Pub Date : 2025-12-01Epub Date: 2025-11-20DOI: 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 : 2025-12-01Epub 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":"2025-12-01","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}
In this study we compare three methods for manipulating cell function: RNA interference (RNAi), CRISPR-Cas9 gene knock-out, and antibody-mediated loss-of-function. We have focused on analyzing changes in cell-matrix adhesion via targeting two key regulators, Talin1 (TLN1) and Kindlin-2 (KD2). Adhesion-relevant phenotypic assays revealed distinct temporal onset dynamics for each method. RNAi and CRISPR-Cas9 effectively reduced target mRNA and protein levels. In contrast, antibody transfection induced phenotypic changes without altering target expression, suggesting direct intracellular antibody-target interaction. Transcriptome analysis demonstrated that antibody transfection and CRISPR-Cas9 induced fewer deregulated mRNAs than RNAi. Furthermore, transfected antibodies and sgRNAs shared 30 % and 70 % of deregulated transcripts to their negative controls, respectively. Whereas only 10 % of overlap was recorded between targeting and control siRNAs. Our findings emphasize the importance of considering method-specific temporal dynamics of on-target phenotype appearance and off-target manifestation. Additionally, they highlight intracellular delivered antibodies as a valuable alternative for validating and complementing genetic approaches.
{"title":"Comparative analysis of antibody-mediated loss-of-function versus gene knock-out and knock-down","authors":"Marie Buck-Wiese , Sally Liechocki , Holger Erfle , Vytaute Starkuviene","doi":"10.1016/j.slasd.2025.100283","DOIUrl":"10.1016/j.slasd.2025.100283","url":null,"abstract":"<div><div>In this study we compare three methods for manipulating cell function: RNA interference (RNAi), CRISPR-Cas9 gene knock-out, and antibody-mediated loss-of-function. We have focused on analyzing changes in cell-matrix adhesion via targeting two key regulators, Talin1 (TLN1) and Kindlin-2 (KD2). Adhesion-relevant phenotypic assays revealed distinct temporal onset dynamics for each method. RNAi and CRISPR-Cas9 effectively reduced target mRNA and protein levels. In contrast, antibody transfection induced phenotypic changes without altering target expression, suggesting direct intracellular antibody-target interaction. Transcriptome analysis demonstrated that antibody transfection and CRISPR-Cas9 induced fewer deregulated mRNAs than RNAi. Furthermore, transfected antibodies and sgRNAs shared 30 % and 70 % of deregulated transcripts to their negative controls, respectively. Whereas only 10 % of overlap was recorded between targeting and control siRNAs. Our findings emphasize the importance of considering method-specific temporal dynamics of on-target phenotype appearance and off-target manifestation. Additionally, they highlight intracellular delivered antibodies as a valuable alternative for validating and complementing genetic approaches.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"37 ","pages":"Article 100283"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145304879","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 : 2025-10-01Epub Date: 2025-09-03DOI: 10.1016/j.slasd.2025.100269
Xiaohua Douglas Zhang
High-throughput screening (HTS) assays are pivotal in modern biomedical research, particularly in drug discovery and functional genomics. Ensuring the quality and reliability of HTS data is critical, especially when dealing with the small sample sizes that are typical in such assays. This study explores the integration of two powerful statistical metrics—Strictly Standardized Mean Difference (SSMD) and Area Under the Receiver Operating Characteristic Curve (AUROC)—for quality control (QC) in HTS. SSMD offers a standardized, interpretable measure of effect size, while AUROC provides a threshold-independent assessment of discriminative power. By establishing the theoretical and empirical relationships between AUROC and SSMD, we demonstrate how these metrics complement each other and enhance QC practices. We provide parametric, semi-parametric, and non-parametric estimation methods, and demonstrate the utility of the integrated framework in real HTS datasets. Our findings support the joint application of SSMD and AUROC as a robust and interpretable approach to improving QC in HTS, particularly under constraints of limited sample sizes of positive and negative controls.
