Pub Date : 2024-11-08DOI: 10.1016/j.slasd.2024.100193
Antje Pommereau , Francesca Sassone , Alessandro Poli , Marcella De Silvestris , Lia Scarabottolo , Yasmin Zuschlag , Thomas Licher , Felix Bärenz
GLUT9/SLC2A9 is a urate transporter and takes a fundamental role in the maintenance of normal serum urate levels. GLUT9 is the sole transporter of reabsorbed urate from renal epithelial cells to blood, thus making it an ideal pharmacological target for the development of urate-lowering drugs. None of the three currently available assays for studying GLUT9 pharmacological inhibition can support a high throughput drug discovery screening campaign. In this manuscript we present two novel assay technologies which can be used in a drug discovery screening cascade for GLUT9: a GLUT9 membrane potential assay for primary screening; and a solid-supported membrane (SSM)-based supported electrophysiological assay for secondary screening.
{"title":"The development of a novel high-throughput membrane potential assay and a solid-supported membrane (SSM)-based electrophysiological assay to study the pharmacological inhibition of GLUT9/SLC2A9 isoforms in a drug discovery program","authors":"Antje Pommereau , Francesca Sassone , Alessandro Poli , Marcella De Silvestris , Lia Scarabottolo , Yasmin Zuschlag , Thomas Licher , Felix Bärenz","doi":"10.1016/j.slasd.2024.100193","DOIUrl":"10.1016/j.slasd.2024.100193","url":null,"abstract":"<div><div>GLUT9/SLC2A9 is a urate transporter and takes a fundamental role in the maintenance of normal serum urate levels. GLUT9 is the sole transporter of reabsorbed urate from renal epithelial cells to blood, thus making it an ideal pharmacological target for the development of urate-lowering drugs. None of the three currently available assays for studying GLUT9 pharmacological inhibition can support a high throughput drug discovery screening campaign. In this manuscript we present two novel assay technologies which can be used in a drug discovery screening cascade for GLUT9: a GLUT9 membrane potential assay for primary screening; and a solid-supported membrane (SSM)-based supported electrophysiological assay for secondary screening.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100193"},"PeriodicalIF":2.7,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-05DOI: 10.1016/j.slasd.2024.100191
Blaž Andlovic , Alexander Wolf , Malgorzata Hiltmann , Bert M. Klebl , Jan Eickhoff , Christian Ottmann
The Hippo pathway plays an important role in organ size control and tissue homeostasis. Dysregulation is involved in many pathologies, including cancer, which has attracted interest in targeting the Hippo pathway. Since the upstream components are bona fide tumor suppressors, it is feasible to target oncogenic downstream targets such as TAZ, a key downstream effector in the Hippo pathway. Its activity is regulated by phosphorylation on multiple sites, with Ser89 playing a critical role in regulation of TAZ activity. Phosphorylation of TAZ at Ser89 promotes binding to 14–3–3 scaffolding proteins, preventing nuclear translocation and abolishing target gene transcription. Here we describe the development of a cell-based assay suitable for high-throughput screening, based on a split NanoLuc luciferase, for monitoring interactions between 14 3–3 and TAZ in living cells. We have validated the assay by screening of a kinase-biased library. The assay can be quickly adapted for higher throughput and thus offers a valuable tool to study new signal inputs involved in regulation of TAZ activity as well as for identification of molecules that modulate the Hippo pathway.
