Pub Date : 2025-12-01DOI: 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.
{"title":"PCIM: Learning pixel attributions via pixel-wise channel isolation mixing in high content imaging","authors":"Daniel Siegismund, Mario Wieser, Stephan Heyse, 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}
Pub Date : 2025-12-01DOI: 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.
{"title":"Fluorescent probe-based detection of outer membrane damage of Gram-negative bacteria","authors":"Bingchen Li, Qionglu Duan, Wenjing Shi, Yinghong Li, Yuanjuan Wei, Shuyi Si, Yucheng Wang, Minghua Wang, 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}
Pub Date : 2025-12-01DOI: 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.
{"title":"High-throughput screening identifies non-nucleoside inhibitors of the SARS-CoV-2 polymerase with novel mechanisms","authors":"Colin R. Woodford, Kristine E. Frank, Haizhong Zhu, Gilman Dionne, Michael R. Schrimpf, Sujatha M. Gopalakrishnan, Nathaniel L. Elsen","doi":"10.1016/j.slasd.2025.100289","DOIUrl":"10.1016/j.slasd.2025.100289","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"37 ","pages":"Article 100289"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145582479","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}
Pub Date : 2025-11-29DOI: 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.
{"title":"Streamlining cellular thermal shift assay for ultra-high throughput screening","authors":"Pascal Lambertz, Loretta Hamacher, Jana Flegel, Philipp Pflüger, Torsten Feller, Mike Küster, Tom Stockter, Tommaso Mari, Yousef Morcos, 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":"2025-11-29","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}
Pub Date : 2025-11-25DOI: 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.
{"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 , Michael J. Lynch , Kurni Kurniyati , Chunhao Li , 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":"2025-11-25","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}
Pub Date : 2025-11-22DOI: 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.
{"title":"MICRO-TAG enzyme complementation enables quantification of cellular drug-target engagement in temperature series","authors":"Ivan Babic , Nikolas Bryan , Claire Cunningham , Avery Sampson , Daniel Starczynowski , 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":"2025-11-22","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}
Pub Date : 2025-11-19DOI: 10.1016/j.slasd.2025.100288
Glauco R. Souza, Evan Cromwell, Madhu Nag
{"title":"Protocols and research articles in 3D biology: technologies and methodologies reshaping 3D cell culture","authors":"Glauco R. Souza, Evan Cromwell, 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":"2025-11-19","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}
Pub Date : 2025-11-12DOI: 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.
{"title":"High-content immunofluorescence assay detecting PD-L1 expression changes in head and neck cancer patient-derived cultures","authors":"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","doi":"10.1016/j.slasd.2025.100286","DOIUrl":"10.1016/j.slasd.2025.100286","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"37 ","pages":"Article 100286"},"PeriodicalIF":2.7,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145524715","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}
Pub Date : 2025-10-25DOI: 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.
{"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, 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":"2025-10-25","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}
Pub Date : 2025-10-21DOI: 10.1016/j.slasd.2025.100284
Thomas S. Dexheimer , Nathan P. Coussens , Thomas Silvers , Poorva Juneja , Eric Jones , Steven D. Gore , Mark W. Kunkel , James H. Doroshow , Beverly A. Teicher
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.
g -四链（G4s）是一种调节关键细胞过程的四链核酸结构，是肿瘤学中有前景的治疗靶点。为了研究3种G4配体pidnarulex、APTO-253和braco -19的治疗潜力，我们对31个来自患者肿瘤和已建立的癌细胞系的多细胞型肿瘤球体进行了高通量联合筛选。这些三维球体模拟肿瘤微环境的关键特征，包括恶性、内皮细胞和间充质细胞群。根据G4s在复制和基因组稳定性中的作用，选择的化合物包括与G4生物学机制相关的药物，如DNA损伤反应（DDR）抑制剂、复制应激和染色质调节。通过细胞活力测定和纵向明场成像来监测球体形态和生长动态来评估组合反应。使用Bliss独立性评分和活力表面下的体积来量化药物相互作用，提供协同作用和总体反应的补充指标。在G4配体中，pidnarulex具有最广泛的单药活性，而APTO-253和BRACO-19作用有限。通过与PARP、DDR激酶抑制剂（ATM、ATR、DNA-PK）和细胞周期调节剂（WEE1、PIM1）的联合，观察到模型特异性协同作用。有趣的是，pidnarulex在8种胰腺腺癌模型之一（966289-007-R4-J1）中跨多种ddr靶向组合表现出一致的协同作用。在一些模型中也观察到与HDAC抑制剂的联合相互作用。亮场成像证实了协同组合增强的球体生长抑制。这些发现强调了G4配体的环境依赖性活性，并支持在生理相关的3D癌症模型中使用综合功能和基于成像的方法来表征潜在的治疗组合。
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