{"title":"Integrating AUROC and SSMD for quality control in high-throughput screening assays","authors":"Xiaohua Douglas Zhang","doi":"10.1016/j.slasd.2025.100269","DOIUrl":"10.1016/j.slasd.2025.100269","url":null,"abstract":"<div><div>High-throughput screening (HTS) assays are pivotal in modern biomedical research, particularly in drug discovery and functional genomics. Ensuring the quality and reliability of HTS data is critical, especially when dealing with the small sample sizes that are typical in such assays. This study explores the integration of two powerful statistical metrics—Strictly Standardized Mean Difference (SSMD) and Area Under the Receiver Operating Characteristic Curve (AUROC)—for quality control (QC) in HTS. SSMD offers a standardized, interpretable measure of effect size, while AUROC provides a threshold-independent assessment of discriminative power. By establishing the theoretical and empirical relationships between AUROC and SSMD, we demonstrate how these metrics complement each other and enhance QC practices. We provide parametric, semi-parametric, and non-parametric estimation methods, and demonstrate the utility of the integrated framework in real HTS datasets. Our findings support the joint application of SSMD and AUROC as a robust and interpretable approach to improving QC in HTS, particularly under constraints of limited sample sizes of positive and negative controls.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100269"},"PeriodicalIF":2.7,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145006970","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 : 2025-10-01Epub Date: 2025-09-30DOI: 10.1016/j.slasd.2025.100279
Fengyu Cheng , Yuanyuan Ming , Yunsong Pan , Tianpeng Zhang , Mingxing Li , Rui Zhang , Hui Zhou , Hui Shi
Glioma, particularly glioblastoma (GBM), stands as the most prevalent and formidable tumor in neurosurgery, marked by relatively poor prognosis and high recurrence rates. Despite significant advancements in multimodal therapies—encompassing surgical intervention, radiotherapy, and systemic treatments such as chemotherapy and targeted agents—the overall survival rate for GBM still remains around 5%. In high-grade gliomas, dysregulated inflammatory pathways critically undermine the efficacy of surgical resection and subsequent targeted chemoradiotherapy, which are pivotal for managing these aggressive malignancies. Even with refined surgical techniques and chemotherapeutic mainstays like temozolomide (TMZ), patient prognoses persist in bleak territory, as survival rates languish far below clinical expectations. The NLRP3 inflammatory signaling pathway, extensively studied in this context, drives pathogenesis through allosteric activation and assembly into the NLRP3 inflammasome, catalyzing the maturation of IL-1β and IL-18, thereby triggering cascading inflammatory responses and pyroptosis. The NLRP3 signaling pathway plays a pivotal role in tumor progression and inflammatory responses, and targeted drugs have already entered Phase I clinical trials. Clinically, NLRP3 can serve as a prognostic biomarker, while the combined application of NLRP3-targeted drugs with novel materials and their technical specifics require further exploration. This review explores the NLRP3 pathway’s mechanistic role in glioma progression, reviews cutting-edge clinical research on NLRP3-targeting therapeutics, and evaluates the transformative potential of modulating this pathway in glioma treatment—offering referential insights into its clinical implications and the inflammatory microenvironment’s interplay with tumor dynamics.