{"title":"Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ","authors":"Blaž Andlovic , Alexander Wolf , Malgorzata Hiltmann , Bert M. Klebl , Jan Eickhoff , Christian Ottmann","doi":"10.1016/j.slasd.2024.100191","DOIUrl":"10.1016/j.slasd.2024.100191","url":null,"abstract":"<div><div>The Hippo pathway plays an important role in organ size control and tissue homeostasis. Dysregulation is involved in many pathologies, including cancer, which has attracted interest in targeting the Hippo pathway. Since the upstream components are <em>bona fide</em> tumor suppressors, it is feasible to target oncogenic downstream targets such as TAZ, a key downstream effector in the Hippo pathway. Its activity is regulated by phosphorylation on multiple sites, with Ser89 playing a critical role in regulation of TAZ activity. Phosphorylation of TAZ at Ser89 promotes binding to 14–3–3 scaffolding proteins, preventing nuclear translocation and abolishing target gene transcription. Here we describe the development of a cell-based assay suitable for high-throughput screening, based on a split NanoLuc luciferase, for monitoring interactions between 14 3–3 and TAZ in living cells. We have validated the assay by screening of a kinase-biased library. The assay can be quickly adapted for higher throughput and thus offers a valuable tool to study new signal inputs involved in regulation of TAZ activity as well as for identification of molecules that modulate the Hippo pathway.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100191"},"PeriodicalIF":2.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-04DOI: 10.1016/j.slasd.2024.100189
Ryan Chan , Christian Shema Mugisha , Vorada Chuenchob , Stephanie A. Moquin , Ujjini H. Manjunatha , Nadine Jarrousse , Vineet D. Menachery , Xuping Xie , Erika L. Flannery , Richard T. Eastman
Over the past 25 years, the global community has faced challenges posed by three distinct outbreaks of coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of a novel alphacoronavirus canine CoV (CCoV-HuPn2018) in human patients in Malaysia underscores the potential for crossover infections to humans. The threat of the ever-evolving nature of viral infections as well as the lingering health and socioeconomic effects of the recent SARS-CoV-2 pandemic emphasize the urgent need for advanced antiviral drug screening tools that can be quickly implemented to strengthen preparedness and preventive measures against future outbreaks. Here, we present the development and validation of a novel RNA-fluorescence in situ hybridization (FISH) imaging assay as a 384-well, high-throughput rapid response platform for antiviral drug discovery. RNA-FISH is a powerful tool to visualize specific mRNA in cultured cells using a high-content imaging platform. The flexibility of RNA-FISH probe sets allows for the rapid design of viral genome-specific probes, enabling in vitro assay development to test for inhibition of viral replication by either biologic or small molecule inhibitors. Screening of 170 antiviral compounds in concentration-response demonstrates a strong correlation between the RNA-FISH assay and an immunofluorescence assay (IFA) for both human coronaviruses HCoV-OC43 and HCoV-229E. Additionally, we successfully applied this methodology in the context of CCoV strain 1–71, proving rapid development and deployment, opening new avenues for the evaluation of antiviral drugs to potential future emerging threats.
{"title":"Rapid-response RNA-fluorescence in situ hybridization (FISH) assay platform for coronavirus antiviral high-throughput screening","authors":"Ryan Chan , Christian Shema Mugisha , Vorada Chuenchob , Stephanie A. Moquin , Ujjini H. Manjunatha , Nadine Jarrousse , Vineet D. Menachery , Xuping Xie , Erika L. Flannery , Richard T. Eastman","doi":"10.1016/j.slasd.2024.100189","DOIUrl":"10.1016/j.slasd.2024.100189","url":null,"abstract":"<div><div>Over the past 25 years, the global community has faced challenges posed by three distinct outbreaks of coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of a novel alphacoronavirus canine CoV (CCoV-HuPn2018) in human patients in Malaysia underscores the potential for crossover infections to humans. The threat of the ever-evolving nature of viral infections as well as the lingering health and socioeconomic effects of the recent SARS-CoV-2 pandemic emphasize the urgent need for advanced antiviral drug screening tools that can be quickly implemented to strengthen preparedness and preventive measures against future outbreaks. Here, we present the development and validation of a novel RNA-fluorescence <em>in situ</em> hybridization (FISH) imaging assay as a 384-well, high-throughput rapid response platform for antiviral drug discovery. RNA-FISH is a powerful tool to visualize specific mRNA in cultured cells using a high-content imaging platform. The flexibility of RNA-FISH probe sets allows for the rapid design of viral genome-specific probes, enabling <em>in vitro</em> assay development to test for inhibition of viral replication by either biologic or small molecule inhibitors. Screening of 170 antiviral compounds in concentration-response demonstrates a strong correlation between the RNA-FISH assay and an immunofluorescence assay (IFA) for both human coronaviruses HCoV-OC43 and HCoV-229E. Additionally, we successfully applied this methodology in the context of CCoV strain 1–71, proving rapid development and deployment, opening new avenues for the evaluation of antiviral drugs to potential future emerging threats.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100189"},"PeriodicalIF":2.7,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142578690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-31DOI: 10.1016/j.slasd.2024.100192
Paul E. Belcher , Anna Moberg , Michael B. Murphy
The number of peer-reviewed publications that feature biosensor data increases every year. A search of PubMed using common technique terminology, including bio-layer interferometry (BLI), surface plasmon resonance (SPR) and grating-coupled interferometry (GCI) generated more than 2500 scientific papers from 2022. Compared to 2009, when David Myszka and Rebecca Rich presented their most recent review of biosensor literature (Rich and Myszka, 2011), this number has nearly doubled. With this increasing number of publications comes an increasing need for standardization of the way biosensor data is reported in journals to allow for replication of the experiments that were performed. Biosensor data is often poorly described in papers which makes it difficult, if not impossible, to replicate the experiment. Critical information typically missing includes sample preparation, method settings, and data evaluation details. We have also found published work in which the authors have failed to report the type of sensor that was used, or which biosensor instrumentation was used. To come to terms with this growing problem, we propose a standardization of the way biosensor data is reported in scientific journals. We call this standard STROBE, standards for reporting optical biosensor experiments.
