2.7,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145574916","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"From Large to Small Cytokine Receptor Antagonists","authors":"Pawel Leznicki,&nbsp;Karl Deacon,&nbsp;Bohdan Waszkowycz,&nbsp;John F Unitt","doi":"10.1016/j.slasd.2025.100280","DOIUrl":"10.1016/j.slasd.2025.100280","url":null,"abstract":"<div><div>Here, we review general trends in biological and small molecule cytokine drug discovery, highlighting key learnings from two successful case studies (TNFα and IL-17), which have helped shape our understanding of how best to identify new small molecule cytokine antagonists.</div><div>Over the past three decades, biological drugs have revolutionized the treatment of a wide range of diseases, from oncology to autoimmune diseases. Their ability to modulate extracellular targets, often inaccessible to conventional small molecule drugs due to their complex protein-protein interactions, has enabled the clinical validation of numerous novel cytokine targets.</div><div>Despite their success, biological drugs have significant limitations. Challenges include poor oral bioavailability, tissue penetration and access to intracellular drug targets, as well as high manufacturing costs. These constraints have catalyzed efforts to develop small molecule equivalents that replicate the therapeutic efficacy of biologicals while overcoming their poor delivery and high production costs.</div><div>Over the past twenty years, advances in structural biology, computational modeling, disease biology, hit-finding technologies, and medicinal chemistry have converged to collectively enable the identification and subsequent progression of small molecule cytokine modulators into clinical development.</div><div>Looking ahead, we speculate on the future drug discovery landscape of this field with the likely emergence of small molecules drugs for a range of clinically validated cytokines like TSLP and TL1A. This evolution will be accelerated by the advent of novel modalities like extracellular degraders, oral peptide drugs and the development of next-generation biological drugs with multi-valency and improved delivery.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"38 ","pages":"Article 100280"},"PeriodicalIF":2.7,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145240333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MICRO-TAG enzyme complementation enables quantification of cellular drug-target engagement in temperature series","authors":"Ivan Babic ,&nbsp;Nikolas Bryan ,&nbsp;Claire Cunningham ,&nbsp;Avery Sampson ,&nbsp;Daniel Starczynowski ,&nbsp;Elmar Nurmemmedov","doi":"10.1016/j.slasd.2025.100291","DOIUrl":"10.1016/j.slasd.2025.100291","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"38 ","pages":"Article 100291"},"PeriodicalIF":2.7,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145598270","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput combination screening of Pidnarulex and other G-quadruplex ligands in multi-cell type tumor spheroids","authors":"Thomas S. Dexheimer ,&nbsp;Nathan P. Coussens ,&nbsp;Thomas Silvers ,&nbsp;Poorva Juneja ,&nbsp;Eric Jones ,&nbsp;Steven D. Gore ,&nbsp;Mark W. Kunkel ,&nbsp;James H. Doroshow ,&nbsp;Beverly A. Teicher","doi":"10.1016/j.slasd.2025.100284","DOIUrl":"10.1016/j.slasd.2025.100284","url":null,"abstract":"<div><div>G-quadruplexes (G4s) are four-stranded nucleic acid structures that regulate key cellular processes and represent promising therapeutic targets in oncology. To investigate the therapeutic potential of three G4 ligands—pidnarulex, APTO-253, and BRACO-19—a high-throughput drug combination screen was conducted in thirty-one multi-cell type tumor spheroids derived from patient tumors and established cancer cell lines. These 3D spheroids mimic key features of the tumor microenvironment, comprising malignant, endothelial, and mesenchymal cell populations. Compounds selected for combination screening included agents with mechanistic relevance to G4 biology, such as inhibitors of DNA damage response (DDR), replication stress, and chromatin regulation, based on the proposed roles of G4s in replication and genome stability. Combination responses were assessed using cell viability assays and supported by longitudinal brightfield imaging to monitor spheroid morphology and growth dynamics. Drug interactions were quantified using Bliss independence scores and the volume under the viability surface, providing complementary metrics of synergy and overall response. Among the G4 ligands, pidnarulex demonstrated the broadest single-agent activity, while APTO-253 and BRACO-19 showed limited effects. Model-specific synergy was observed from combinations with inhibitors of PARP, DDR kinases (ATM, ATR, DNA-PK), and cell cycle regulators (WEE1, PIM1). Interestingly, pidnarulex exhibited consistent synergy in one of eight pancreatic adenocarcinoma models (966289-007-R4-J1) across multiple DDR-targeted combinations. Combination interactions were also observed with HDAC inhibitors in a subset of models. Brightfield imaging corroborated enhanced spheroid growth suppression from synergistic combinations. These findings underscore the context-dependent activity of G4 ligands and support the use of integrated functional and imaging-based approaches to characterize potential therapeutic combinations in physiologically relevant 3D cancer models.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"37 ","pages":"Article 100284"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145357056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PCIM: Learning pixel attributions via pixel-wise channel isolation mixing in high content imaging","authors":"Daniel Siegismund,&nbsp;Mario Wieser,&nbsp;Stephan Heyse,&nbsp;Stephan Steigele","doi":"10.1016/j.slasd.2025.100287","DOIUrl":"10.1016/j.slasd.2025.100287","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"37 ","pages":"Article 100287"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145582507","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescent probe-based detection of outer membrane damage of Gram-negative bacteria","authors":"Bingchen Li,&nbsp;Qionglu Duan,&nbsp;Wenjing Shi,&nbsp;Yinghong Li,&nbsp;Yuanjuan Wei,&nbsp;Shuyi Si,&nbsp;Yucheng Wang,&nbsp;Minghua Wang,&nbsp;Yan Li","doi":"10.1016/j.slasd.2025.100290","DOIUrl":"10.1016/j.slasd.2025.100290","url":null,"abstract":"<div><div>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 <em>Escherichia coli</em> (<em>E. coli</em>), 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 <em>E. coli</em> 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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"37 ","pages":"Article 100290"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145590058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validation, optimization, and quality assessment of a neuro-behavioral in vivo assay for phenotypic high-content evaluation of anesthetics","authors":"Günther K.H. Zupanc,&nbsp;Mariam Ahmed","doi":"10.1016/j.slasd.2025.100285","DOIUrl":"10.1016/j.slasd.2025.100285","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"37 ","pages":"Article 100285"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145384593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}