{"title":"The NLRP3 signaling pathway is a potential target for clinical translation in glioma treatment","authors":"Fengyu Cheng , Yuanyuan Ming , Yunsong Pan , Tianpeng Zhang , Mingxing Li , Rui Zhang , Hui Zhou , Hui Shi","doi":"10.1016/j.slasd.2025.100279","DOIUrl":"10.1016/j.slasd.2025.100279","url":null,"abstract":"<div><div>Glioma, particularly glioblastoma (GBM), stands as the most prevalent and formidable tumor in neurosurgery, marked by relatively poor prognosis and high recurrence rates. Despite significant advancements in multimodal therapies—encompassing surgical intervention, radiotherapy, and systemic treatments such as chemotherapy and targeted agents—the overall survival rate for GBM still remains around 5%. In high-grade gliomas, dysregulated inflammatory pathways critically undermine the efficacy of surgical resection and subsequent targeted chemoradiotherapy, which are pivotal for managing these aggressive malignancies. Even with refined surgical techniques and chemotherapeutic mainstays like temozolomide (TMZ), patient prognoses persist in bleak territory, as survival rates languish far below clinical expectations. The NLRP3 inflammatory signaling pathway, extensively studied in this context, drives pathogenesis through allosteric activation and assembly into the NLRP3 inflammasome, catalyzing the maturation of IL-1β and IL-18, thereby triggering cascading inflammatory responses and pyroptosis. The NLRP3 signaling pathway plays a pivotal role in tumor progression and inflammatory responses, and targeted drugs have already entered Phase I clinical trials. Clinically, NLRP3 can serve as a prognostic biomarker, while the combined application of NLRP3-targeted drugs with novel materials and their technical specifics require further exploration. This review explores the NLRP3 pathway’s mechanistic role in glioma progression, reviews cutting-edge clinical research on NLRP3-targeting therapeutics, and evaluates the transformative potential of modulating this pathway in glioma treatment—offering referential insights into its clinical implications and the inflammatory microenvironment’s interplay with tumor dynamics.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100279"},"PeriodicalIF":2.7,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145214683","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 : 2025-10-01Epub Date: 2025-10-08DOI: 10.1016/j.slasd.2025.100281
Longfei Zhang, Moustafa T. Gabr
Chitinase-3-like 1 (CHI3L1) protein is a secreted glycoprotein involved in various normal physiological processes, while its abnormal elevation is closely associated to carcinogenesis. CHI3L1 recruits and polarizes immune cells into the tumor microenvironment to maintain an immune suppressive environment, and directly stimulate cancer cells to promote their proliferation and migration. Recent studies demonstrated the feasibility of CHI3L1 deletion in cancer treatment on animal models, however, only a limited number of molecular modulators have been developed. To address this gap, a TRIC-based high-throughput screening (HTS) platform was developed, and a library of 5280 molecules was screened. From the screen, 11 hits (hit rate: 0.21 %) were identified as CHI3L1 binders, and 3 compounds (9N05, 11C19, and 3C13) were validated using surface plasmon resonance (SPR). Among them, 9N05 demonstrated the strongest binding affinity towards CHI3L1, and the Kd value was measured as 202.3 ± 76.6 μM. In summary, this proof-of-concept study demonstrates the feasibility of TRIC-based screening for CHI3L1-targeted molecules, and provide a potent tool for the future CHI3L1 molecular modulator development.
{"title":"Temperature-related intensity change (TRIC)-based high throughput screening platform for the discovery of CHI3L1-targeted small molecules","authors":"Longfei Zhang, Moustafa T. Gabr","doi":"10.1016/j.slasd.2025.100281","DOIUrl":"10.1016/j.slasd.2025.100281","url":null,"abstract":"<div><div>Chitinase-3-like 1 (CHI3L1) protein is a secreted glycoprotein involved in various normal physiological processes, while its abnormal elevation is closely associated to carcinogenesis. CHI3L1 recruits and polarizes immune cells into the tumor microenvironment to maintain an immune suppressive environment, and directly stimulate cancer cells to promote their proliferation and migration. Recent studies demonstrated the feasibility of CHI3L1 deletion in cancer treatment on animal models, however, only a limited number of molecular modulators have been developed. To address this gap, a TRIC-based high-throughput screening (HTS) platform was developed, and a library of 5280 molecules was screened. From the screen, 11 hits (hit rate: 0.21 %) were identified as CHI3L1 binders, and 3 compounds (<strong>9N05, 11C19</strong>, and <strong>3C13</strong>) were validated using surface plasmon resonance (SPR). Among them, <strong>9N05</strong> demonstrated the strongest binding affinity towards CHI3L1, and the <em>K</em><sub>d</sub> value was measured as 202.3 ± 76.6 μM. In summary, this proof-of-concept study demonstrates the feasibility of TRIC-based screening for CHI3L1-targeted molecules, and provide a potent tool for the future CHI3L1 molecular modulator development.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100281"},"PeriodicalIF":2.7,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145269032","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 : 2025-10-01Epub Date: 2025-10-12DOI: 10.1016/j.slasd.2025.100282