{"title":"Standards for reporting optical biosensor experiments (STROBE): Improving standards in the reporting of optical biosensor-based data in the literature","authors":"Paul E. Belcher , Anna Moberg , Michael B. Murphy","doi":"10.1016/j.slasd.2024.100192","DOIUrl":"10.1016/j.slasd.2024.100192","url":null,"abstract":"<div><div>The number of peer-reviewed publications that feature biosensor data increases every year. A search of PubMed using common technique terminology, including bio-layer interferometry (BLI), surface plasmon resonance (SPR) and grating-coupled interferometry (GCI) generated more than 2500 scientific papers from 2022. Compared to 2009, when David Myszka and Rebecca Rich presented their most recent review of biosensor literature (Rich and Myszka, 2011), this number has nearly doubled. With this increasing number of publications comes an increasing need for standardization of the way biosensor data is reported in journals to allow for replication of the experiments that were performed. Biosensor data is often poorly described in papers which makes it difficult, if not impossible, to replicate the experiment. Critical information typically missing includes sample preparation, method settings, and data evaluation details. We have also found published work in which the authors have failed to report the type of sensor that was used, or which biosensor instrumentation was used. To come to terms with this growing problem, we propose a standardization of the way biosensor data is reported in scientific journals. We call this standard STROBE, standards for reporting optical biosensor experiments.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100192"},"PeriodicalIF":2.7,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142565046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-28DOI: 10.1016/j.slasd.2024.100190
Chayanit Chaweewannakorn , Khin The Nu Aye , Joao N. Ferreira
Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.
{"title":"Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay","authors":"Chayanit Chaweewannakorn , Khin The Nu Aye , Joao N. Ferreira","doi":"10.1016/j.slasd.2024.100190","DOIUrl":"10.1016/j.slasd.2024.100190","url":null,"abstract":"<div><div>Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100190"},"PeriodicalIF":2.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142570581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-19DOI: 10.1016/j.slasd.2024.100188
Rama Balakrishnan , Ellen L. Berg , Christopher C. Butler , Alex M. Clark , Sheryl P. Denker , Isabella Feierberg , Jason Harris , Timothy P. Ikeda , Samantha Jeschonek , Vladimir A. Makarov , Christopher Southan , Dana Vanderwall , Peter Winstanley
We present a standardized metadata template for assays used in pharmaceutical drug discovery research, according to the FAIR principles. We also describe the use of an automated tool for annotating assays from a variety of sources, including PubChem, commercial assay providers, and the peer-reviewed literature, to this metadata template. Adoption of a standardized metadata template will allow drug discovery scientists to better understand and compare the increasing amounts of assay data becoming available, and will facilitate the use of artificial intelligence tools and other computational methods for analysis and prediction. Since bioassays drive advances in biomedical research, improvements in assay metadata can improve productivity in discovery of new therapeutics, platform technologies, and assay methods.