Hannah A. Strobel , Manohar P. Bhandari , Eva Borras , Mitchell M. McCartney , Sarah M. Moss , Cristina E Davis , James B. Hoying
Identifying microbial contamination early in cell culture processing can save time, money, and reagents. However, standard strategies for performing contamination testing often take days, leaving production vulnerable to potential spread of bacteria, mold, and mycoplasma. Volatile organic compounds (VOCs) are released by every living organism. These VOCs are increasingly being used to identify cell pathologies, study cell metabolomics, and assess other biological processes. Often, gas chromatography with mass spectrometry (GC–MS) is used to perform these analyses. Here, we utilized gas chromatography with ion mobility spectrometry (GC-IMS) to detect bacteria, mold, and mycoplasma in both cell and tissue cultures. Traditional GC–MS was used to validate the detection of microbes in cell cultures using GC-IMS. In most cases, headspace samples were collected just two hours after inoculations. GC-IMS was able to detect as low as 10 CFU of 5 different industry standard microbes, including both bacteria and mold species. In addition, mycoplasma presence, which is notoriously difficult to test, was detectable at 24 h post-inoculation. GC-IMS is highly sensitive, has a small footprint, requires minimal training, and can provide results in as little as 20 min per sample. Combined, this makes it an ideal strategy for detecting contamination in cell and tissue production workflows. Such rapid detection could save substantial amounts of time, money, and valuable reagents, and reduce risks to patient safety.
{"title":"VOC analysis for rapid, early detection of bacteria, mold, and mycoplasma in cell and tissue cultures","authors":"Hannah A. Strobel , Manohar P. Bhandari , Eva Borras , Mitchell M. McCartney , Sarah M. Moss , Cristina E Davis , James B. Hoying","doi":"10.1016/j.slasd.2025.100282","DOIUrl":"10.1016/j.slasd.2025.100282","url":null,"abstract":"<div><div>Identifying microbial contamination early in cell culture processing can save time, money, and reagents. However, standard strategies for performing contamination testing often take days, leaving production vulnerable to potential spread of bacteria, mold, and mycoplasma. Volatile organic compounds (VOCs) are released by every living organism. These VOCs are increasingly being used to identify cell pathologies, study cell metabolomics, and assess other biological processes. Often, gas chromatography with mass spectrometry (GC–MS) is used to perform these analyses. Here, we utilized gas chromatography with ion mobility spectrometry (GC-IMS) to detect bacteria, mold, and mycoplasma in both cell and tissue cultures. Traditional GC–MS was used to validate the detection of microbes in cell cultures using GC-IMS. In most cases, headspace samples were collected just two hours after inoculations. GC-IMS was able to detect as low as 10 CFU of 5 different industry standard microbes, including both bacteria and mold species. In addition, mycoplasma presence, which is notoriously difficult to test, was detectable at 24 h post-inoculation. GC-IMS is highly sensitive, has a small footprint, requires minimal training, and can provide results in as little as 20 min per sample. Combined, this makes it an ideal strategy for detecting contamination in cell and tissue production workflows. Such rapid detection could save substantial amounts of time, money, and valuable reagents, and reduce risks to patient safety.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100282"},"PeriodicalIF":2.7,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145287836","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 : 2025-10-01Epub Date: 2025-08-28DOI: 10.1016/j.slasd.2025.100268
Suganya Sivagurunathan , Patrick Byrne , Alán F. Muñoz , John Arevalo , Anne E. Carpenter , Shantanu Singh , Maria Kost-Alimova , Beth A. Cimini
Background
Cell Painting, the leading image-based profiling assay, involves staining plated cells with six dyes that mark the different compartments in a cell. Such profiles can then be used to discover connections between samples (whether different cell lines, different genetic treatments, or different compound treatments) as well as to assess particular features impacted by each treatment. Researchers may wish to vary the standard dye panel to assess particular phenotypes, or image cells live while maintaining the ability to cluster profiles overall.
Methods
In this study, we evaluate the performance of dyes that can either replace or augment the traditional Cell Painting dyes or enable tracking live cell dynamics. We perturbed U2OS cells with 90 different compounds and subsequently stained them with either standard Cell Painting dyes (Revvity), or with MitoBrilliant (Tocris) replacing MitoTracker or Phenovue phalloidin 400LS (Revvity) replacing phalloidin. We also tested the live-cell compatible ChromaLive dye (Saguaro).
Results
All dye sets effectively separated biological replicates of the same sample vs. negative controls (phenotypic activity), although separating from replicates of all other compounds (phenotypic distinctiveness) proved challenging for all dye sets. While individual dye substitutions within the standard Cell Painting panel had minimal impact on assay performance, the live cell dye exhibited distinct performance profiles across different compound classes compared to the standard panel, with later time points more distinct than earlier ones.
Discussion
Substituting MitoBrilliant or Phenovue phalloidin 400LS for standard mitochondrial or actin dyes minimally impacted Cell Painting assay performance. Phenovue phalloidin 400LS offers the advantage of isolating actin features from Golgi or plasma membrane while accommodating an additional 568 nm dye. Live cell imaging, enabled by ChromaLive dye, provides real-time assessment of compound-induced morphological changes. Combining this with the standard Cell Painting assay significantly expands the feature space for enhanced cellular profiling. Our findings provide data-driven options for researchers selecting dye sets for image-based profiling.
{"title":"Alternate dyes for image-based profiling assays","authors":"Suganya Sivagurunathan , Patrick Byrne , Alán F. Muñoz , John Arevalo , Anne E. Carpenter , Shantanu Singh , Maria Kost-Alimova , Beth A. Cimini","doi":"10.1016/j.slasd.2025.100268","DOIUrl":"10.1016/j.slasd.2025.100268","url":null,"abstract":"<div><h3>Background</h3><div>Cell Painting, the leading image-based profiling assay, involves staining plated cells with six dyes that mark the different compartments in a cell. Such profiles can then be used to discover connections between samples (whether different cell lines, different genetic treatments, or different compound treatments) as well as to assess particular features impacted by each treatment. Researchers may wish to vary the standard dye panel to assess particular phenotypes, or image cells live while maintaining the ability to cluster profiles overall.</div></div><div><h3>Methods</h3><div>In this study, we evaluate the performance of dyes that can either replace or augment the traditional Cell Painting dyes or enable tracking live cell dynamics. We perturbed U2OS cells with 90 different compounds and subsequently stained them with either standard Cell Painting dyes (Revvity), or with MitoBrilliant (Tocris) replacing MitoTracker or Phenovue phalloidin 400LS (Revvity) replacing phalloidin. We also tested the live-cell compatible ChromaLive dye (Saguaro).</div></div><div><h3>Results</h3><div>All dye sets effectively separated biological replicates of the same sample vs. negative controls (phenotypic activity), although separating from replicates of all other compounds (phenotypic distinctiveness) proved challenging for all dye sets. While individual dye substitutions within the standard Cell Painting panel had minimal impact on assay performance, the live cell dye exhibited distinct performance profiles across different compound classes compared to the standard panel, with later time points more distinct than earlier ones.</div></div><div><h3>Discussion</h3><div>Substituting MitoBrilliant or Phenovue phalloidin 400LS for standard mitochondrial or actin dyes minimally impacted Cell Painting assay performance. Phenovue phalloidin 400LS offers the advantage of isolating actin features from Golgi or plasma membrane while accommodating an additional 568 nm dye. Live cell imaging, enabled by ChromaLive dye, provides real-time assessment of compound-induced morphological changes. Combining this with the standard Cell Painting assay significantly expands the feature space for enhanced cellular profiling. Our findings provide data-driven options for researchers selecting dye sets for image-based profiling.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100268"},"PeriodicalIF":2.7,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144981862","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 : 2025-10-01Epub Date: 2025-09-16DOI: 10.1016/j.slasd.2025.100278