{"title":"Bioassay protocol metadata annotation: Proposed standards adoption","authors":"Rama Balakrishnan , Ellen L. Berg , Christopher C. Butler , Alex M. Clark , Sheryl P. Denker , Isabella Feierberg , Jason Harris , Timothy P. Ikeda , Samantha Jeschonek , Vladimir A. Makarov , Christopher Southan , Dana Vanderwall , Peter Winstanley","doi":"10.1016/j.slasd.2024.100188","DOIUrl":"10.1016/j.slasd.2024.100188","url":null,"abstract":"<div><div>We present a standardized metadata template for assays used in pharmaceutical drug discovery research, according to the FAIR principles. We also describe the use of an automated tool for annotating assays from a variety of sources, including PubChem, commercial assay providers, and the peer-reviewed literature, to this metadata template. Adoption of a standardized metadata template will allow drug discovery scientists to better understand and compare the increasing amounts of assay data becoming available, and will facilitate the use of artificial intelligence tools and other computational methods for analysis and prediction. Since bioassays drive advances in biomedical research, improvements in assay metadata can improve productivity in discovery of new therapeutics, platform technologies, and assay methods.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100188"},"PeriodicalIF":2.7,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142482706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.slasd.2024.100187
Suzhen Wei , Qiang Wu , Chunlai Cao , Zhuoni Yang , Jianrui Shi , Jingqun Huang , Hua He , Yongjie Lai , Jing Li
Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs.
{"title":"A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies","authors":"Suzhen Wei , Qiang Wu , Chunlai Cao , Zhuoni Yang , Jianrui Shi , Jingqun Huang , Hua He , Yongjie Lai , Jing Li","doi":"10.1016/j.slasd.2024.100187","DOIUrl":"10.1016/j.slasd.2024.100187","url":null,"abstract":"<div><div>Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 7","pages":"Article 100187"},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142402302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.slasd.2024.100185
Mozhgan Dehghan Harati , Jim King , Simon Langer , Florian Binder , Ralf Heilker
Human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) present a valuable substitute for monocyte-derived macrophages (MDMs) in order to study inflammation pathways in vitro. Through optimization of an IDM differentiation protocol, a six-fold increase in the production yield of myeloid progenitors was achieved. The derived IDMs were further characterized with respect to nucleotide-binding oligomerization domain (NOD) and receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signaling, a key regulatory pathway for autoimmune diseases. The IDM cells recapitulated MDM biology with respect to the proinflammatory chemokine and inflammatory cytokine fingerprint more closely than THP-1 cells. When assessing RIPK2 modulation effect on tumor necrosis factor α (TNF-α), a cardinal mediator of inflammation, a similar pharmacological effect of RIPK2 inhibitors was observed in IDMs and MDMs. Additionally, IDMs and MDMs displayed a similar transcription and pathway profile in response to NOD1/2 stimulation and pharmacological inhibition of RIPK2. In summary, the enhanced myeloid production yield in the improved IDM differentiation protocol offers new opportunities for utilizing physiologically relevant macrophage models in the context of inflammatory diseases.
{"title":"Recapitulation of NOD/RIPK2 signaling in iPSC-derived macrophages","authors":"Mozhgan Dehghan Harati , Jim King , Simon Langer , Florian Binder , Ralf Heilker","doi":"10.1016/j.slasd.2024.100185","DOIUrl":"10.1016/j.slasd.2024.100185","url":null,"abstract":"<div><div>Human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) present a valuable substitute for monocyte-derived macrophages (MDMs) in order to study inflammation pathways in vitro. Through optimization of an IDM differentiation protocol, a six-fold increase in the production yield of myeloid progenitors was achieved. The derived IDMs were further characterized with respect to nucleotide-binding oligomerization domain (NOD) and receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signaling, a key regulatory pathway for autoimmune diseases. The IDM cells recapitulated MDM biology with respect to the proinflammatory chemokine and inflammatory cytokine fingerprint more closely than THP-1 cells. When assessing RIPK2 modulation effect on tumor necrosis factor α (TNF-α), a cardinal mediator of inflammation, a similar pharmacological effect of RIPK2 inhibitors was observed in IDMs and MDMs. Additionally, IDMs and MDMs displayed a similar transcription and pathway profile in response to NOD1/2 stimulation and pharmacological inhibition of RIPK2. In summary, the enhanced myeloid production yield in the improved IDM differentiation protocol offers new opportunities for utilizing physiologically relevant macrophage models in the context of inflammatory diseases.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 7","pages":"Article 100185"},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142334048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.slasd.2024.100186