Laura L. Vollmer , Presley Roberts , Samantha L. Eicher , Marta Wołosowicz , Priyal Patel , Joseph R. Figura , Ella R. Donahue , Josh Berkowitz , Dillon Gavlock , Peter Wipf , Matt LaPorte , Steven J. Mullett , Amelle Shillington , Gregg E. Homanics , Michael J. Palladino , Andreas Vogt
Triosephosphate Isomerase deficiency (TPI-Df) is a devastating untreatable childhood metabolic disease resulting in anemia, severe locomotor impairment, and premature death. Numerous single amino acid substitutions in TPI are pathogenic and result in rapidly progressing multisystem disease. Importantly, all known pathogenic TPI-Df mutations result in a protein that retains function, and pathogenesis is known to result from decreased steady state levels of the functioning protein. There are no small molecule therapies for TPI-Df; current treatments are limited to symptomatic support and dietary interventions. We reasoned that a phenotypic screen was most appropriate to capture agents that stabilize mutant TPI and developed a human cellular TPI-Df assay based on a cellular model of the “common” TPIE105D mutant protein fused with a GFP and a fluorescent ROS biosensor. The assay was implemented for high-content, high-throughput imaging, optimized to full HTS standards, and used to screen a 2,560 compound pilot library and the 220,700 compound NIH MLSMR compound collection to identify candidate compounds for development into small molecule TPI-Df therapies. Hits were validated in dose-response, TPI-Df patient cells, and various orthogonal assays. Limited SAR revealed three promising compound series, which were evaluated for potential mechanisms of action. The lead series had previously been identified as inducers of HIF1 alpha, spawning a novel hypothesis that HIF1 alpha activation might be a potential avenue to treat TPI-Df patients. A lead molecule was moved into preliminary mouse studies to evaluate pharmacokinetics and tissue distribution and was shown to be moderately brain-penetrant. The lead compound is now positioned for target identification studies and efficacy testing in vivo TPI Df models, including a newly validated mouse model.
{"title":"Discovery and validation of small molecule stabilizers of mutant triose phosphate isomerase (TPI) as potential lead candidates for TPI deficiency","authors":"Laura L. Vollmer , Presley Roberts , Samantha L. Eicher , Marta Wołosowicz , Priyal Patel , Joseph R. Figura , Ella R. Donahue , Josh Berkowitz , Dillon Gavlock , Peter Wipf , Matt LaPorte , Steven J. Mullett , Amelle Shillington , Gregg E. Homanics , Michael J. Palladino , Andreas Vogt","doi":"10.1016/j.slasd.2025.100278","DOIUrl":"10.1016/j.slasd.2025.100278","url":null,"abstract":"<div><div><em>Triosephosphate Isomerase</em> deficiency (TPI-Df) is a devastating untreatable childhood metabolic disease resulting in anemia, severe locomotor impairment, and premature death. Numerous single amino acid substitutions in <em>TPI</em> are pathogenic and result in rapidly progressing multisystem disease. Importantly, all known pathogenic TPI-Df mutations result in a protein that retains function, and pathogenesis is known to result from decreased steady state levels of the functioning protein. There are no small molecule therapies for TPI-Df; current treatments are limited to symptomatic support and dietary interventions. We reasoned that a phenotypic screen was most appropriate to capture agents that stabilize mutant TPI and developed a human cellular TPI-Df assay based on a cellular model of the “common” TPI<sup>E105D</sup> mutant protein fused with a GFP and a fluorescent ROS biosensor. The assay was implemented for high-content, high-throughput imaging, optimized to full HTS standards, and used to screen a 2,560 compound pilot library and the 220,700 compound NIH MLSMR compound collection to identify candidate compounds for development into small molecule TPI-Df therapies. Hits were validated in dose-response, TPI-Df patient cells, and various orthogonal assays. Limited SAR revealed three promising compound series, which were evaluated for potential mechanisms of action. The lead series had previously been identified as inducers of HIF1 alpha, spawning a novel hypothesis that HIF1 alpha activation might be a potential avenue to treat TPI-Df patients. A lead molecule was moved into preliminary mouse studies to evaluate pharmacokinetics and tissue distribution and was shown to be moderately brain-penetrant. The lead compound is now positioned for target identification studies and efficacy testing in vivo TPI Df models, including a newly validated mouse model.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100278"},"PeriodicalIF":2.7,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145088523","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 : 2025-09-01Epub Date: 2025-07-30DOI: 10.1016/j.slasd.2025.100255