Thomas S. Dexheimer , Nathan P. Coussens , Thomas Silvers , Eric M. Jones , Li Chen , Jianwen Fang , Joel Morris , Jeffrey A. Moscow , James H. Doroshow , Beverly A. Teicher
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes of drug transporters and metabolic enzymes to detoxify small molecule xenobiotics. It has a complex role in cancer biology, influencing both the progression and suppression of tumors by modulating malignant properties of tumor cells and anti-tumor immunity, depending on the specific tumor type and developmental stage. This has led to the discovery and development of selective AhR modulators, including BAY 2416964 which is currently in clinical trials. To identify small molecule anticancer agents that might be combined with AhR antagonists for cancer therapy, a high-throughput combination screen was performed using multi-cell type tumor spheroids grown from malignant cells, endothelial cells, and mesenchymal stem cells. The AhR selective antagonists BAY 2416964, GNF351, and CH-223191 were tested individually and in combination with twenty-five small molecule anticancer agents. As single agents, BAY 2416964 and CH-223191 showed minimal activity, whereas GNF351 reduced the viability of some spheroid models at concentrations greater than 1 µM. The activity of most combinations aligned well with the single agent activity of the combined agent, without apparent contributions from the AhR antagonist. All three AhR antagonists sensitized tumor spheroids to TAK-243, an E1 ubiquitin-activating enzyme inhibitor. These combinations were active in spheroids containing bladder, breast, ovary, kidney, pancreas, colon, and lung tumor cell lines. The AhR antagonists also potentiated pevonedistat, a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit, in several tumor spheroid models. In contrast, the AhR antagonists did not enhance the cytotoxicity of the proteasome inhibitor bortezomib.
{"title":"Combination screen in multi-cell type tumor spheroids reveals interaction between aryl hydrocarbon receptor antagonists and E1 ubiquitin-activating enzyme inhibitor","authors":"Thomas S. Dexheimer , Nathan P. Coussens , Thomas Silvers , Eric M. Jones , Li Chen , Jianwen Fang , Joel Morris , Jeffrey A. Moscow , James H. Doroshow , Beverly A. Teicher","doi":"10.1016/j.slasd.2024.100186","DOIUrl":"10.1016/j.slasd.2024.100186","url":null,"abstract":"<div><div>The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes of drug transporters and metabolic enzymes to detoxify small molecule xenobiotics. It has a complex role in cancer biology, influencing both the progression and suppression of tumors by modulating malignant properties of tumor cells and anti-tumor immunity, depending on the specific tumor type and developmental stage. This has led to the discovery and development of selective AhR modulators, including BAY 2416964 which is currently in clinical trials. To identify small molecule anticancer agents that might be combined with AhR antagonists for cancer therapy, a high-throughput combination screen was performed using multi-cell type tumor spheroids grown from malignant cells, endothelial cells, and mesenchymal stem cells. The AhR selective antagonists BAY 2416964, GNF351, and CH-223191 were tested individually and in combination with twenty-five small molecule anticancer agents. As single agents, BAY 2416964 and CH-223191 showed minimal activity, whereas GNF351 reduced the viability of some spheroid models at concentrations greater than 1 µM. The activity of most combinations aligned well with the single agent activity of the combined agent, without apparent contributions from the AhR antagonist. All three AhR antagonists sensitized tumor spheroids to TAK-243, an E1 ubiquitin-activating enzyme inhibitor. These combinations were active in spheroids containing bladder, breast, ovary, kidney, pancreas, colon, and lung tumor cell lines. The AhR antagonists also potentiated pevonedistat, a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit, in several tumor spheroid models. In contrast, the AhR antagonists did not enhance the cytotoxicity of the proteasome inhibitor bortezomib.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 7","pages":"Article 100186"},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142373664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.slasd.2024.100184
Stephanie E. Doyle, Courtney N. Cazzola, Cynthia M. Coleman
Inducing osteogenic differentiation in vitro is useful for the identification and development of bone regeneration therapies as well as modelling bone disorders. To couple in vitro models with high throughput screening techniques retains the assay's relevance in research while increasing its therapeutic impact. Miniaturizing, automating and/or digitalizing in vitro assays will reduce the required quantity of cells, biologic stimulants, culture/output assay reagents, time and cost. This review highlights the design and workflow considerations for creating a high throughput screen-compatible model of osteogenesis, comparing and contrasting osteogenic cell type, assay fabrication and culture methodology, osteogenic induction approach and repurposing existing output techniques.
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