Natalie Fuchs , Katarzyna Kuncewicz , Farida El Gaamouch , Moustafa T. Gabr
Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor implicated in both neurodegenerative diseases and cancer. Depending on the context, TREM2 agonists or inhibitors hold therapeutic potential. To date, the majority of TREM2-targeted strategies have centered on monoclonal antibodies (mAbs), which face limitations such as poor tissue penetration and potential immunogenic side effects. To overcome these challenges and expand the chemical space for TREM2-targeting agents, we developed a high-throughput screening (HTS) platform to identify novel small molecule TREM2 binders. Using temperature-related intensity change (TRIC) technology in a 384-well plate format (NanoTemper Dianthus), we screened two focused compound libraries comprising over 1,200 molecules. From this screen, 18 preliminary hits (1.44 % hit rate) were identified and subsequently validated by dose-response binding studies using microscale thermophoresis (MST), yielding four validated hits (0.32 % hit rate) with binding affinities in the high to medium micromolar range (e.g., T2337, KD = 22.4 µM). The binding of the top hit, T2337, was further validated using surface plasmon resonance (SPR). Additionally, we assessed the functional activity of all four validated hits in a cellular assay measuring TREM2-mediated Syk phosphorylation in HEK293 cells co-expressing human TREM2 and its adaptor protein DAP12. These findings establish a robust and scalable platform for the discovery of small molecule TREM2 modulators and serve as a proof-of-concept for broader HTS campaigns targeting TREM2.
{"title":"TREM2 hit discovery using temperature-related intensity change (TRIC) technology: A proof-of-concept high-throughput screening approach","authors":"Natalie Fuchs , Katarzyna Kuncewicz , Farida El Gaamouch , Moustafa T. Gabr","doi":"10.1016/j.slasd.2025.100255","DOIUrl":"10.1016/j.slasd.2025.100255","url":null,"abstract":"<div><div>Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor implicated in both neurodegenerative diseases and cancer. Depending on the context, TREM2 agonists or inhibitors hold therapeutic potential. To date, the majority of TREM2-targeted strategies have centered on monoclonal antibodies (mAbs), which face limitations such as poor tissue penetration and potential immunogenic side effects. To overcome these challenges and expand the chemical space for TREM2-targeting agents, we developed a high-throughput screening (HTS) platform to identify novel small molecule TREM2 binders. Using temperature-related intensity change (TRIC) technology in a 384-well plate format (NanoTemper Dianthus), we screened two focused compound libraries comprising over 1,200 molecules. From this screen, 18 preliminary hits (1.44 % hit rate) were identified and subsequently validated by dose-response binding studies using microscale thermophoresis (MST), yielding four validated hits (0.32 % hit rate) with binding affinities in the high to medium micromolar range (e.g., <strong>T2337</strong>, <em>K</em><sub>D</sub> = 22.4 µM). The binding of the top hit, <strong>T2337</strong>, was further validated using surface plasmon resonance (SPR). Additionally, we assessed the functional activity of all four validated hits in a cellular assay measuring TREM2-mediated Syk phosphorylation in HEK293 cells co-expressing human TREM2 and its adaptor protein DAP12. These findings establish a robust and scalable platform for the discovery of small molecule TREM2 modulators and serve as a proof-of-concept for broader HTS campaigns targeting TREM2.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100255"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144763963","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号